Patent

Date

September 24, 2020

Right

Case

2016(Wa)25436

Court

Tokyo District Court, 29th
Civil Division

number- A case in which a claim seeking an injunction and a claim for compensation fordamages based on the patent right for an invention titled "Process for constructing
amino acid-producing bacterium and process for producing amino acid by thefermentation method using the constructed amino acid-producing bacterium" and thepatent right for an invention titled "L-glutamic acid-producing bacterium and process
for producing L-glutamic acid" were partially affirmed.
Summary of the Judgment
In this case, the Plaintiff holds the patent right of Patent No. 3651002 for an
invention titled "Process for constructing amino acid-producing bacterium and process
for producing amino acid by the fermentation method using the constructed amino acidproducing bacterium" (hereinafter referred to as "Patent Right 1") and the patent rightof Patent No. 5343303 for an invention titled "L-glutamic acid-producing bacterium
and process for producing L-glutamic acid" (hereinafter referred to as "Patent Right 2").
The Plaintiff alleged that the processes for producing the monosodium glutamate
imported and sold by the Defendant (hereinafter the relevant monosodium glutamate isrespectively referred to as "Defendant's Products 1 through 4" and the relevant
processes are respectively referred to as "Defendant's Production Processes 1 through4") fall within the technical scope of the invention claimed in Patent 1 or 2 (hereinafter
respectively referred to as "Invention 1 or 2"), and that the Defendant's act of importing,transferring, and offering to transfer the Defendant's Products produced by the
Defendant's Production Processes, by itself or jointly with its foreign affiliated company,
constitutes infringement of Patent Right 1 with regard to Defendant's Products 1 and 3,
and Patent Right 2 with regard to Defendant's Products 1 through 4. Based on thisallegation, the Plaintiff made a claim seeking an injunction against transfer, import, andoffer to transfer of the Defendant's Products, a claim seeking disposal of the Defendant's
Products, and a claim for compensation for damages in tort due to patent infringement(a claim for damages under Article 102, paragraph (2) or (3) of the Patent Act andattorneys' fees).
The major issues of this case are as follows: [i] whether Defendant's Production
Processes fall within the technical scope of Invention 1 or 2 (literal infringement /infringement under the doctrine of equivalents); [ii] whether there are grounds foriinvalidation of the respective Patents (with regard to Patent 1, lack of an inventive step,
violation of the enablement requirement, or violation of the support requirement; withregard to Patent 2, lack of novelty, lack of an inventive step, violation of the enablement
requirement, violation of the support requirement, or violation of the clarity
requirement); [iii] whether re-defense of correction of the respective Patents can beestablished; [iv] occurrence or non-occurrence and the amount of damages; and [v]whether or not the claim seeking an injunction and the claim seeking disposal areappropriate.
In this judgment, [i] the court held that Defendant's Production Processes 1 and 3
literally fall within the technical scope of Invention 1, and Defendant's Production
Processes 1 through 3 fall within that of Invention 2, and that Defendant's Production
Process 4, which has differences with the configuration specified in Invention 2, suchas a difference in the species of bacterium from which the introduced mutant -type geneis derived, also falls within the technical scope of Invention 2 as an equivalent to theconfiguration specified in Invention 2. Although [ii] the court found that Invention 1
has a ground for invalidation, which is violation of the support requirement, [iii] it
affirmed establishment of re-defense of correction except for some parts due to reasons
such as that this ground for invalidation was eliminated by correction and no othergrounds for invalidation are found at least in corrected Invention 1. The court also foundestablishment of re-defense of correction for Invention 2 on the basis that no grounds
for invalidation are found at least in the corrected invention.
In this judgment, [iv] the court found the Defendant's act of importing, transferring,and offering to transfer as constituting the working of the Inventions for which the
Defendant is liable for tort with regard to the Defendant's Products sold by the
Defendant by itself, while finding the Defendant's act of offering to transfer in Japan as
constituting the same with regard to the Defendant's Products relating to the portion
sold by the affiliated company, and found establishment of joint tort by the Defendant
and the affiliated company with regard to these acts of working the Inventions. In
calculating the amount of damages under Article 102, paragraph (2) of the Patent Act
relating to these acts, the court found that the presumption has been rebutted for a partof the marginal profit based on sale of competitive products, but holding that the amountof damages that takes the rebuttal into consideration exceeds the amount of the partial
claim made by the Plaintiff in this case, the court entirely affirmed the amount claimedby the Plaintiff (the amount of the partial claim). [v] With regard to the claim seeking
an injunction and a claim seeking disposal, the court denied the claims based on Patent
Right 1 due to expiration of the patent term, and partially affirmed the claim seeking aniiinjunction and the claim seeking disposal based on Patent Right 2.

iii

Judgment rendered on September 24, 2020 Original received on the same date by the
court clerk
2016 (Wa) 25436 Appeal case of seeking injunction against patent right infringement
Date of conclusion of oral argument: March 19, 2020
Judgment
Indication of the parties: As stated in Attachment 1 "List of Parties."
Main text
1. The Defendant shall not transfer, import, or offer to transfer (including displaying forthe purpose of transfer) the respective products specified in 1. and 2. of Attachment 17"List of Products Subject to Injunction."
2. The Defendant shall dispose of the respective products specified in the preceding
paragraph.
3. The Defendant shall pay to the Plaintiff 990 million yen and an amount accrued thereonat the rate of 5% per annum for the period from August 10, 2016, until the completion of
the payment.
4. The Plaintiff’s other claims shall be dismissed.
5. The court costs shall be divided into 20, and the Plaintiff shall bear one-twentieth
thereof and the Defendant shall bear the remaining amount.
6. This judgment may be provisionally executed as far as paragraphs (1) and (3) areconcerned.
Facts and reasons[Table of Contents]
No. 1 Claims ..................................................................................................... 5
No. 2 Outline of the case .................................................................................. 6
1. Summary of the case................................................................................................. 6(1) Outline of the Plaintiff's claims........................................................................... 6(2) Outline of the Defendant's allegations ................................................................ 7
2. Basic facts, etc. ......................................................................................................... 8(1) Parties, etc. .......................................................................................................... 8(2) Production of monosodium glutamate by the fermentation method ................... 8(3) Patent Rights ....................................................................................................... 9(4) Claims, etc. of the invention claimed in Patent 1 ............................................. 10(5) Claims, etc. of the invention claimed in Patent 2 ..............................................11(6) Descriptions, etc. of the Patents ........................................................................ 12(7) Defendant's Products and Defendant's Production Processes ........................... 12(8) Prior art documents ........................................................................................... 13
1

3. Issues ...................................................................................................................... 14(omitted)
No. 4 Judgment of this court .......................................................................................... 15
1. Regarding Issue 1 (use or non-use, the periods of use, etc. of the Defendant's
Production Processes) ................................................................................................. 15(1) Regarding the period from January 2011 through May 2014 ........................... 15(2) Regarding the period from August 2015 through June 2016 ............................ 16(3) Regarding other periods .................................................................................... 18(4) Summary of the periods of use of the Defendant's Production Processes ........ 19
2. Regarding Issue 2-1 (whether Defendant's Production Processes 1 and 3 literally
fall within the technical scope of Invention 1) ........................................................... 20
3. Regarding Issue 2-2 (whether Defendant's Production Processes 1 through 3
literally fall within the technical scope of Invention 2) .............................................. 20(1) Regarding Defendant's Production Process 1 ................................................... 20(2) Regarding Defendant's Production Processes 2 and 3 ...................................... 20
4. Regarding Issue 2-3 (whether Defendant's Production Process 4 falls within thetechnical scope of Invention 2 as an equivalent thereto) ............................................ 21(1) Regarding the criteria for determination of infringement under the doctrineof equivalents .......................................................................................................... 21(2) Contents of Invention 2..................................................................................... 22(3) Regarding the configuration using the 19-type mutation ................................. 28(4) Regarding the strains to be used in Defendant's Production Process 4 ............ 31(5) Comparison between Defendant's Production Process 4 and Invention 2 ........ 34(6) Regarding the first requirement (non-essential part) ........................................ 35(7) Regarding the second requirement (replaceability) .......................................... 40(8) Regarding the third requirement (ease of replacement) .................................... 41(9) Regarding the fourth requirement (whether the subject process can be easily
presumptively conceived of) .................................................................................. 46(10) Regarding the fifth requirement (special circumstances) ............................... 46(11) Summary on the existence or non-existence of infringement under thedoctrine of equivalents............................................................................................ 48
5. Regarding Issue 3-3 (whether or not Invention 1 violates the enablement
requirement or the support requirement) .................................................................... 48(1) Statements concerning the process for producing arginine in Description 1 .... 48
2

(2) Regarding violation of the enablement requirement and the support
requirement by the process for producing arginine ................................................ 53(3) Summary on the grounds for invalidation regarding Invention 1 ..................... 55
6. Regarding Issue 5 (Whether re-defense of correction of Patent 1 can beestablished) ................................................................................................................... 55(1) Contents of Corrected Invention 1 ............................................................................ 55(2) Whether or not Defendant's Production Processes 1 and 3 fall within the technicalscope of Corrected Invention 1 .................................................................................... 58(3) Regarding Exhibit Otsu 6 Invention ......................................................................... 58(4) Regarding Exhibit Otsu 9 Invention ......................................................................... 62(5) Regarding matters described in publicly known documents other than Exhibit
Otsu 6 Document and Exhibit Otsu 9 Document ......................................................... 68(6) Whether or not Corrected Invention 1 lacks an inventive step based on Exhibit
Otsu 6 Invention as the primary cited document.......................................................... 75(7) Whether or not Corrected Invention 1 lacks an inventive step based on Exhibit
Otsu 9 Invention as the primary cited document.......................................................... 83(8) Whether or not Corrected Invention 1 violates the enablement requirement or the
support requirement...................................................................................................... 87(9) Summary on the defense of invalidity concerning Patent Right 1............................ 95
7. Regarding Issue 6 (whether re-defense of correction of Patent 2 (Correction 2) canbe established) .............................................................................................................. 97(1) Whether Correction 2 satisfies the requirements for correction ............................... 97(2) Regarding whether the Defendant's Production Processes fall within the technicalscope of Corrected Invention 2 .................................................................................... 99(3) Whether or not Corrected Invention 2 lacks novelty based on Exhibit Otsu 42
Invention..................................................................................................................... 100(4) Whether or not Corrected Invention 2 lacks an inventive step based on Exhibit
Otsu 24 Invention as the primary cited document...................................................... 101(5) Whether or not Corrected Invention 2 violates the enablement requirement or the
support requirement.................................................................................................... 104(6) Whether or not Corrected Invention 2 violates the clarity requirement ..................113(7) Summary on the defense of invalidity concerning Patent Right 2...........................113
8. Regarding Issue 8 (occurrence or non-occurrence and the value of damages) .........113(1) Organization of the periods of use of the Defendant's Production Processes andthe correspondence with the Patent Rights .................................................................114(2) Scope of working of inventions for which the Defendant is liable for tort .............115
3

(3) Regarding the value of damages under Article 102, paragraph (2) of the Patent
Act for the entire portion sold by the Defendant, etc. (Attachment 11-1 "Allegationon the Value of Damages 1") ...................................................................................... 123(4) Regarding lawyer fees and patent attorney fees ..................................................... 132(5) Summary on the claim for compensation for damages ........................................... 132
9. Regarding Issue 9 (whether or not the claim seeking injunction and the claim
seeking disposal are appropriate) ............................................................................... 133(1) Regarding the claims based on Patent Right 1........................................................ 133(2) Regarding the claims based on Patent Right 2........................................................ 133
10. Conclusion .............................................................................................................. 134
List of Attachments ....................................................................................................... 134
Attachment 1 "List of Parties" ...................................................................................... 135
Attachment 2 "List of the Defendant's Products" ......................................................... 136
Attachment 3 "List of the Defendant's Production Processes" ..................................... 137
Attachment 4-1 "Claims (Patent 1)" ............................................................................. 138
Attachment 4-2 "Corrected Claims (Patent 1)" ............................................................ 139
Attachment 4-3 "Division into Constituent Features (Invention 1)" ............................ 140
Attachment 4-4 "Division into Constituent Features (Corrected Invention 1)" ........... 141
Attachment 5-1 "Claims (Patent 2)" ............................................................................. 142
Attachment 5-2 "Corrected Claims (Patent 2)" ............................................................ 145
Attachment 5-3 "Re-corrected Claims (Patent 2)" ....................................................... 148
Attachment 5-4 "Division into Constituent Features (Invention 2)" ............................ 150
Attachment 5-5 "Division into Constituent Features (Corrected Invention 2)" ........... 153
Attachment 5-6 "Division into Constituent Features (Re-corrected Invention 2)" ...... 156
Attachment 6-1 "Comparison between Defendant's Production Processes 1 and 3 andInvention 1" ................................................................................................................ 158
Attachment 6-2 "Comparison between Defendant's Production Processes 1 and 3 andCorrected Invention 1" ............................................................................................... 162
Attachment 7-1 "Comparison between Defendant's Production Processes 1 through 3
and Invention 2" ......................................................................................................... 165
Attachment 7-2 "Comparison between Defendant's Production Processes 1 through 3
and Corrected Invention 2" ........................................................................................ 168
Attachment 7-3 "Comparison between Defendant's Production Processes 1 through 3
and Re-corrected Invention 2" ................................................................................... 172
Attachment 8-1 "Comparison between Defendant's Production Process 4 andInvention 2" ................................................................................................................ 174
4

Attachment 8-2 "Comparison between Defendant's Production Process 4 andCorrected Invention 2" ............................................................................................... 176
Attachment 8-3 "Comparison between Defendant's Production Process 4 and Recorrected Invention 2" ................................................................................................ 178
Attachment 9 "Comparison Table of Allegations concerning Processes for Producing
the MSG in Question" ................................................................................................ 180
Attachment 10 "List of Sales Values/Volumes"............................................................ 182
Attachment 11-1 "Allegations on the Value of Damages 1" (Value of Damages under
Article 102, Paragraph (2) of the Patent Act for the Overall Portion Sold by the
Defendant, etc.) .......................................................................................................... 183
Attachment 11-2 "Allegations on the Value of Damages 2" (Value of Damages under
Article 102, paragraph (3) of the Patent Act for the Overall Portion Sold by the
Defendant, etc.) .......................................................................................................... 192
Attachment 11-3 "Allegations on the Value of Damages 3" (Value of Damages under
Article 102, paragraph (3) of the Patent Act for the Portion Sold by the Defendant
and under Paragraph (2) of that Article for the Portion Sold by CJ Indonesia) ......... 195
Attachment 11-4 "Allegations on the Value of Damages 4" (Value of Damages under
Article 102, Paragraph (3) of the Patent Act for the Portion Sold by the Defendant
and under Paragraph (2) of That Article for the Defendant's Commissions for thePortion Sold by CJ Indonesia) .................................................................................... 199
Attachment 12 "Statements in Description 1" .............................................................. 202
Attachment 13 "Statements in Description 2" .............................................. 219
Attachment 14 "Drawings of Cited Documents" ......................................................... 266
Attachment 15 "Calculation Table of the Value of Damages under Article 102,
Paragraph (2) of the Patent Act for the Portion Sold by the Defendant, etc." ............ 272
Attachment 16 "Situation of Import of Monosodium Glutamate During the Period of
Use of the Defendant's Production Processes" ........................................................... 273
Attachment 17 "List of Products Subject to Injunction" .............................................. 274

No. 1 Claims
1. The Defendant shall not transfer, import, or offer to transfer (including displaying forthe purpose of transfer) the monosodium glutamate specified in 1. through 4. of
Attachment 2 "List of the Defendant's Products."
2. The Defendant shall dispose of the monosodium glutamate specified in 1. through 4.
of Attachment 2 "List of the Defendant's Products" which are under the Defendant's
5possession.
3. The same as paragraph (3) of the main text.
No. 2 Outline of the case
1. Summary of the case(1) Outline of the Plaintiff's claims
In this case, the Plaintiff holds the patent right of Patent No. 3651002 for an invention
titled "Process for constructing amino acid-producing bacterium and process for
producing amino acid by the fermentation method using the constructed amino acidproducing bacterium" (hereinafter, this patent right is referred to as "Patent Right 1" andthis patent is referred to as "Patent 1") and the patent right of Patent No. 5343303 for an
invention titled "L-glutamic acid-producing bacterium and process for producing Lglutamic acid" (hereinafter, this patent right is referred to as "Patent Right 2" and thispatent is referred to as "Patent 2"; in addition, Patent Right 1 and Patent Right 2 arecollectively referred to as the "Patent Rights" and Patent 1 and Patent 2 are collectively
referred to as the "Patents"). The Plaintiff alleged that the processes for producing themonosodium glutamate specified in 1. through 4. of Attachment 2 "List of the Defendant's
Products" (hereinafter, each monosodium glutamate is referred to as "Defendant's Product1" or the like according to the number assigned to it on that list, and they are collectively
referred to as the "Defendant's Products") respectively by the corresponding processes
specified in Attachment 3 "List of the Defendant's Production Processes" (hereinafter,
each process is referred to as "Defendant's Production Process 1" or the like according to
the number assigned to it on that list, and they are collectively referred to as the"Defendant's Production Processes") fall within the technical scopes of the inventions
claimed in Patents 1 and 2, and that the Defendant's act of importing, transferring, andoffering to transfer the Defendant's Products produced by the Defendant's Production
Processes, by itself or jointly with its affiliated company, constitutes infringement ofPatent Right 1 with regard to Defendant's Products 1 and 3, as well as Patent Right 2 withregard to Defendant's Products 1 through 4. Based on this allegation, the Plaintiff madethe following claims.
A. Under Article 100, paragraph (1) of the Patent Act, a claim seeking an injunction
against transfer, import, and offer to transfer (including displaying for the purpose oftransfer) of the Defendant's Products (paragraph (1) of the object of claims)
B. Under Article 100, paragraph (2) of the Patent Act, a claim seeking disposal of the
Defendant's Products which are under the Defendant's possession (paragraph (2) of theobject of claims)
C. Based on the right to claim compensation for damages in tort due to patent6

infringement, a claim for payment of damages in the amount of 990 million yen (a totalof 900 million yen as partial claims for the damages under Article 102, paragraph (2) or(3) of the Patent Act and 90 million yen as attorneys' fees), with delay damages accrued
thereon at the rate of 5% per annum as prescribed in the Civil Code (the Code prior to theamendment by Act No. 44 of 2017; the same applies hereinafter) for the period from
August 10, 2016 (the day following the date of service of the complaint), which is a day
after the act of tort, until the completion of the payment (paragraph (3) of the object ofclaims)The period subject to the claim for compensation for damages is the period fromJanuary 1, 2007, to December 31, 2017, for the infringement of Patent Right 1 (hereinafterreferred to as the "subject period") and the period from August 23, 2013, which is theregistration date of Patent Right 2, to December 31, 2017, for the infringement of Patent
Right 2 (hereinafter, this period may be referred to as the "subject period for Patent Right2"), and the abovementioned amount was selectively claimed as partial claims of theclaims for compensation for damages due to the respective patent infringements in these
periods.
(2) Outline of the Defendant's allegations
The Defendant, while recognizing that Defendant's Production Process 1 was being
used in a part of the subject period, disputes the Plaintiff's allegations with regard to theuse or non-use, the periods of use, etc. of the Defendant's Production Processes. In
addition, in regard to whether the Defendant's Production Processes fall within thetechnical scopes of the inventions claimed in the Patents, the Defendant disputes theexistence or non-existence of infringement of Patent Right 2 under the doctrine ofequivalents mainly regarding Defendant's Product 4. Moreover, the Defendant allegesthat both Patent 1 and Patent 2 had grounds for invalidation before and after thecorrections, and hence the Defendant's Production Processes have not infringed the Patent
Rights.
Furthermore, the Defendant disputes the necessity of an injunction on the basis thatthe Defendant's Products are currently not produced. Also, with regard to the existence or
non-existence and the scope of acts of working the inventions claimed in the Patents forwhich the Defendant should be held liable for compensation for damages in connection
with the claim for compensation for damages caused by acts conducted during the subject
period, the Defendant alleges that, whereas the Defendant's Products were produced in
the Republic of Indonesia (hereinafter referred to as "Indonesia"), the Defendant's
Products exported to Japan include a portion directly sold to Japanese customers by theIndonesian corporation which produced the products, and argues that this portion does7

not constitute working of the inventions in Japan because the act of sale was conducted
outside Japan, and that the Defendant should not be held liable for compensation fordamages with regard to this portion. Thus, the Defendant disputes the Plaintiff's methodof calculation of damages.
2. Basic facts, etc. (facts not disputed between the parties or facts that can easily be foundbased on the evidence mentioned below (hereinafter, any branch numbers of documentary
evidence are omitted unless otherwise indicated) and the entire import of oral arguments,
etc.)(1) Parties, etc.
A. The Plaintiff
The Plaintiff is a company engaged in the production, processing, purchase and sale,
import and export, research and development, etc. of products of seasonings, sweeteners,and various amino acids, as well as their ingredients, byproducts, and related goods in thecourse of trade, and produces monosodium glutamate (MSG) products named"Ajinomoto" (the entire import of oral arguments).
B. The Defendant and the corporate group to which the Defendant belongs(A) The Defendant is a Japanese corporation engaged in the processing and sale ofseasonings and spices including monosodium glutamate and nucleic acids, processing andsale of feeds and feed additives, sale of seasonings and food additives applying
fermentation chemistry, organic chemistry, and inorganic chemistry, etc. in the course of
trade (the entire import of oral arguments).
(B) The Defendant belongs to a corporate group centered on a South Korean corporation,
CJ CheilJedang Corporation (hereinafter, the corporation is referred to as "CJCJ" and thegroup is referred to as the "CJ Group"). In the CJ Group, the group companies share roles
in producing and selling CJ-brand and other products in countries around the world.
The Defendant is a wholly owned subsidiary company of a South Korean corporation"CJ CORP." (hereinafter referred to as "CJ Company"), which is the holding company,
and CJ Company holds 44% of CJCJ's shares (Exhibit Otsu 108; the entire import of oral
arguments).
(C) "PT CHEIL JEDANG INDONESIA" (hereinafter referred to as "CJ Indonesia") is an
Indonesian corporation which belongs to the CJ Group and is a wholly owned subsidiarycompany of CJCJ (Exhibit Otsu 108; the entire import of oral arguments; hereinafter, theDefendant and CJ Indonesia may be collectively referred to as the "Defendant, etc.").
(2) Production of monosodium glutamate by the fermentation method
One process for producing glutamic acid and other amino acids is the amino acidfermentation method (hereinafter referred to as the "fermentation method") whereby8ingredients such as molasses are added to a medium for culturing microorganisms tocause the microorganisms to produce amino acids as they propagate.
Well-known glutamic acid-producing bacteria used for the fermentation method
include bacteria of the genus Corynebacterium, which are coryneform bacteria.
One of the general production processes of monosodium glutamate using thefermentation method is as follows: glutamic acid-producing bacteria are placed in afermentation vessel (fermentation tank) containing major ingredients, such as the carbon
source represented by molasses, and are propagated; L-glutamic acid (hereinafter, "Lglutamic acid" is simply referred to as "glutamic acid" unless otherwise indicated) is
produced by having the carbon source (sugar) converted into glutamic acid throughmetabolic reactions within the bacterial cell; after the end of this fermentation process,crystals are precipitated from the fermented liquid and glutamic acid crystals are collectedby a separation operation; the collected glutamic acid is neutralized by caustic soda andthen decolorized with activated carbon; and monosodium glutamate crystals are obtained(Exhibits Ko 8 and 9, Exhibit Otsu 2).
The Patent Rights relate to processes for producing glutamic acid by the fermentation
method.
(3) Patent Rights
The Plaintiff is the patentee of the Patent Rights, and the filing dates, etc. of the Patent
Rights are as shown below (Exhibits Ko 1 through 4). The term of Patent Right 1 expired
on September 22, 2019.
A. Patent Right 1
Patent number: Patent No. 3651002
Filing date: July 8, 2004
Application number: Patent Application No. 2004-202121
Registration date: March 4, 2005
Filing date of the original application: September 22, 1999
Priority date: September 25, 1998 (hereinafter referred to as "Priority Date 1")
Priority claim country: Japan
Title of the invention: Process for constructing amino acid-producing bacterium andprocess for producing amino acid by the fermentation method using the constructedamino acid-producing bacterium
B. Patent Right 2
Patent number: Patent No. 5343303
Filing date: December 28, 2005
Application number: Patent Application No. 2005-379259
9

Registration date: August 23, 2013
Priority date: December 28, 2004 (hereinafter referred to as "Priority Date 2")
Priority claim country: Japan
Title of the invention: L-glutamic acid-producing bacterium and process for producing Lglutamic acid(4) Claims, etc. of the invention claimed in Patent 1
A. Claims before corrections
Claims 1 through 4 of the claims of Patent 1 are as shown in Attachment 4-1 "Claims(Patent 1)."
The inventions claimed in Claims 1 through 4 (hereinafter, these inventions arereferred to as "Invention 1-1," "Invention 1-2," "Invention 1-3," and "Invention 1-4" inthe respective order, and collectively as "Invention 1") can be divided into constituent
features as shown in Attachment 4-3 "Division into Constituent Features (Invention 1)"(hereinafter, each divided constituent feature is referred to as "Constituent Feature 1-A 1" or the like according to the code assigned at the beginning of each constituent feature;
the same applies to the division of claims in Attachments 4-4, 5-4, 5-5, and 5-6).
B. Requests for correction
On July 7, 2017, the Plaintiff filed a request for correction of Patent 1 (hereinafterreferred to as "Correction 1") in the proceedings of a trial for patent invalidation filed by
the Defendant (Invalidation Trial No. 2017-800021) (Exhibit Ko 93, Exhibit Otsu 69).
Claims 1, 2, and 4 (Claim 3 was deleted) of the claims corrected by Correction 1 are
as shown in Attachment 4-2 "Corrected Claims (Patent 1)."
The inventions claimed in Claims 1, 2, and 4 after the correction (hereinafter, theseinventions are referred to as "Corrected Invention 1-1" (Claim 1), "Corrected Invention
1-2" (Claim 2), and "Corrected Invention 1-3" (Claim 4) in the respective order, andcollectively referred to as "Corrected Invention 1") can be divided into constituent
features as shown in Attachment 4-4 "Division into Constituent Features (Corrected
Invention 1)."As the corrections in Correction 1 are all made within the scope of the matters statedin the description, claims, or drawings attached to the written application, for the purposeof restricting the claims or clarifying an ambiguous statement (items (i) and (iii) of Article134-2, paragraph (1) of the Patent Act), and do not substantially enlarge or alter the claims,
they satisfy the requirements for correction (Article 134-2, paragraph (9) and Article 126,
paragraphs (5) and (6) of the Patent Act) (Exhibits Ko 50-1 and 93; the entire import oforal arguments).
C. JPO decision, etc.
10With regard to the request for a trial for patent invalidation referred to in B. above,
the JPO rendered a trial decision on January 8, 2019, affirming Correction 1, holding thatthe patent is to be maintained with regard to the portion other than that deleted by thecorrection (Exhibit Ko 93). In response, the Defendant filed a lawsuit seeking rescissionof the JPO decision, and as of the time of conclusion of the oral argument of this case, theabovementioned JPO decision has yet to become final and binding (the entire import oforal arguments).
(5) Claims, etc. of the invention claimed in Patent 2
A. Claims before the corrections
Claims 1, 4, 6, 10, and 11 of the claims of Patent 2 are as shown in Attachment 5-1"Claims (Patent 2)."The inventions claimed in Claims 1, 4, 6, 10, and 11 (hereinafter, these inventions arereferred to as "Invention 2-1" (Claim 1), "Invention 2-2" (Claim 4), "Invention 2-3"
(Claim 6), "Invention 2-4" (Claim 10), and "Invention 2-5" (Claim 11) in the respective
order, and collectively referred to as "Invention 2") can be divided into constituent
features as shown in Attachment 5-4 "Division into Constituent Features (Invention 2)."
B. Request for correction
On July 7, 2017, the Plaintiff filed a request for correction of Patent 2 (hereinafterreferred to as "Correction 2") in the proceedings of a trial for patent invalidation filed by
the Defendant (Invalidation Trial No. 2017-800022) (Exhibit Ko 94, Exhibit Otsu 70).
Claims 1, 4, 6, 10, and 11 of the claims corrected by Correction 2 are as shown in
Attachment 5-2 "Corrected Claims (Patent 2)."The inventions claimed in Claims 1, 4, 6, 10, and 11 after the correction (hereinafter,these inventions are referred to as "Corrected Invention 2-1" (Claim 1), "Corrected
Invention 2-2" (Claim 4), "Corrected Invention 2-3" (Claim 6), "Corrected Invention 24" (Claim 10), and "Corrected Invention 2-5" (Claim 11) in the respective order, andcollectively referred to as "Corrected Invention 2") can be divided into constituent
features as shown in Attachment 5-5 "Division into Constituent Features (Corrected
Invention 2)."
C. JPO decision, etc.
With regard to the request for a trial for patent invalidation referred to in B. above,
the JPO rendered a trial decision on January 8, 2019, affirming Correction 2, holding thatthe patent is to be maintained with regard to the portion other than that deleted by thecorrection (Exhibit Ko 94). In response, the Defendant filed a lawsuit seeking rescissionof the JPO decision, and as of the time of conclusion of the oral argument of this case, theabovementioned JPO decision has yet to become final and binding (the entire import of11

oral arguments).
D. Plan for further correction
As an alternative claim to the claim based on Correction 2 regarding Patent 2, thePlaintiff asserts that it plans to make further correction that differs from Correction 2(hereinafter, that further correction may be referred to as "re-correction of Patent 2") when
it becomes procedurally possible to do so, but as of the time of conclusion of the oral
argument of this case, a request for such correction has not been filed (the entire importof oral arguments).
Claims 4, 13, and 14 of the claims to be corrected by re-correction of Patent 2 are asshown in Attachment 5-3 "Re-corrected Claims (Patent 2)" (Exhibit Ko 53).
The inventions claimed in Claims 4, 13, and 14 after the re-correction of Patent 2(hereinafter, these inventions are referred to as "Re-corrected Invention 2-2" (Claim 4),"Re-corrected Invention 2-3" (Claim 13), and "Re-corrected Invention 2-6" (Claim 14) inthe respective order, and collectively referred to as " Re-corrected Invention 2") can bedivided into constituent features as shown in Attachment 5-6 "Division into Constituent
Features (Re-corrected Invention 2)."
(6) Descriptions, etc. of the Patents
The description and drawings of Patent 1 (Exhibit Ko 3) are referred to as"Description 1," and the description and drawings of Patent 2 (Exhibit Ko 4) are referred
to as "Description 2."
In addition, unless otherwise indicated, when describing working examples,paragraph numbers (to be described as [0001] or the like), or drawings or table numbers(to be described as [Table 1], [Figure 1], or the like) contained in the detailed explanationof the invention of the description with regard to the contents of the inventions claimed
in the Patents, they respectively indicate the corresponding parts of Description 1 for theinventions claimed in Patent 1 and the corresponding parts of Description 2 for theinventions claimed in Patent 2. Meanwhile, as paragraph [0033] of Description 2 has beencorrected by Correction 2, when citing that part, a clear indication is made as to whether
the cited contents are those before or after the correction.
(7) Defendant's Products and Defendant's Production Processes
A. Import and sale of monosodium glutamate products by the Defendant
The Defendant imported monosodium glutamate with the product name "MIPOONG" (hereinafter referred to as the "MSG in question") into Japan from Indonesia,
offered to transfer it and transferred it from 2007 to at least until the end of 2017.
The MSG in question was produced by the fermentation method by CJ Indonesia in afactory in Indonesia (Exhibit Otsu 2, the entire import of oral arguments).
12

The Defendant alleges that the MSG in question which was exported from Indonesia
to Japan during the subject period and is distributed in Japan includes not only the portion
imported and sold by the Defendant (hereinafter referred to as the "portion sold by the
Defendant"), but also the portion directly sold by CJ Indonesia to Japanese customers(hereinafter referred to as the "portion sold by CJ Indonesia," and together with theportion sold by the Defendant, they are referred to as the "portion sold by the Defendant,etc."), and, as mentioned below, there is a dispute between the parties as to whether the
Defendant is liable for tort due to infringement of the Patent Rights with regard to the
portion sold by CJ Indonesia.
B. Scope, etc. of products subject to the Plaintiff's claims
Of the MSG in question, the scope of products specified by the Plaintiff as thosesubject to the Plaintiff's claims are the Defendant's Products described in Attachment 2"List of the Defendant's Products" that were produced by the Defendant's Production
Processes described in Attachment 3 "List of the Defendant's Production Processes."
With regard to the relationship between the Defendant's Production Processes and theinventions claimed in the Patents, the Plaintiff alleges as follows: both before and after
the corrections, Defendant's Production Process 1 falls within the technical scope of theinventions claimed in Patents 1 and 2, Defendant's Production Process 2 falls within thetechnical scope of the inventions claimed in Patent 2, Defendant's Production Process 3
falls within the technical scope of the inventions claimed in Patents 1 and 2, andDefendant's Production Process 4 falls within the technical scope of the inventions
claimed in Patent 2 as an equivalent thereto.
(8) Prior art documents
A. Regarding Patent 1
The following documents, etc. existed before September 25, 1998, which is Priority
Date 1.
(A) Microbiology (1996) 142, p. 1297–1309 (Exhibit Otsu 6; hereinafter, the document
is referred to as "Exhibit Otsu 6 Document" and the invention described in Exhibit Otsu
6 Document is referred to as "Exhibit Otsu 6 Invention"; similarly, unless otherwise
indicated, the following documents are also respectively referred to as "Exhibit Otsu 6
Document" or the like according to the corresponding Exhibit Otsu number, and theinventions described in those documents are described as "Exhibit Otsu 6 Invention" or
the like)(B) Microbiology (1994) 140, p. 1817–1828 (Exhibit Otsu 8, Exhibit Ko 25)(C) International Publication No. 95/34672 (Exhibit Otsu 9)(D) EMBO J. (1982) 1 (7), p. 875–881 (Exhibit Otsu 10)
13

(E) J. Biol. Chem.(1985) 260 (6), p. 3539–3541 (Exhibit Otsu 11)(F) Unexamined Patent Application Publication No. 1991–147792 (Exhibit Otsu 12)(G) Unexamined Patent Application Publication No. 1988–214189 (Exhibit Otsu 35)
B. Regarding Patent 2
The following documents, etc. existed before December 28, 2004, which is Priority
Date 2.
(A) Eur. J. Biochem. (1997) 247, p. 572–580 (Exhibit Otsu 24)(B) FEMS Microbiol. Lett. (2003), 218, p. 305–309 (Exhibit Otsu 26)(C) Science (2002) 298 (22), p. 1582–1587 (Exhibit Otsu 29)(D) EMBO J. (2003) 22 (1), p. 36–46 (Exhibit Otsu 30)(E) Biophysical J. (2004. Nov.) 87, p. 3050–3065 (Exhibit Otsu 31)(F) International Publication No. 2003/046123 (Exhibit Otsu 41-1), U.S. Patent
Application Publication No. 2005/0003494 Description (Exhibit Otsu 41-2; it is the U.S.
patent application publication corresponding to the international application of Exhibit
Otsu 41-1, and together with Exhibit Otsu 41-1 it is referred to as "Exhibit Otsu 41
Document")(G) European Patent Application Publication No. 1108790 Description (Exhibit Otsu 42)
3. Issues(1) Use or non-use, the periods of use, etc. of the Defendant's Production Processes (Issue
1)(2) Whether the Defendant's Production Processes fall within the technical scopes of theinventions claimed in the Patents (Issue 2)
A. Whether Defendant's Production Processes 1 and 3 literally fall within the technicalscope of Invention 1 (Issue 2-1)
B. Whether Defendant's Production Processes 1 through 3 literally fall within thetechnical scope of Invention 2 (Issue 2-2)
C. Whether Defendant's Production Process 4 falls within the technical scope of Invention
2 as an equivalent thereto (Issue 2-3)
(3) Whether Patent 1 should be invalidated by a trial for patent invalidation (Issue 3)
A. Whether or not Invention 1 lacks an inventive step based on Exhibit Otsu 6 Invention
as the primary cited document (Issue 3-1)
B. Whether or not Invention 1 lacks an inventive step based on Exhibit Otsu 9 Invention
as the primary cited document (Issue 3-2)
C. Whether or not Invention 1 violates the enablement requirement or the support
requirement (Issue 3-3)(4) Whether Patent 2 should be invalidated by a trial for patent invalidation (Issue 4)14

A. Whether or not Invention 2 lacks novelty based on Exhibit Otsu 42 Invention (Issue 41)
B. Whether or not Invention 2 lacks an inventive step based on Exhibit Otsu 24 Invention
as the primary cited document (Issue 4-2)
C. Whether or not Invention 2 violates the enablement requirement or the support
requirement (Issue 4-3)
D. Whether or not Invention 2 violates the clarity requirement (Issue 4-4)
(5) Whether re-defense of correction of Patent 1 can be established (Issue 5)(6) Whether re-defense of correction of Patent 2 (Correction 2) can be established (Issue
6)(7) Whether re-defense of re-correction of Patent 2 can be established (alternative claim
to the re-defense of Issue 6) (Issue 7)(8) Occurrence or non-occurrence and the amount of damages (Issue 8)(9) Whether or not the claim seeking injunction and the claim seeking disposal areappropriate (Issue 9)(omitted)
No. 4 Judgment of this court
1. Regarding Issue 1 (use or non-use, the periods of use, etc. of the Defendant's Production
Processes)(1) Regarding the period from January 2011 through May 2014
A. There is no dispute between the parties concerning the fact that the MSG in question
was being produced by Defendant's Production Process 1 during the period from January
2011 through May 2014, during which the Defendant alleges using Strain [ii].
Therefore, the strain being used for producing the MSG in question during this period
was Corynebacterium glutamicum, in which genes on the chromosome have thecharacteristics described in 1. of Attachment 2 "List of the Defendant's Products" (thesame characteristics as those described for Strain [ii] in the "Defendant's allegations"column of Attachment 9 "Comparison Table of Allegations concerning Processes for
Producing the MSG in Question").
B. Regarding analysis results of PROMATE2012
According to the evidence (Exhibits Ko 15 and 22) and the entire import of oral
arguments, on November 30, 2012, which is a date included in the relevant period, thePlaintiff obtained and analyzed PROMATE produced in CJ Indonesia's factory(PROMATE is livestock feed made by utilizing used bacterial cells produced when15

producing the MSG in question; hereinafter, this PROMATE obtained by the Plaintiff isreferred to as "PROMATE2012"), and as a result, confirmed that the strain contained in
PROMATE2012 had the characteristics referred to in A. above.
(2) Regarding the period from August 2015 through June 2016
A. With regard to this period in which the Defendant alleges using Strain [x], the Plaintiff
alleges, based on the analysis results (Exhibit Ko 49) of the obtained PROMATE2015,that the same strain as that in the period referred to in (1) above was being used, whereas
the Defendant alleges that a strain having different characteristics from that in (1) above
was being used.
B. The Plaintiff's analysis results and the credibility thereof(A) According to the evidence (Exhibits Ko 49, 66, and 67) and the entire import of oral
arguments, it was found that, on December 30, 2015, the Plaintiff obtained and analyzed
PROMATE2015 produced by CJ Indonesia, and that according to the analysis results(Exhibit Ko 49), the following mutations were detected in the DNA extracted from
PROMATE2015, as in the case with those detected in PROMATE2012: a mutation thatalters the -35 region of the promoter sequence of the GDH gene to TTGTCA and the -10
region to TATAAT; a mutation that alters the -10 region of the promoter sequence of the
CS gene to TATAAT; and a mutation in which alanine at position 100 in the amino acidsequence encoded by the yggB gene is substituted with threonine.
(B) The Defendant disputes the credibility of the Plaintiff's analysis results (Exhibit Ko49), alleging that it could not confirm those analysis results in the results of CJCJ's
analysis of PROMATE which was produced on the same date as PROMATE2015 (Exhibit
Otsu 75). However, CJCJ's analysis failed to conduct sequence analysis with regard to apart of the analysis of the promoter region of the GDH gene and the analysis of thepromoter region of the CS gene and the yggB gene, due to reasons such as weak signals,
and in spite of obtaining such results, there is no sign of CJCJ having conducted analysis,
etc. again under different conditions. Therefore, it should be said that CJCJ's analysis
lacks credibility as analysis conducted by a person skilled in the art of processes for
producing the MSG in question (Exhibit Otsu 2), and that it is not sufficient to overturnthe credibility of the Plaintiff's analysis results.
Consequently, it should be said that the Plaintiff's analysis results which indicated thatthe mutations referred to in (A) above, which are the same as those detected in the analysisof PROMATE2012, were detected in the DNA derived from PROMATE2015 are credible.
C. Regarding inclusion of a strain used in the past
According to the Plaintiff's analysis results (Exhibit Ko 49), it is found that, in the
promoter sequence of the GDH gene of PROMATE2015, [i] sequences having the same16

mutations as those in PROMATE2012, [ii] wild-type sequences, and [iii] sequences thatdiffer from both of the former two were detected, and in regard to their percentages, [i]above accounted for the highest percentage whereas [ii] accounted for the lowest
percentage, while the ratio of their signal intensities was 5:1:2 in the order of their
numbers.
The Defendant alleges that, because powder made by crushing PROMATE that wasused in the past is added as seed when producing PROMATE, and also because someresidues remain within the production equipment, components derived from the paststrain are present along with components derived from the most recently used strain.
According to the evidence (Exhibit Otsu 88) and the entire import of oral arguments,
it is found that, at the stage of production of PROMATE, not only the strain that was being
used for producing the MSG in question at the time was used as an ingredient, but also
past PROMATE that had been produced earlier was added as seed for promoting
granulation. However, while the Defendant explains that the percentage of seed added
was typically 30%, the sequences having the same mutations as those in PROMATE2012
were detected from PROMATE2015 at the highest percentage, whereas the wild-typesequences, which the Defendant alleges to have been the sequences of the strain used at
the time, were detected at the lowest percentage, as mentioned above, and hence, thesequences having the abovementioned mutations are unlikely to have been added as seed.
Although the Defendant also indicates the effect of residues within the production
equipment, the fact that the sequences having the same mutations as those in
PROMATE2012 were detected at the highest percentage as mentioned above is difficult
to explain by the effect of residues.
In addition, the Defendant indicates that PROMATE is livestock feed, and it is notproduced in a manner such that bacterial cells are distributed evenly among the particles,
but as the Defendant has not shown analysis results that indicate different results from thePlaintiff's analysis results with regard to the percentage of sequences having the same
mutations as those in PROMATE2012, it cannot be said that this sufficiently explains thereason for which the past strain is detected more strongly than the present strain.
In this way, even if the points indicated by the Defendant are taken into consideration,
it is reasonable to find that the mutations referred to in B. (A) above detected from
PROMATE2015 were derived from the strain that was being used for producing the MSG
in question as of December 30, 2015, and there is no sufficient evidence to overturn thisfinding.
D. Consequently, it is reasonable to presume that the strain that was being used aroundDecember 30, 2015, when PROMATE2015 was produced, was a strain which has the17same characteristics as Strain [ii] and which was used in Defendant's Production Process
1, and that this strain was being used at least during the period from around August 2015
through around June 2016 in which the Defendant alleges using Strain [x].
As the Defendant admits that the MSG in question was produced by the fermentation
method, and does not specifically dispute points other than the type of the strain withregard to the use or non-use of Defendant's Production Process 1 during the relevant
period, it is found that the MSG in question produced during the relevant period was
Defendant's Product 1 produced by Defendant's Production Process 1.
(3) Regarding other periods
A. The Plaintiff asserts that Defendant's Production Process 1 was being used not only inthe periods referred to in (1) and (2) above, but also in the respective periods of Strain
Unknown Period [i] and the periods in which the Defendant alleges using Strains [iii]through [ix] and Strains [xi] through [xiv], but there are no materials directly supporting
the fact that Defendant's Production Process 1 was being used in these periods.
In addition, as mentioned in (2) C. above, when producing PROMATE, components
derived from a strain that had been used for producing the MSG in question earlier may
be mixed in, and according to the Plaintiff's analysis results of PROMATE2015,
sequences other than the sequences having the same mutations as those in
PROMATE2012 were also detected in the promoter sequence of the GDH gene. This isconsistent with the Defendant's allegation that, when producing the MSG in question, thesame strain was not always being used, but different strains were being used according to
the period.
Regarding the characteristics of the genes of the strains that were being used, theDefendant makes allegations as shown in Attachment 9 "Comparison Table of Allegations
concerning Processes for Producing the MSG in Question," except for Strain Unknown
Period [i], and the Defendant has disclosed analysis results that are consistent with thosecharacteristics with regard to strains that were used in some periods (Exhibit Otsu 74).
When considering these facts, it cannot be presumed based on the analysis results of
PROMATE2012 and PROMATE2015, which were examined in (1) and (2) above, that
Defendant's Production Process 1 was also being used in periods other than those referred
to in (1) and (2) above, and there is no other evidence sufficient for affirming the Plaintiff's
allegation regarding this point.
Accordingly, it is reasonable to find that Corynebacterium glutamicum, which has thecharacteristics described in the "Defendant's allegations" column of Attachment 9"Comparison Table of Allegations concerning Processes for Producing the MSG in
Question," was being used in the respective periods of Strain Unknown Period [i] and in18

the periods during which the Defendant alleges using Strains [iii] through [ix] and Strains[xi] through [xiv].
B. As the Defendant admits that the MSG in question was produced by the fermentation
method, and does not specifically dispute points other than the type of the strain withregard to the use or non-use of the Defendant's Production Processes, it is reasonable tofind that the state of use of the Defendant's Production Processes in production of the
MSG in question during the periods of use of the respective strains that were examined
in A. above was the same as that in the "Plaintiff's alternative claims" column ofAttachment 9, and the Defendant's Production Processes are not found to have been usedin Strain Unknown Period [i] and the period of use of Strain [xiv].
C. The Plaintiff asserts that it is unconceivable that the Defendant does not possess
records of strains that were used only six years ago with regard to Strain Unknown Period[i], and that, as long as the Defendant cannot prove that it was using a bacterium thatdiffers from Strain [ii], the Defendant should naturally be presumed to have been using
the same bacterium as Strain [ii]. However, considering that the analysis results of
PROMATE2015 also suggest that different strains were being used depending on the
period for producing the MSG in question, as mentioned in A. above, it cannot naturally
be presumed that the Defendant was using the same strain as Strain [ii] in Strain Unknown
Period [i] only based on the fact that the Defendant has not alleged or proved the specificcharacteristics of the strain that it was using.
In addition, with regard to Strains [xii] and [xiii], the Plaintiff alleges that if thesequence of the yggB gene had been altered from earlier strains, as alleged by the
Defendant, an application for the safety assessment required under the Food Sanitation
Act should have been filed, but as the application has not been filed, the Defendant's
allegation cannot be trusted. However, according to the Plaintiff's allegation, theDefendant should have filed an application for the abovementioned safety assessment
also for the MSG produced by using strains having a mutant-type yggB gene with an
A100T-type mutation (Strains [ii] through [xi]), but the Defendant failed to do so.
Therefore, even if the Defendant did not file an application for the abovementioned safetyassessment in the periods before and after the use of Strains [xii] and [xiii], it cannot be
said that the same mutant-type yggB gene as earlier was being used for Strains [xii] and[xiii] based on the Defendant's failure to file the application, and this point also isinsufficient for overturning the determination in B. above.
(4) Summary of the periods of use of the Defendant's Production Processes
According to (1) through (3) above, with regard to the use or non-use of the
Defendant's Production Processes in the respective periods, it is found that Defendant's
19

Production Process 1 was being used during the period of use of Strains [ii] and [x]described in Attachment 9 "Comparison Table of Allegations concerning Processes for
Producing the MSG in Question" and, during other periods, the state of use was the sameas that described in the "Plaintiff's alternative claims" column of that Attachment.
2. Regarding Issue 2-1 (whether Defendant's Production Processes 1 and 3 literally fallwithin the technical scope of Invention 1)
According to the entire import of oral arguments, it is found that, when Defendant's
Production Processes 1 and 3 are compared with Invention 1, the results of the comparison
will be the same as the description in Attachment 6-1 "Comparison between Defendant's
Production Processes 1 and 3 and Invention 1," and therefore Defendant's Production
Process 1 literally falls within the technical scopes of Inventions 1-1 through 1-4, andDefendant's Production Process 3 literally falls within the technical scopes of Inventions
1-1 and 1-4.
3. Regarding Issue 2-2 (whether Defendant's Production Processes 1 through 3 literally
fall within the technical scope of Invention 2)(1) Regarding Defendant's Production Process 1
According to the analysis results of PROMATE2012 (Exhibit Ko 22), it is found that,with regard to Strain [ii], an A100T mutation is introduced into the amino acid sequenceof SEQ ID NO: 6 of the yggB gene, and the amino acid sequence of the yggB gene after
the mutation coincides with the amino acid sequence of SEQ ID NO: 22. In addition,according to Working Example 8 in Description 2 and evidence (Exhibit Ko 37), theintroduction of the mutant-type yggB gene is found to cause the glutamic acid-producing
ability of the strain to be enhanced as compared to a non-modified strain (it can be
confirmed based on Working Example 8 and Exhibit Ko 37 that the glutamic acidproducing ability is enhanced in a configuration using the 19-type mutation that uses amutant-type yggB gene having an amino acid sequence of SEQ ID NO: 22, as mentioned
in 4. (3) B. below).
According to the points mentioned above and the entire import of oral arguments, it
is found that, when Defendant's Production Process 1 is compared with Invention 2, theresults of the comparison will be the same as the description in Attachment 7-1"Comparison between Defendant's Production Processes 1 through 3 and Invention 2,"
and therefore the bacteria used in Defendant's Production Process 1 (Strain [ii] and Strain[x] which has the same characteristics as Strain [ii]) fall within the technical scopes ofInventions 2-1, 2-2, 2-3, and 2-4, and Defendant's Production Process 1, which uses thosebacteria, falls within the technical scope of Invention 2-5.
(2) Regarding Defendant's Production Processes 2 and 3
20

The mutant-type yggB genes of the strains used in Defendant's Production Processes
2 and 3 (Strains [iii] through [ix] and [xi]) are those into which an A100T mutation has
been introduced, as in the case with the yggB genes of the strains used in Defendant's
Production Process 1 (Strains [ii] and [x]). Moreover, the Defendant has not alleged any
differences between them with regard to the amino acid sequence of any other portionsof the yggB genes. Therefore, also in regard to the mutant-type yggB genes of the strainsused in Defendant's Production Processes 2 and 3 (Strains [iii] through [ix] and [xi]), it isfound that the amino acid sequence before the mutation coincides with the amino acidsequence of SEQ ID NO: 6, and the amino acid sequence after the mutation coincides
with the amino acid sequence of SEQ ID NO: 22, as in the case with the yggB gene of
the strain used in Defendant's Production Process 1.
Accordingly, the results of comparison between the bacteria used in Defendant's
Production Processes 2 and 3 and Invention 2 will be the same as those of the comparison
between Defendant's Production Process 1 and Invention 2 mentioned in (1) above, andthe bacteria used in Defendant's Production Processes 2 and 3 (Strains [iii] through [ix]and [xi]) all fall within the technical scopes of Inventions 2-1, 2-2, 2-3, and 2-4, while
Defendant's Production Processes 2 and 3, which use those bacteria, both fall within thetechnical scope of Invention 2-5.
4. Regarding Issue 2-3 (whether Defendant's Production Process 4 falls within thetechnical scope of Invention 2 as an equivalent thereto)(1) Regarding the criteria for determination of infringement under the doctrineof equivalents
Even if, within the configuration indicated in the claims, there is a part which isdifferent from the product to be produced, etc. or the process to be used by thecounterparty (hereinafter referred to as the "subject product, etc."), if [i] this part is not
the essential part of the patented invention, [ii] the purpose of the patented invention canbe achieved and an identical function and effect can be obtained by replacing this partwith a part in the subject product, etc., [iii] a person with ordinary skill in the art of theinvention (person skilled in the art) could have easily conceived of the abovementioned
replacement at the time of the production, etc. of the subject product, etc., [iv] the subject
product, etc. is not identical to publicly known art at the time of the patent application of
the patented invention nor could have been easily conceived at that time by a person
skilled in the art, and [v] there were no special circumstances such as the fact that the
subject product, etc. had been intentionally excluded from the claims in the course of thepatent application procedure of the patented invention, it is reasonable to construe thatthe subject product, etc. falls within the technical scope of the patented invention as being21

an equivalent to the configuration indicated in the claims (see the judgment of the Third
Petty Bench of the Supreme Court, 1994 (O) 1083, rendered on February 24, 1998,
Minshu Vol. 52, No. 1, at 113 (hereinafter referred to as the "1998 Supreme Court
Judgment"), and the judgment of the Second Petty Bench of the Supreme Court, 2016
(Ju) 1242, rendered on March 24, 2017, Minshu Vol. 71, No. 3, at 359 (hereinafter referred
to as the "2017 Supreme Court Judgment"); hereinafter, the requirements in [i] through[v] above are respectively referred to as "first requirement" through "fifth requirement").
In addition, regarding the burden of alleging and proving the fulfillment of the first to
fifth requirements, a person who alleges that the subject product, etc. is equivalent to apatented invention should be considered to have the burden of allegation and proof forthe first to third requirements, while a person who denies the application of the doctrineof equivalents in relation to the subject product, etc. should be considered to have theburden of allegation and proof for the fourth and fifth requirements, which are related tothe cases where the application of the doctrine of equivalents should be eliminated, even
if the subject product, etc. is within the scope of equivalents under the first to third
requirements (see the judgment of the Special Division of the Intellectual Property High
Court, 2015 (Ne) 10014 rendered on March 25, 2016, Hanji No. 2306, at 87).
The following part examines whether Defendant's Production Process 4 falls within
the technical scope of Invention 2 as an equivalent thereto, by first considering andcomparing the contents of Invention 2 and the configuration using the 19-type mutation
comprised therein and the contents of Defendant's Production Process 4, and thenexamining the first to fifth requirements of the doctrine of equivalents in order.
(2) Contents of Invention 2
A. Contents described in Description 2
The statements in the detailed explanation of the invention in Description 2
concerning Invention 2 are outlined as described in Attachment 13 "Statements in
Description 2."
While paragraph [0033] of Description 2 has been corrected by Correction 2, as thecorrection of that part is found to be correction of errors as mentioned in 7. (1) B. below,
the corrected statements are hereinafter used unless otherwise indicated.
B. Outline of Invention 2
According to the claims of Invention 2 (Attachment 5-1), the statements in
Description 2 referred to in A. above, and the entire import of oral arguments, the outline
of Invention 2 is found to be as follows.
(A) Technical field/background art
Invention 2 relates to the fermentation industry, and relates to a process for producing
22

L-glutamic acid, which is widely used as seasoning ingredients, etc., and further relates
to a bacterium used in the process (paragraph [0001]).
L-glutamic acid has been conventionally produced on an industrial scale by thefermentation method using coryneform bacteria which have L-glutamic acid-producing
ability, such as bacteria belonging to the genus Brevibacterium or the genus
Corynebacterium (paragraph [0002]).
Generally, a wild-type strain of a coryneform bacterium does not produce glutamic
acid under the condition of the presence of biotin. Accordingly, the production of Lglutamic acid by a coryneform bacterium is typically performed under a glutamic acidproduction-inducing condition such as a biotin-limited condition, a surfactant-added
condition, and a penicillin-added condition. Further, as strains which can produce Lglutamic acid in the presence of sufficient biotin without applying these methods, asurfactant-temperature-sensitive strain, a penicillin-sensitive strain, a cerulenin-sensitive
strain, and a lysozyme-sensitive strain, etc. have been developed. However, it was highly
probable that these strains caused reduced adaptation to environmental changes inexchange for L-glutamic acid production. Therefore, considerable efforts have beenrequired for the development of a strain capable of accumulating a significant amount of
L-glutamic acid (paragraphs [0003] and [0004]).
On the other hand, a strain which produces L-glutamic acid in the presence ofsufficient biotin can be achieved by deleting a gene encoding α-ketoglutarate
dehydrogenase. However, the α-ketoglutarate dehydrogenase gene-deficient strain blocks
the TCA cycle in the midway and thus grows slowly. Therefore, it was difficult to surely
obtain a sufficient amount of bacterial cells, which was one of the problems (paragraph[0005]).
The yggB gene of a coryneform bacterium is a homologue of the yggB gene of
Escherichia coli (E. coli), and has been analyzed as a kind of mechanosensitive channel,
but the effect of the yggB gene on L-glutamic acid production was not known (paragraph[0006]).
(B) Problem to be solved by Invention 2
The problem to be solved by Invention 2 is to provide novel art to enhance the Lglutamic acid-producing ability of a coryneform bacterium when L-glutamic acid is
produced by using the coryneform bacterium (paragraph [0007]).
(C) Means for solving the problem, etc.
a. Modification using the yggB gene
Invention 2 has clarified that the yggB gene is involved in the production of Lglutamic acid by a coryneform bacterium, and has found that the L-glutamic acid23

producing ability can be greatly enhanced by modifying the coryneform bacterium byusing the yggB gene (paragraph [0008]).
The coryneform bacterium of Invention 2 is a coryneform bacterium having an Lglutamic acid-producing ability, wherein the coryneform bacterium has an enhanced Lglutamic acid-producing ability as compared to a non-modified strain as a result of being
modified by using the yggB gene (paragraph [0011]).
Modification using the yggB gene includes a method of enhancing the expression
level of the yggB gene and a method of introducing a mutant-type yggB gene, andInvention 2 uses the latter method (paragraph [0029]).
b. Introduction of a mutant-type yggB geneSpecific examples of the mutant-type yggB gene are shown below, but the mutanttype yggB gene is not particularly limited so far as it has a mutation that could enhance
the L-glutamic acid producing ability of a coryneform bacterium under a condition inwhich an excess of biotin is present (paragraph [0069]).
(a) C-terminal side mutation
One mode of mutation that is introduced into the yggB gene in Invention 2 is a Cterminal side mutation, which is a mutation introduced into the C-terminal region of theyggB gene. This is a mutation which is introduced into a portion of the nucleotide
sequence in the region encoding the sequence of amino acid numbers 419–533 of SEQ
ID NOs: 6, 68, 84, and 85, or the sequence of amino acid numbers 419–529 of SEQ ID
NO: 62 (paragraph [0070]). Examples of the C-terminal side mutation include [i] amutation in which a transposable element is introduced (2A-1-type mutation) and [ii] amutation in which the proline residue is substituted with another amino acid (66-type
mutation, 22-type mutation) (Claim 1, paragraphs [0070] through [0072], Working
Examples 2 through 6, Working Example 12, Working Example 13, and Working
Example 17).
(b) Mutations in transmembrane regions
Another mode of mutation that is introduced into the yggB gene in Invention 2 is amutation which is introduced into a portion encoding the five transmembrane regions
which the YggB protein encoded by the yggB gene is presumed to have (in the aminoacid sequence of the wild-type YggB protein of SEQ ID NOs: 6, 62, 68, 84, and 85, thetransmembrane regions correspond to amino acid numbers 1–23 (the first transmembraneregion), amino acid numbers 25–47 (the second transmembrane region), amino acidnumbers 62–84 (the third transmembrane region), amino acid numbers 86–108 (the fourthtransmembrane region), and amino acid numbers 110–132 (the fifth transmembraneregion)). Examples of these mutations include [i] a mutation in the first transmembrane24

region (A1-type mutation), [ii] a mutation in the fourth transmembrane region (19-type
mutation), and [iii] a mutation in the fifth transmembrane region (L30-type mutation, 8type mutation) (Claim 1, paragraphs [0073] through [0076], Working Examples 7 through11, Working Example 15, and Working Example 16).
(D) Effect of Invention 2
L-glutamic acid can be efficiently produced by using a coryneform bacterium which
has been modified by using the yggB gene of Invention 2 (paragraph [0010]).
C. Significance of Invention 2 (the principle for solving the problem)
In light of the above, it should be said that, when compared with the prior art describedin Description 2, the specific technical idea in Invention 2 that cannot be found in theprior art (the principle for solving the problem) was to focus on the yggB gene of acoryneform bacterium, whose effect on production of glutamic acid had been unknown
in the past, modify the YggB protein, which is a type of a mechanosensitive channel, byusing a mutant-type yggB gene into which a C-terminal side mutation or mutations intransmembrane regions have been introduced, and thereby provide novel art to enhance
the glutamic acid-producing ability of the bacterium.
D. Prior art as of Priority Date 2
Regarding prior art as of Priority Date 2, the Defendant alleges among other mattersthat, according to Exhibit Otsu 24 Document, it had already been known at that time thatthe osmoregulated channel (YggB) of Corynebacterium glutamicum contributed to effluxof glutamic acid.
(A) Contents described in Exhibit Otsu 24 Document
The contents described in Exhibit Otsu 24 Document include the following (Exhibit
Otsu 24, the entire import of oral arguments; for Table 1, see Attachment 14 "Drawingsof Cited Documents").
a. Abstract(a) Lines 1 through 6 on page 572"Bacteria respond to hypoosmotic stress by releasing low-molecular-mass solutes in orderto maintain constant turgor pressure. We have studied the function of osmoregulatedchannel(s) in Corynebacterium glutamicum, which are responsible for efflux of various
solutes upon sudden decrease in osmotic pressure. The channels preferentially mediatedefflux of compatible solutes such as glycine betaine and proline. The release of moleculesof similar size, e.g. glutamate or lysine, was restricted, and ATP was completely retainedeven after severe osmotic shock."(b) Lines 13 through 14 on page 572"These results indicate the presence of an osmoregulated channel in C. glutamicum
25

similar to the mechanosensitive channel(s) of Escherichia coli."b. Results(a) Table 1 (page 575)
"Table 1. Specificity of solute efflux after hypoosmotic shock in C. glutamicum. Cells
were loaded with solutes under hyperosmotic conditions (1860 mOsm and 2100 mOsm,
respectively) either by accumulation of labeled or unlabeled compatible solutes (glycine
betaine, proline, ectoine), or by addition of dipeptides (lysine, alanine). The cells werediluted as given in the table. Efflux was measured 1 min after the hypoosmotic shock inboth the supernatant and the pellet of the silicone oil centrifugation using scintillation
counting (sc. count.), HPLC, enzymatic assay, NMR, luciferin/luciferase assay, or flame
photometry (flame ph.). The values are in μmol·mgdm-1, since correcting to the true
internal concentration according to the changed cytoplasmic volume would givemisleading results of the net changes. All values are means of at least three experiments.
For isoosmolar dilution and for hypoosmotic shock to 540 mOsm, relative values ofretained solutes are given."(b) Last line of the right column on page 575 through line 1 of the left column on page576
"efflux of glutamate and lysine was significantly restricted"
c. Discussion(a) Lines 38 through 49 of the left column on page 578"As a soil bacterium, C. glutamicum is equipped with effective mechanisms
responding to a frequent case of emergency, i.e. a hypoosmotic shock. The mechanicalstress acting on the membrane due to osmotic changes became obvious by quantifying
the significant change in cell size. The efflux system which we have studied, responds tohypoosmotic shock in a manner qualitatively similar to mechanosensitve channels whichhave previously been described in E. coli and in L. plantarum (Berrier et al., 1992;
Schleyer et al., 1993; Glaasker et al., 1996). However, the C. glutamicum channel showsa number of specific properties, both with respect to transfer specificity and to regulationof efflux activity."(b) Lines 30 through 33 of the right column on page 578"Molecules related in size, e.g. glutamate, or even small inorganic ions (Na+, K+),
obviously do not use this channel to the same extent as glycine betaine or proline."(c) Lines 36 through 40 of the right column on page 578"As long as the molecular identity of the channel in C. glutamicum is not known,
however, the observed sequence of permeability through the C. glutamicum channel canalso be explained by a multiplicity of channels with different specificities."26

(d) Lines 46 through 55 of the right column on page 578"The lack of sensitivity of this channel to Gd3+, an unspecific channel blocker (Berrier
et al., 1992; Schleyer et al., 1993; Hase et al., 1995), furthermore defines it as being
different from the Gd3+-sensitive MscL channel, described and cloned in E. coli
(Sukharev et al., 1994). There are, however, other examples where this unspecificblocking agent did not work (Berrier et al., 1996). Thus, the channel in C. glutamicum
may be similar to another type identified in E. coli by using electrophysiological
techniques, i.e. the MscS channel (Martinac et al., 1987, 1990; Zoratti and Petronilli,
1988; Berrier et al., 1996)."(e) Lines 39 through 46 of the left column on page 579"Finally, it should be emphasized here that the described efflux channel is not related
to the well-known glutamate efflux observed in C. glutamicum under particular metabolic
conditions. Although glutamate efflux under conditions of continuous production of thisamino acid seems to respond to osmotic changes with respect to its activity (Lambert etal., 1995), it is mediated by an energy-dependent, specific carrier system, as has beenshown before (Gutmann et al., 1992; Kramer, 1994)."(B) Examination
As mentioned above, Table 1 of Exhibit Otsu 24 Document describes that, as a resultof measuring efflux of solutes when hypoosmotic shock was applied to Corynebacterium
glutamicum, efflux of glutamic acid was observed under hypoosmotic conditions.
However, the efflux was only 20% at 540 mOsm, which greatly differed from the efflux
rates of substances such as glycine betaine (70% efflux under the same conditions) andproline (71% efflux under the same conditions). The abovementioned efflux rate ofglutamic acid was the lowest following ATP, for which efflux was hardly observed, among
the total of 11 types of solutes examined, and it was assessed that "efflux of glutamate
and lysine was significantly restricted."
In light of such results, Exhibit Otsu 24 Document states that osmoregulated channels
in Corynebacterium glutamicum preferentially mediated efflux of glycine betaine andproline, but that the channels were unrelated to efflux of glutamic acid, and derived aconclusion that efflux of glutamic acid is mediated not by osmoregulated channels, but
by a carrier system as had been indicated before.
Accordingly, although Exhibit Otsu 24 Document suggests that Corynebacterium
glutamicum has osmoregulated channels having similar characteristics asmechanosensitive channels of E. coli, it does not show that the osmoregulated channels
contribute to efflux of glutamic acid.
In addition, although other documents cited by the Defendant as common general27

technical knowledge as of Priority Date 2 (Exhibits Otsu 28 and 37 through 40) also havesome parts that mention the need to increase the amount of amino acid efflux to outsideof the bacterial cell when producing amino acid by using a coryneform bacterium, they
are not found to mention the relevance of the osmoregulated channel (YggB) of acoryneform bacterium to glutamic acid efflux.
Consequently, the statements on prior art in Description 2, including the fact that theyggB gene of a coryneform bacterium is a homologue of the yggB gene of E. coli, andhas been analyzed as a kind of mechanosensitive channel, but the effect of the yggB geneon L-glutamic acid production was not known, (paragraphs [0002] through [0006]) are
not found to be insufficient from an objective viewpoint.
(3) Regarding the configuration using the 19-type mutation
A. Contents of the configuration using the 19-type mutation
The configuration using the 19-type mutation is a configuration comprised in
Invention 2-5, and it is the process for producing glutamic acid claimed in Claim 11 ofPatent 2, which uses a coryneform bacterium into which the mutant-type yggB gene
mentioned in (e) of Claim 6, which cites Claim 1 or 4, has been introduced.
Among the mutations introduced in the yggB gene in Invention 2, the 19-type
mutation introduces a mutation in the portion encoding the fourth transmembrane regionof the YggB protein (paragraph [0075]). Specifically, it is a mutation in which alanine atposition 100 in the amino acid sequence of SEQ ID NO: 6 encoded by the yggB genederived from Corynebacterium glutamicum is substituted with threonine (A100T
mutation), and the amino acid sequence encoded by the yggB gene after the mutation is
SEQ ID NO: 22 (paragraphs [0033], [0075], and [0119]).
B. Effect of the configuration using the 19-type mutation
As the Defendant alleges that, among the configurations comprised in Invention 2,
the configuration using the 19-type mutation only marginally affects the glutamic acidproduction amount if at all, the effect of the configuration using the 19-type mutation is
examined below.
(A) Regarding Working Example 10
In Working Example 10, in all cases under a glutamic acid production-inducing
condition, such as a surfactant (Tween40)-added condition or a biotin-limited condition,
the glutamic acid production amount was found to increase as compared to a wild-typestrain (non-modified strain) due to the configuration using the 19-type mutation ([Table
9] and [Table 10]).
(B) Regarding Working Example 8
a. Regarding statements in Description 2
28

Working Example 8 is an experiment performed, as in the case with Working Example
2, under a non-inducing condition where no surfactant, etc. is added, in the presence of
300 μg/l biotin, which corresponds to "a condition with an excess of biotin" (paragraph[0032]) (paragraphs [0120] and [0097]).
[Table 7] of Working Example 8 shows that the glutamic acid production amount ofa wild-type strain (non-modified strain) was 0.5 g/L, whereas that of a strain into which
the 19-type mutation was introduced was 0.7 g/L. Regarding this result, paragraph [0120]
contains the description that the glutamic acid accumulation in the culture solution was
greatly enhanced for the strain into which the 19-type mutation was introduced ascompared to the wild-type strain.
b. The Defendant alleges that the glutamic acid production amount of the abovementioned
strain into which the 19-type mutation was introduced is so low that it would match theblank values in Working Example 2 and Working Example 3, and the difference with thewild type is also only within the margin of error.
According to paragraph [0097] and Exhibit Ko 37, it can be found that the blank value
indicates the value of the glutamic acid which is derived from the soybean hydrolysate
added to the medium as a nutrient for culturing the bacterial cell and which is containedin the medium at the start of culture, but in [Table 1], [Table 2], [Table 4], and [Table 5]of Working Examples 2, 3, and 6, all of which are said to have adopted the culture methodof Working Example 2, different values from 0.4 g/L to 0.7 g/L are indicated as blankvalues. Accordingly, even if the same culture method of Working Example 2 is adopted,
the blank value could differ depending on the experiment, and hence it cannot be said that
there was no enhancement in the glutamic acid production amount of the 19-type
mutation-introduced strain in Working Example 8 by comparing the amount with blankvalues in different experiments. In addition, it also cannot be said that the difference inthe glutamic acid production amount between the 19-type mutation-introduced strain andthe wild-type strain in Working Example 8 was within the margin of error, based on blankvalues in different experiments or based on the culture results of the same strain in
different experiments.
c. Regarding the experiment in Exhibit Otsu 44
The Defendant alleges that, in the experiment of Exhibit Otsu 44 that was performedon request by the Defendant or CJCJ, no enhancement in the glutamic acid-producing
ability caused by the 19-type mutation was observed.
Exhibit Otsu 44 contains a description that, as a result of performing a culture
experiment under the same medium conditions as Working Example 8, using the samestrains as in Working Example 8, namely, a wild-type ATCC13869 strain and a29

ATCC13869 strain into which the 19-type mutation-introduced yggB gene wasintroduced, it was confirmed that neither strain produces glutamic acid.
While Working Example 8 adopts the same culture method as Working Example 2(paragraph [0120]), Working Example 2 contains a description of "shaking culture" in a"flask medium," but it does not specify the type of flask or the shaking speed (paragraph[0097]), and there is no description in Working Example 8 that specifies them either.
According to the evidence (Exhibits Ko 37 through 43) and the entire import of oral
arguments, it is found, as the common general technical knowledge as of Priority Date 2,that the glutamic acid production by fermentation will be inhibited unless the oxygen
supply amount is sufficient, and that, while a conical flask, baffled conical flask, andSakaguchi flask are typically used for shaking culture of aerobic bacteria, in order tosupply sufficient oxygen, higher speed shaking will be required for a conical flask
compared to a Sakaguchi flask.
While in the experiment of Exhibit Otsu 44, the shaking culture is found to have been
performed at a speed of 115 rpm using a conical flask (Exhibit Otsu 44; the entire importof oral arguments), Description 2 has a statement in paragraph [0146] of Working
Example 15 about an experiment in which shaking culture was performed at a speed of
115 rpm, but as it is stated that a Sakaguchi flask was used in the experiment, it cannot be
said that the abovementioned shaking speed in Exhibit Otsu 44 was appropriate based on
the statement in paragraph [0146].
In the experiment in 1. of Exhibit Ko 37 conducted by the Plaintiff, shaking culture
was performed at a speed of 115 rpm using the same medium as in Working Example 8
and using a Sakaguchi flask. As a result, it was confirmed that the glutamic acidproducing ability of a 19-type mutation-introduced ATCC13869 strain was enhanced
compared to a wild-type ATCC13869 strain. Moreover, when the same experiment was
conducted by changing the conditions to those of Exhibit Otsu 44, where a conical flask
is used and a 115 rpm shaking speed is adopted, it was confirmed that the glutamic acidproduction amount of the 19-type mutation-introduced ATCC13869 strain substantiallydeclined as compared to the abovementioned experiment results using a Sakaguchi flask.
Taking these factors into consideration, it can be considered that the glutamic acidproduction was inhibited in the experiment of Exhibit Otsu 44 due to the low shaking
speed, so the results of that experiment cannot be regarded to affect the contents ofWorking Example 8 described in a. above.
d. Consequently, as described in Working Example 8, the glutamic acid production isfound to increase as compared to a wild-type strain (non-modified strain) due to theconfiguration using the 19-type mutation under a condition with an excess of biotin.
30

(C) According to (A) and (B) above, the configuration using the 19-type mutation is foundto have an effect of solving the problem to be solved by Invention 2, and the Defendant's
argument that its effect is marginal cannot be adopted.
(4) Regarding the strains to be used in Defendant's Production Process 4
A. Regarding introduction of the A98T mutation and V241I mutation into a yggB genederived from Corynebacterium callunae
According to the evidence (Exhibit Ko 54, Exhibit Otsu 4) and the entire import oforal arguments, it is found that a mutant-type yggB gene derived from wild-type
Corynebacterium callunae is introduced into Strain [xiii], and that the mutant-type yggB
gene is one into which the following mutations have been introduced: a mutation in whichalanine at position 98 in the amino acid sequence encoded by the yggB gene of wild-type
Corynebacterium callunae (the amino acid sequence of YggB of the DSM20147 strain,
which is wild-type Corynebacterium callunae) is substituted with threonine (A98T
mutation) and a mutation in which valine at position 241 in that amino acid sequence issubstituted with isoleucine (V241I mutation).
The Defendant alleges that, although it has not submitted analysis results concerning
the yggB gene derived from Corynebacterium callunae, which was introduced into Strain[xii], the yggB gene also had the A98T mutation and V241I mutation. As the Defendant
has not asserted that there were any other points that differed from the abovementioned
mutant-type yggB gene which was introduced into Strain [xiii], it is reasonable to findthat the same mutant-type yggB gene was being introduced into Strain [xii] as well.
B. Introduction of mutations into a yggB gene derived from Corynebacterium glutamicum
According to the evidence (Exhibit Ko 54, Exhibits Otsu 2 and 4) and the entire
import of oral arguments, it is also found that a sequence of a yggB gene derived from
Corynebacterium glutamicum, which differed from the mutant-type yggB gene in A.
above, also existed in Strains [xii] and [xiii], and that the amino acid sequence encodedby the yggB gene derived from Corynebacterium glutamicum had characteristics of ●(omitted)●.
C. Effect of introduction of each mutation on the glutamic acid-producing ability
While Strains [xii] and [xiii] are both Corynebacterium glutamicum (the entire importof oral arguments), how their glutamic acid-producing ability is affected by the
introduction of the mutations mentioned in A. and B. above into the yggB genes of thesestrains is examined below.
(A) Regarding the A98T mutation and V241I mutation
a. Results of the experiment of Exhibit Ko 92 conducted by the Plaintiff
In the experiment conducted by the Plaintiff, when [i] a mutant-type yggB gene31

obtained by introducing only the A98T mutation into a yggB gene derived from
Corynebacterium callunae (DSM20147 strain) was introduced on the chromosome of
Corynebacterium glutamicum, and shaking culture was performed at a speed of 200 rpm(the same speed as in the Defendant's experiment mentioned in c. below) using the same
medium as in Working Example 8 (a condition with an excess of biotin) and using abaffled conical flask, it was confirmed that the glutamic acid production amount of thatstrain increased compared to Corynebacterium glutamicum into which a wild-type yggB
gene derived from Corynebacterium callunae has been introduced (Experiment 1 of
Exhibit Ko 92).
In addition, as a result of performing shaking culture using the abovementioned Strain[i] and using the same medium as in Working Example 10 (a condition in which surfactant
Tween 40 has been added), it was confirmed that the glutamic acid production amount of
the abovementioned Strain [i] increased compared to Corynebacterium glutamicum into
which a wild-type yggB gene derived from Corynebacterium callunae has been
introduced (Experiment 2 of Exhibit Ko 92).
b. Results of the experiment of Exhibit Ko 54 conducted by the Plaintiff
In the experiment conducted by the Plaintiff, when [ii] a mutant-type yggB geneobtained by introducing the A98T mutation and V241I mutation into a yggB gene derived
from Corynebacterium callunae (DSM20147 strain) and [iii] a mutant-type yggB geneobtained by introducing only the V241I mutation into a yggB gene derived from
Corynebacterium callunae (DSM20147 strain) were each introduced into
Corynebacterium glutamicum as plasmids, and shaking culture was performed at a speedof 115 rpm using the same medium as in Working Example 8 (a condition with an excess
of biotin) and using a Sakaguchi flask, it was confirmed that, compared with
Corynebacterium glutamicum into which a wild-type yggB gene derived from
Corynebacterium callunae has been introduced as a plasmid, the glutamic acid production
amount of the abovementioned Strain [iii] remained the same with both strains hardly
producing any glutamic acid, but the glutamic acid production amount of theabovementioned Strain [ii] greatly increased (Experiment 2 of Exhibit Ko 54).
c. Results of the experiment of Exhibit Otsu 97
In an experiment conducted by the Defendant's affiliated research institution, when amutant-type yggB gene obtained by introducing only the A98T mutation into a yggB genederived from Corynebacterium callunae was introduced into Corynebacterium
glutamicum as a plasmid, and shaking culture was performed at a speed of 200 rpm using
a medium containing an excess of biotin (300 μg/l) and using a baffled conical flask, no
glutamic acid production was confirmed in the strain (Exhibit Otsu 97).
32

d. Examination(a) Comparison between the Plaintiff's experiment and the Defendant side's experiment
As mentioned in a. and c. above, different results are indicated in the Plaintiff's
experiment (Exhibit Ko 92) and the Defendant side's experiment (Exhibit Otsu 97)
concerning whether a mutant-type yggB gene obtained by introducing only the A98T
mutation into a yggB gene derived from Corynebacterium callunae has an effect ofenhancing the glutamic acid-producing ability of the strain under a condition in which an
excess of biotin is present.
With regard to this difference, the report of the Plaintiff's experiment mentioned above
states in its discussion part that, in the Defendant side's experiment, a mutant-type yggB
gene was introduced as a plasmid, but no confirmation was made as to whether the
introduced plasmid was maintained in a stable manner within the bacterial cell, so it isprobable that the effect of the mutant-type yggB gene could not be confirmed as a resultof the plasmid becoming unstable (Exhibit Ko 92).
While the method to introduce a mutant-type yggB gene in Invention 2 includes amethod to introduce the yggB gene on the chromosome of a bacterium and a method to
introduce a plasmid containing a mutant-type yggB gene into the bacterial cell (paragraph[0050]), it has been pointed out that generally when introducing a genetic mutation byusing a plasmid, there are problems such as loss of the plasmid itself (paragraphs [0002]
and [0003] of Description 1). Also taking into consideration that the Defendant has notoffered specific allegations or proof concerning the stability of the mutant-type yggB gene
introduced as a plasmid in the Defendant side's experiment above, the Defendant side's
experiment results (Exhibit Otsu 97) cannot be adopted with regard to the effect of thecase where only the A98T mutation is introduced into a yggB gene derived from
Corynebacterium callunae.
(b) Effect of introduction of the A98T mutation and V241I mutation
Thus, according to the experiment results in a. and b. above, the introduction of amutant-type yggB gene obtained by introducing the A98T mutation into a yggB genederived from Corynebacterium callunae (DSM20147 strain) is found to cause theglutamic acid-producing ability of Corynebacterium glutamicum to be enhanced under acondition with an excess of biotin, whereas the introduction of the V241I mutation into a
yggB gene derived from Corynebacterium callunae (DSM20147 strain) is found not tocontribute to enhancement of the glutamic acid-producing ability of Corynebacterium
glutamicum under a condition with an excess of biotin.
The Defendant asserts that the introduction of only the A98T mutation into a yggB
gene derived from Corynebacterium callunae does not cause the glutamic acid-producing
33

ability to be enhanced, but that the glutamic acid-producing ability increases only when
the V241I mutation is added to the A98T mutation. However, in light of the experimentresults in a. and b. above, this assertion cannot be adopted.
(B) Regarding introduction of a mutation into a yggB gene derived from
Corynebacterium glutamicum
According to the evidence (Exhibit Ko 54), it is found that when, after introducing
the mutation mention in B. above into a yggB gene derived from Corynebacterium, thismutant-type yggB gene was introduced into Corynebacterium glutamicum, and shaking
culture was performed at a speed of 115 rpm using the same medium as in Working
Example 8 (a condition with an excess of biotin) and using a Sakaguchi flask, the glutamic
acid-producing ability could not be confirmed in this strain, indicating the same result as
for a strain without the yggB gene; and with regard to this experiment result, aconsideration has been made that the amino acid sequence encoded by theabovementioned mutant-type yggB gene does not function as YggB. Consequently, it canbe said that the introduction of this mutation does not contribute to enhancement of theglutamic acid-producing ability under a condition in which an excess of biotin is present.
(5) Comparison between Defendant's Production Process 4 and Invention 2
A. In light of the examination made in (4) above, it can be said that Strains [xii] and [xiii]
used in Defendant's Production Process 4 are both Corynebacterium glutamicum into
which a mutant-type yggB gene obtained by introducing the A98T mutation and V241I
mutation into a yggB gene derived from Corynebacterium callunae (DSM20147 strain)
was introduced, and their glutamic acid-producing ability under a condition in which an
excess of biotin is present has been enhanced as compared to a non-modified strain.
Consequently, Strains [xii] and [xiii] used in Defendant's Production Process 4literally fulfill Constituent Features 2-A and 2-B of Invention 2-1 but do not fulfill
Constituent Feature 2-C of Invention 2-1, and they do not fulfill any of the constituent
features (Constituent Features 2-D, 2-E, 2-F-1, 2-F-2, and 2-F-3) of Inventions 2-2 and2-3. In addition, according to the entire import of oral arguments, as in the case withDefendant's Production Processes 1 through 3, Defendant's Production Process 4 is foundto literally fulfill Constituent Feature 2-H of Invention 2-5, except for the parts citing
Claims 1 through 10, and fulfill Constituent Features 2-I and 2-J of Invention 2-5.
B. Differences between Defendant's Production Process 4 and the configuration using the
19-type mutation
In light of the examination made in (3) and (4) above, the differences between
Defendant's Production Process 4 and the configuration using the 19-type mutation
comprised in Defendant's Invention 2-5 (the process for producing glutamic acid claimed34

in Claim 11 of Patent 2, which uses a coryneform bacterium into which the mutant-type
yggB gene mentioned to in (e) of Claim 6, which cites Claim 1 or 4, has been introduced)
lie in the parts relating to Constituent Features 2-C, 2-D, 2-E, 2-F-1, 2-F-2, 2-F-3, and 2H (literally non-fulfilling parts mentioned in A. above), and specifically, the following
differences are found with regard to the strains being used.
Difference 1: The mutant-type yggB gene introduced is one derived from
Corynebacterium glutamicum (the amino acid sequence before the mutation is SEQ ID
NO: 6) in the case of the strain with the configuration using the 19-type mutation, whereas
it is one derived from Corynebacterium callunae (DSM20147 strain) in the case of thestrains used in Defendant's Production Process 4 (Strains [xii] and [xiii]).
Difference 2: The mutation introduced into the yggB gene is one in which alanine atposition 100 in the amino acid sequence encoded by the yggB gene is substituted withthreonine (A100T mutation) in the case of the strain with the configuration using the 19type mutation, whereas it is one in which alanine at position 98 in the amino acid sequence
encoded by the yggB gene is substituted with threonine (A98T mutation) in the case of
the strains used in Defendant's Production Process 4.
Difference 3: In the case of the configuration using the 19-type mutation, only the
A100T mutation is introduced into the yggB gene, whereas in the case of the strains usedin Defendant's Production Process 4, not only the A100T mutation, but also a mutation inwhich valine at position 241 in the amino acid sequence encoded by the yggB gene issubstituted with isoleucine (V241I mutation) is introduced into the yggB gene.
As mentioned in (4) B. above, apart from the abovementioned mutant-type yggB genederived from Corynebacterium callunae, Strains [xii] and [xiii] have a sequence of a
yggB gene derived from Corynebacterium glutamicum, and the amino acid sequence
encoded by it is ●(omitted)● . However, as mentioned in (4) C. (B) above, it isconsidered that the sequence of this yggB gene does not contribute to enhancement of theglutamic acid-producing ability under a condition in which an excess of biotin is present,
and that the amino acid sequence encoded by it does not function as YggB, and neither
party argues that the presence of that sequence is a difference that should be considered
in determining whether or not infringement under the doctrine of equivalents isestablished. Therefore, it cannot be said that the presence of this sequence is a difference
that should be considered in addition to Differences 1 through 3 above.
(6) Regarding the first requirement (non-essential part)A. Essential part of Invention 2 (configuration using the 19-type mutation)
As mentioned above, in this case, the reasonable approach to determine whether or
not Defendant's Production Process 4 falls within the technical scope of Invention 2 as an35

equivalent thereof would be to compare Defendant's Production Process 4 with theconfiguration using the 19-type mutation comprised in Invention 2, thus, also in
examining the essential part of Invention 2 in relation to fulfillment of the firstrequirement, specifically, the essential part of the configuration using the 19-type
mutation will be examined as follows.
(A) The substantial value of an invention which the Patent Act intends to protect exists inthe disclosure, with a specific structure, to society of a means for solving a technicalproblem that could not have been solved by prior art, which is based on a unique technicalidea that is not seen in prior art. The essential part of a patented invention should beunderstood as the characteristic part which constitutes a unique technical idea that is notseen in prior art in the statements in the claims of the patented invention (see theabovementioned Intellectual Property High Court judgment of March 25, 2016).
(B) As mentioned in (2) C. above, when compared with the prior art described in
Description 2, the unique technical idea in Invention 2 that is not seen in prior art (theprinciple for solving the problem) should be considered to be the following: to focus on
the yggB gene of a coryneform bacterium, whose effect on production of glutamic acidhad been unknown in the past, modify the YggB protein, which is a type of amechanosensitive channel, by using a mutant-type yggB gene into which a C-terminalside mutation or mutations in transmembrane regions are introduced, and thereby provide
novel art to enhance the glutamic acid-producing ability of the bacterium. In addition, as
examined in (2) D. above, the statements on prior art in Description 2 are not found to be
insufficient from an objective viewpoint.
(C) As mentioned in (3) A. above, the configuration using the 19-type mutation iscomprised in Invention 2-5, and it uses a coryneform bacterium into which the mutanttype yggB gene mentioned in (e) of Claim 6, which cites Claim 1 or 4, has been introduced.
In light of the unique technical idea or the principle for solving the problem in Invention
2 mentioned in (B) above, the essential part of the configuration using the 19-type
mutation is found to be "to introduce into a yggB gene derived from a coryneformbacterium a mutation equivalent to the A100T mutation in the yggB gene derived from
Corynebacterium glutamicum, modify the coryneform bacterium by using that mutanttype yggB gene, and enhance the glutamic acid-producing ability of the bacterium evenunder a condition in which an excess of biotin is present.
(D) The Defendant alleges that, in light of the application history, the state of the art as ofPriority Date 2, and the effect of the configuration using the 19-type mutation, when
determining the essential part of the configuration using the 19-type mutation, thestatements of the claims should not be adopted as a generic concept, but instead should36

be limited to "the A100T mutation in the specific amino acid sequence of SEQ ID NO:
22, which is the sequence of the yggB gene after the mutation" described in the claims.
However, according to the contents of Description 2 described in (2) A. and B. above,
Invention 2 does not target a problem that only exists in a coryneform bacterium having
a yggB gene with a specific sequence, and the principle for solving the problem was tointroduce novel art of modifying YggB protein, which is a type of a mechanosensitive
channel, by using a mutant-type yggB gene into which a C-terminal side mutation or
mutations in transmembrane regions are introduced, in order to enhance the glutamic
acid-producing ability. Even by taking into consideration that Claim 1 or Claim 4 ofInvention 2 lists the amino acid sequences of the yggB gene before introduction of themutation, and Claim 6 of Invention 2 lists the amino acid sequences of the yggB geneafter the mutation, when determining Invention 2 and the essential part of theconfiguration using the 19-type mutation comprised therein, it is reasonable to find thatthe species of the coryneform bacterium from which the yggB gene is derived, the specificsequence of the entire yggB gene before the mutation, or the specific sequence of theyggB gene after the introduction of a mutation equivalent to the A100T mutation is notan essential part. The same applies even if consideration is given to the following facts,
which are pointed out by the Defendant: the fact that initial Claim 1 at the time of filingthe application for Patent 2 did not specify the species of the coryneform bacterium fromwhich the yggB gene is derived or the amino acid sequence of the yggB gene before andafter the mutation, but rather, as a result of correction, the amino acid sequences of theyggB gene were specified as those of SEQ ID NOs: 6, 62, 68, 84, and 85, which arederived from Corynebacterium glutamicum (including Brevibacterium flavum) or
Corynebacterium melassecola, as in current Claim 1 (paragraph [0033]; Exhibits Otsu 80
through 84); and the fact that the amino acid sequences of SEQ ID NOs: 6, 62, 68, 84,
and 85, which are described in Claim 1, are mutually highly homologous (Exhibit Otsu
85). In addition, the Defendant alleges that the statements of the claims after the recorrection of Patent 2 should also be taken into consideration in relation to the application
history, but it should be said that the contents of that correction at least do not affect thedetermination of the essential part of Invention 2 before the correction.
Moreover, the Defendant's allegations regarding the state of the art as of Priority Date
2 and the effect of the configuration using the 19-type mutation cannot be adopted as
mentioned in (2) D. and (3) B. above, and it cannot be said that the essential part of theconfiguration using the 19-type mutation should be limited to the specific sequence of theyggB gene before and after the mutation described in the claims based on thoseallegations. Thus, the Defendant's allegations on those points do not affect the37determination mentioned in (C) above either.
B. Regarding Difference 1
As mentioned in A. (D) above, with regard to the essential part of the configuration
using the 19-type mutation, when considering that it is reasonable to find that the speciesof the coryneform bacterium from which the yggB gene is derived, the specific sequenceof the entire yggB gene before the mutation, or the specific sequence of the yggB geneafter the introduction of a mutation equivalent to the A100T mutation is not an essential
part of the configuration, and when also considering the points in (A) and (B) below, itcannot be said that the differences relating to Difference 1, that is, the difference in thespecies of bacterium from which the introduced mutant-type yggB gene is derived andthe resulting difference in the specific sequence of the yggB gene constitute the essential
part of the configuration using the 19-type mutation.
(A) Characteristics of Corynebacterium callunae
According to the evidence (Exhibits Ko 54 through 56 and 78, Exhibit Otsu 86) andthe entire import of oral arguments, it is found that, Corynebacterium callunae, whencompared with Corynebacterium glutamicum, belongs to the same genus
Corynebacterium and is also a closely related bacterium in that category, and as in thecase with Corynebacterium glutamicum, it is known as a microorganism used whenindustrially producing glutamic acid. In Description 2 as well, it is mentioned as an
example of the coryneform bacterium in Invention 2 (paragraph [0012]). Exhibit Otsu 96
describes that, although the two bacteria belong to the same genus Corynebacterium, theircharacteristics are varied, but it does not indicate that there are specific differences
between the characteristics of Corynebacterium callunae and those of Corynebacterium
glutamicum in relation to glutamic acid production, and thus does not affect theabovementioned determination.
(B) Identity and homology levels of the specific sequence of the mutant-type yggB gene
According to the evidence (Exhibits Ko 54, 56, and 71, Exhibit Otsu 85) and the entire
import of oral arguments, when the entire amino acid sequence of the mutant-type yggB
gene derived from Corynebacterium glutamicum used in the configuration using the 19type mutation (the sequence of SEQ ID NO: 22 after the introduction of the A100T
mutation) is compared with the entire amino acid sequence of the yggB gene of Strains[xii] and [xiii], it is found that these amino acid sequences are approximately 64%
identical, and when they are compared by also taking into consideration conservative
substitution with a similar amino acid, the sequences are approximately 91% homologous.
C. Regarding Difference 2
According to the evidence (Exhibit Ko 54, Exhibit Otsu 85) and the entire import of38

oral arguments, it is found that, when comparison is made by a general method forcomparing amino acid sequences for the amino acid sequences of the yggB gene derived
from Corynebacterium glutamicum (the amino acid sequence of SEQ ID NO: 6 before
the mutation) and the yggB gene derived from Corynebacterium callunae (DSM20147
strain), which were introduced into a strain with the configuration using the 19-type
mutation, alanine at position 100 in the former corresponds to alanine at position 98 in
the latter. In addition, when these alanine are compared using three-dimensional protein
structures, they are found to exist at equivalent positions in the transmembrane regions of
the YggB.
Accordingly, with regard to Difference 2, the fact that the position of alanine to be
substituted with threonine in the amino acid sequence encoded by the yggB gene isalanine at position 100 in the case of a strain with the configuration using the 19-type
mutation, whereas it is alanine at position 98 in Strains [xii] and [xiii] of Defendant's
Production Process 4, does not constitute a different in the essential part of theconfiguration using the 19-type mutation.
D. Regarding Difference 3(A) According to the evidence (Exhibits Ko 54 and 56, Exhibit Otsu 85) and the entire
import of oral arguments, when comparison is made by a general method for comparing
amino acid sequences as in C. above, the following results are found: that valine atposition 241 in the yggB gene derived from Corynebacterium callunae (DSM20147
strain) corresponds to valine at position 243 in the yggB gene of a strain with theconfiguration using the 19-type mutation (amino acid sequence of SEQ ID NO: 6 before
the mutation); that these valines do not correspond to the amino acids in thetransmembrane region (paragraph [0073]) and the C-terminal side region (paragraph[0070]) disclosed in Description 2, and even when the three-dimensional protein
structures are examined, they do not exist in the transmembrane regions or the vicinities
thereof; and that as valine and isoleucine have close characteristics, the substitution ofvaline with isoleucine has little effect on the protein structure.
(B) In addition, Description 2 contains a description that, as long as there is a function toenhance the L-glutamic acid-producing ability under a condition in which an excess of
biotin is present, there may be "an amino acid sequence in which one or several aminoacids in addition to amino acids of the ... mutation point" of the C-terminal side mutationor the transmembrane region "are substituted, deleted, inserted, or added," that thissubstitution is "preferably a conservative substitution (neutral mutation)," and that its
examples include substitution of valine (val) with isoleucine (ile) (paragraph [0078]).
Furthermore, Claim 6 (f) indicates that "DNA that encodes a protein having an amino acid39

sequence in which one to five amino acids" in the amino acid sequence of the mutanttype yggB gene (SEQ ID NO: 22) of the strain with the configuration using the 19-type
mutation "are substituted, deleted, inserted, or added, and, through introduction into a
coryneform bacterium, enhances the L-glutamic acid-producing ability of the coryneformbacterium when cultured in a medium containing an excess of biotin" is also comprised
in Invention 2-3.
(C) Moreover, as mentioned in (4) C. above, it is found that, while Strains [xii] and [xiii]
into which the A98T mutation and V241I mutation are introduced show enhancement in
the glutamic acid-producing ability under a condition with an excess of biotin, whichsolves the problem to be solved by Invention 2, what contributes to this is the A98T
mutation, and not the V241I mutation.
(D) In light of these points, the difference relating to Difference 3, that is, the fact that, in
addition to the A98T mutation relating to Difference 2, the V241I mutation is introduced
into the strain of Defendant's Production Process 4, is a difference to make a conservative
substitution to one amino acid at a site unrelated to the mutations in the transmembraneregions or the C-terminal side mutation, which constitutes the principle for solving the
problem to be solved by Invention 2 disclosed in Description 2, and addition of thismutation to the A98T mutation does not affect the solution of the problem. Therefore, thisdifference is not a difference in the essential part of the configuration using the 19-type
mutation.
E. Consequently, as none of the Differences 1 through 3 between the configuration using
the 19-type mutation and Defendant's Production Process 4 are differences in the essential
part of the patented invention, Defendant's Production Process 4 that uses Strains [xii]and [xiii] is found to fulfill the first requirement of the doctrine of equivalents.
(7) Regarding the second requirement (replaceability)
According to (3) B. and (4) C. above, it is found that, Strains [xii] and [xiii] used in
Defendant's Production Process 4 show enhancement in the glutamic acid-producing
ability, which solves the problem to be solved by Invention 2, and have the same functionand effect as the configuration using the 19-type mutation, which is to enhance theglutamic acid-producing ability under a condition with an excess of biotin.
The Defendant alleges that, as the essential part of the configuration using the 19-type
mutation is a specific mutation, SEQ ID NO: 22, and its function and effect are also
limited to the function and effect of a specific amino acid sequence, SEQ ID NO: 22, thefunction and effect of Defendant's Production Process 4 and those of the configuration
using the 19-type mutation are not the same.
However, as mentioned in (6) A. (D) above, in light of the problem to be solved by40

Invention 2 and the principle for solving that problem, it cannot be said that the essential
part of the configuration using the 19-type mutation should be limited to a specificsequence of the yggB gene before and after the mutation, and it cannot be said either thatthe function and effect of the configuration using the 19-type mutation are limited to thoseof the amino acid sequence of SEQ ID NO: 22. Therefore, the Defendant's allegation
cannot be adopted.
Consequently, Defendant's Production Process 4 that uses Strains [xii] and [xiii] isfound to fulfill the second requirement of the doctrine of equivalents.
(8) Regarding the third requirement (ease of replacement)
A. Regarding Difference 1 and Difference 2(A) Statement of Corynebacterium callunae in Description 2
In Description 2, Corynebacterium callunae is mentioned as an example of thecoryneform bacterium in Invention 2, as in the case with Corynebacterium glutamicum(including Brevibacterium flavum) or Corynebacterium melassecola from which SEQ ID
NOs: 6, 62, 68, 84, and 85 of the yggB gene described in Claim 1 are derived (paragraphs[0012] and [0033]).
(B) Common general technical knowledge concerning the characteristics ofCorynebacterium callunae
According to the evidence (Exhibits Ko 54 through 56 and 78, Exhibit Otsu 86) andthe entire import of oral arguments, Corynebacterium callunae is found to have been
known from long ago by persons skilled in the art as a microorganism used whenindustrially producing glutamic acid, with a U.S. patent being registered for a process for
producing glutamic acid by using Corynebacterium callunae in 1963, for example. In
addition, with regard to the relationship between Corynebacterium callunae andCorynebacterium glutamicum, it was already known as of Priority Date 2 that they bothbelong to the same genus Corynebacterium and are also closely related bacteria in thatcategory, and even after the classification was revised due to discovery of new
coryneform bacteria or the like, this point is found to have remained the same. It is foundthat these characteristics of Corynebacterium callunae were already common generaltechnical knowledge as of July 2016, when production by Defendant's Production Process
4 was started.
(C) Status of genomic analysis of Corynebacterium callunae
According to the evidence (Exhibits Ko 56, 74, and 84, Exhibit Otsu 86) and the entire
import of oral arguments, it is found that, the entire genome of the Corynebacterium
callunae DSM20147 strain and the amino acid sequence of the yggB gene had not been
analyzed as of Priority Date 2, but these analyses were completed by March 2013, and41

the results of both analyses were registered on the database in the same month. It is also
found that the results of the abovementioned genomic analysis of the DSM20147 strain
were reported in an article published in January 2015 (Standards in Genomic Sciences(2015) 10：5; Exhibit Otsu 86), which described the relationship with Corynebacterium
glutamicum as shown below, and suggested the possibility that knowledge concerning
Corynebacterium glutamicum may be applied to Corynebacterium callunae due to thecloseness of their genome sequences.
a. Last line of the left column to line 6 of the right column on page 5"As C. callunae was shown to produce l-glutamate in an amount comparable to C.
glutamicum, C. callunae might be considered as a potential candidate for future genome
reduction efforts since the chromosome is already considerably smaller than that of C.
glutamicum and C. efficiens (2.84 Mbp versus 3.21 Mbp and 3.15 Mbp, respectively)."b. Line 10 to line 14 of the left column on page 6"Therefore, this bacterium might be an ideal choice for future development of a platformstrain as the otherwise high degree of similarity of its genome content to the well studied
C. glutamicum would allow an easy transfer of knowledge to the new host."(D) Results of analysis of the yggB gene of Corynebacterium callunae using generalsoftware, etc. after the genomic analysis
According to the evidence (Exhibit Ko 54, Exhibits Otsu 85 and 98) and the entire
import of oral arguments, as mentioned in (C) above, as of 2013, when the yggB gene of
Corynebacterium callunae was registered on the database, it was possible to search, fromamong the yggB genes registered on the database, amino acid sequences that were highly
identical to the amino acid sequence of the yggB gene of the strain with the configuration
using the 19-type mutation before the mutation (SEQ ID NO: 6), by using general search
software (Blast) that can be used via the internet, and the amino acid sequence of theyggB gene of Corynebacterium callunae (DSM20147 strain) that was registered as
mentioned in (C) above is indicated as one of the highly identical sequences (70%
identical at 90% coverage), following a YggB gene derived from Corynebacterium
glutamicum (including Brevibacterium flavum that is classified as Corynebacterium
glutamicum under the current classification). Moreover, when the amino acid sequenceof SEQ ID NO: 6 and the amino acid sequence of the yggB gene of Corynebacterium
callunae (DSM20147 strain) were compared by using the abovementioned software orother general software (ClustalW) that is accessible via the internet, it is found that alanine
at position 100 in SEQ ID NO: 6 could be confirmed to correspond to alanine at position
98 of the DSM20147 strain.
(E) Whether or not there was difficulty, etc. in introducing a mutant-type yggB gene42derived from Corynebacterium callunae
According to the evidence (Exhibits Ko 54 and 92) and the entire import of oral
arguments, it is found that, as of July 2016, when production by Defendant's Production
Process 4 was started, if the amino acid sequence of the yggB gene of Corynebacterium
callunae (DSM20147 strain) and the position of the amino acid into which a mutation is
to be introduced were specified, a person skilled in the art would have been able tointroduce the A98T mutation into that amino acid sequence and further introduce it into
Corynebacterium glutamicum by, for example, using the process described in Description
2, and hence it cannot be found that there was technical difficulty with regard to that point.
In addition, according to the evidence (Exhibits Ko 75 through 77) and the entire
import of oral arguments, it is found to have been known from before Priority Date 2 thatwhen a gene derived from a certain bacterium is introduced into a bacterium of anotherspecies having a similar gene, the bacterium will function in a similar manner. Therefore,
it cannot be said that a person skilled in the art would think introduction of a mutant-type
yggB gene derived from Corynebacterium callunae into Corynebacterium glutamicum
would not produce an effect, only on a basis that the bacterial species are different, and it
also cannot be said that a person skilled in the art had knowledge suggesting that suchconfiguration should be avoided.
(F) Examination
As examined in (A) through (E) above, the following can be found: Description 2
mentioned Corynebacterium callunae as an example of the coryneform bacterium of
Invention 2 ((A) above); it was common general technical knowledge that
Corynebacterium callunae is a glutamic acid-producing bacterium and is closely related
to Corynebacterium glutamicum ((B) above); as the genomic analysis ofCorynebacterium callunae was completed by March 2013 and it became possible tosearch or analyze the amino acid sequence of its yggB gene by using general software, it
became possible to check that the amino acid sequence of the yggB gene of SEQ ID NO:
6 and that of the yggB gene of Corynebacterium callunae (DSM20147 strain) are highly
identical, and that alanine at position 100 of SEQ ID NO: 6 corresponds to alanine atposition 98 of the DSM20147 strain ((C) and (D) above); and if the position of the aminoacid into which a mutation is to be introduced was specified, it would not have beentechnically difficult for a person skilled in the art to introduce the A98T mutation into theamino acid sequence of the DSM20147 strain and further introduce it into
Corynebacterium glutamicum, and it also cannot be said that a person skilled in the art
had knowledge suggesting that use of a mutant-type yggB gene derived from a different
bacterial species should be avoided ((E) above). In light of these findings, it is reasonable43

to find that, as of July 2016, when production by Defendant's Production Process 4 wasstarted, a person skilled in the art could have easily conceived of substituting theconfiguration using the 19-type mutation with the configurations relating to Differences
1 and 2, in other words, replacing Corynebacterium glutamicum used as the strain fromwhich the mutant-type yggB gene is derived with Corynebacterium callunae, andaccordingly changing the position of the alanine to be substituted with threonine in theamino acid sequence of the yggB gene from position 100 to position 98.
The Defendant alleges that the ease in conceiving of the invention as mentioned in
the third requirement should be construed to be the level of ease for any person skilled in
the art to be able to recognize the invention in the same manner as that specified in theclaims, and that such ease did not exist in this case. However, in light of theabovementioned circumstances, it should be said that the ease in conceiving ofDifferences 1 and 2 can be found by deeming an average technician at a glutamic acidfermentation plant using bacteria to which Invention 2 belongs, who is a person skilledin the art, as a standard, and the Defendant's allegation regarding this point cannot be
adopted. In addition, while the Defendant also alleges that the priority order ofintentionally selecting a yggB gene derived from Corynebacterium callunae in place of a
yggB gene derived from Corynebacterium glutamicum was low, this allegation cannot be
considered to overturn the abovementioned conclusion in light of the matters mentioned
in Description 2 as discussed in (A) and (B) above, common general technical knowledge
on Corynebacterium callunae, and the available analysis results mentioned in (D) above.
B. Regarding Difference 3(A) As mentioned in (6) D. (B) above, Description 2 (paragraph [0078]) and Claim 6 (f)of Invention 2 indicated that the problem can be solved in the same manner even if aconservative substitution, such as a substitution of valine with isoleucine, is made to theamino acid sequence of the yggB gene of the strain with the configuration using the 19type mutation (SEQ ID NO: 22).
(B) In addition, as mentioned in (6) D. (A) above, while valine at position 241 in a yggB
gene derived from Corynebacterium callunae (DSM20147 strain) corresponds to valine
at position 243 in the yggB gene that was introduced into the strain with the configuration
using the 19-type mutation (the amino acid sequence of SEQ ID NO: 6 before themutation), according to the evidence (Exhibit Ko 54, Exhibits Otsu 85 and 98) and theentire import of oral arguments, it is found that, in the same manner as in A. (D) above, aperson skilled in the art could check the mutual correspondence between theabovementioned valines by using general software as of 2013, when the yggB gene of
Corynebacterium callunae was registered on the database, and could also recognize that44

the positions of these valines do not correspond to those of amino acids in thetransmembrane regions disclosed in Description 2 (amino acid numbers 1–23, 25–47, 62–
84, 86–108, and 110–132 of SEQ ID NO: 6; [paragraph 0073]) and the C-terminal side
region (amino acid numbers 419–533; SEQ ID NO: 6; [paragraph 0070]).
(C) Moreover, according to the evidence (Exhibit Ko 54) and the entire import of oral
arguments, in the same manner as in A. (E) above, if the amino acid sequence of the yggB
gene of Corynebacterium callunae (DSM20147 strain) and the position of the amino acidinto which a mutation is to be introduced were specified, it would have been possible asof July 2016, when production by Defendant's Production Process 4 was started, for a
person skilled in the art to introduce the V241I mutation along with the A98T mutation
into that amino acid sequence by, for example, using the process described in Description
2, and it cannot be found that there was technical difficulty with regard to that point.
(D) Accordingly, as adopting the configuration relating to Difference 3 in addition to
those relating to Differences 1 and 2 for the configuration using the 19-type mutation, inother words, introducing the V241I mutation, in addition to the A98T mutation, into a
yggB gene derived from Corynebacterium callunae (DSM20147 strain), can beconsidered as introducing a mutation corresponding to the conservative substitution
whose introduction into the yggB gene of the strain with the configuration using the 19type mutation was indicated in Description 2 and Claim 6 (f) as being able to solve theproblem in the same manner, it is reasonable to find that a person skilled in the art could
have easily conceived of the adoption of the relevant configurations as of July 2016, when
production by Defendant's Production Process 4 was started, which was after the yggB
gene of Corynebacterium callunae was registered on the database.
(E) The Defendant alleges that, in Defendant's Production Process 4, the glutamic acidproducing ability increases only when the V241I mutation is added to the A98T mutation,
so it is necessary to combine these two mutations in Defendant's Production Process 4,
but nothing suggested such combination, and hence it was not easy for a person skilled in
the art to adopt the configuration relating to Difference 3 in addition to those relating to
Differences 1 and 2. However, as mentioned in (4) C. above, it cannot be said that theglutamic acid-producing ability increases only when the V241I mutation is added to the
A98T mutation, and the V241I mutation is found to be not contributing to enhancementof the glutamic acid-producing ability under a condition with an excess of biotin, theDefendant's allegation cannot be adopted.
Furthermore, the Defendant also alleges that, because even a substitution of an aminoacid outside the transmembrane regions could affect efflux of glutamic acid, it could not
be said that the V241I mutation clearly had no effect on efflux of glutamic acid. With45

regard to this point, an article published after Priority Date 2, in 2013, as indicated by the
Defendant (Biosci. Biotechnol. Biochem. (2013) 77 (5) 1008–1013; Exhibit Ko 31,
Exhibit Otsu 87) describes that the yggB gene of Corynebacterium glutamicum affects
glutamic acid production as a result of deleting the region of amino acid numbers 221–
232 in the amino acid sequence and deleting the region of amino acid numbers 420–533,
included in the C-terminal side region referred to in Description 2. However, even if suchknowledge existed as of July 2016, given that the deletions at these sites differ from theV241I mutation in terms of their positions and contents of the mutation, it cannot be said
that a person skilled in the art would have considered that introduction of the V241I
mutation would produce a different effect from the conservative substitution indicated in
Description 2 even by taking such knowledge into consideration. Thus, this point is not
sufficient either to overturn the conclusion mentioned in (D) above.
C. Consequently, as it can be found that a person skilled in the art could have easily
conceived of replacing the configuration using the 19-type mutation with theconfigurations relating to Differences 1 through 3 as of July 2016, when production by
Defendant's Production Process 4 was started, Defendant's Production Process 4 using
Strains [xii] and [xiii] fulfills the third requirement of the doctrine of equivalents.
(9) Regarding the fourth requirement (whether the subject process can be easily
presumptively conceived of)With regard to Defendant's Production Process 4, there are no allegations and proof
on existence of circumstances that fall under the fourth requirement based on whichapplication of the doctrine of equivalents should be denied.
(10) Regarding the fifth requirement (special circumstances)
A. Statements of the initial claims, etc. at the time of the filing and the status of corrections
According to the evidence (Exhibits Ko 79 through 82, Exhibits Otsu 80 through 84)
and the entire import of oral arguments, initial Claim 1 at the time of filing the application
for Invention 2 read "a coryneform bacterium having an L-glutamic acid-producing
ability, wherein the coryneform bacterium has an enhanced L-glutamic acid-producing
ability as compared to a non-modified strain as a result of being modified by using theyggB gene" (Exhibit Otsu 80), and Invention 2 included claims that did not specify thestrain from which the mutant-type yggB gene is derived or the specific amino acidsequence before and after the mutation, but subsequently, as a result of making corrections
twice in response to a notice of grounds for rejection, the statement of Claim 1 at the timeof registration read as described in [Claim 1] of Attachment 5-1 "Claims (Patent 2)," andthe numbers of the amino acid sequences of the mutant-type yggB gene before themutation were specified as SEQ ID NOs: 6, 62, 68, 84, and 85 derived from46

Corynebacterium glutamicum (including Brevibacterium flavum) or Corynebacterium
melassecola, while the contents of the mutation to be introduced therein were also
specified. As a result, it is found that a configuration that uses a mutant-type yggB genederived from Corynebacterium callunae, such as in Defendant's Production Process 4,ceased to be literally included within the technical scope of Invention 2.
In addition, according to the evidence (Exhibits Ko 81 and 82, Exhibits Otsu 82
through 84), it is found that, in Description 2 at the time of the filing, Corynebacterium
callunae was only mentioned in paragraphs [0012] and [0013], and these parts were notsubject to corrections at the time of making the abovementioned corrections.
B. Determination on whether or not there are special circumstances(A) Under the fifth requirement, application of the doctrine of equivalents cannot be
asserted if there are special circumstances, such as the fact that the subject product, etc.
falls under those that are intentionally excluded from the claims in the patent application
procedures for the patented invention. The reason is that, for technology which thepatentee had once acknowledged not to fall within the technical scope of the patented
invention, or in relation to which the patentee had behaved as if he/she had objectively so
acknowledged, the patentee is not entitled to make an assertion otherwise afterwards,
since this is against the doctrine of estoppel (see the 1998 Supreme Court Judgment andthe 2017 Supreme Court Judgment).
(B) As shown in the process of the application filing and corrections mentioned in A.
above, Claim 1 at the time of the filing could comprise a configuration that uses a mutanttype yggB gene derived from Corynebacterium callunae, such as in Defendant's
Production Process 4, but such configuration ceased to be literally comprised in Invention
2 as a result of the corrections.
(C) However, as mentioned in (8) A. (C) above, the entire genome of the
Corynebacterium callunae DSM20147 strain and the amino acid sequence of the yggB
gene were analyzed and became available in March 2013, and as of December 28, 2004,
which is Priority Date 2, or as of December 28, 2005, which is the filing date of Patent 2,
it was not possible to specify the amino acid sequence of the yggB gene of
Corynebacterium callunae. Accordingly, as mentioned in (8) A. (B) above, even by taking
into account that it was known from before Priority Date 2 that Corynebacterium callunae
is a glutamic acid-producing bacterium and is closely related to Corynebacterium
glutamicum, it is not found to have been easily possible for the Plaintiff, which is theapplicant, to specify a specific configuration using a mutant-type yggB gene derived from
Corynebacterium callunae, which could solve the problem to be solved by Invention 2,
and state it in the claims in a form that satisfies the support requirement and other47

description requirements at the time of the filing of the application for Invention 2.
(D) Moreover, as mentioned in A. above, the statement of Claim 1 at the time of the filing
did not specify the bacterial species any more than generally stating "a coryneformbacterium having an L-glutamic acid-producing ability," and in particular, it did not statea configuration that uses a mutant-type yggB gene derived from Corynebacterium
callunae. In addition, Description 2 only mentioned Corynebacterium callunae as an
example of a bacterium that could be used as the coryneform bacterium of Invention 2(paragraphs [0012] and [0013]), and no mention is made on a configuration that uses ayggB gene derived from Corynebacterium callunae in Description 2 either before andafter the corrections.
(E) In light of the circumstances mentioned in (C) and (D) above, it cannot be said, basedon the process of the application filing and corrections referred to in (B) above, that a
purport to intentionally exclude a configuration that uses a mutant-type yggB genederived from Corynebacterium callunae from the claims was indicated from an objective
and external viewpoint. Furthermore, no special circumstances are found, based on allevidence in this case, such as a fact that Defendant's Production Process 4 had been
intentionally excluded from the claims in the course of the patent application procedureof the patented invention, in relation to the fifth requirement.
(11) Summary on the existence or non-existence of infringement under thedoctrine of equivalents
According to the above, Defendant's Production Process 4 that uses Strains [xii] and[xiii] is found to satisfy the first through third requirements of the doctrine of equivalents,
and as no circumstances that fall under the fourth and fifth requirements are found, it isreasonable to construe that Defendant's Production Process 4 that uses Strains [xii] and[xiii] falls within the technical scope of Invention 2-5 as an equivalent to the configuration
using the 19-type mutation stated in the claims.
5. Regarding Issue 3-3 (whether or not Invention 1 violates the enablement requirement
or the support requirement)
In light of the case, among grounds for invalidation of Invention 1, determination ismade as to whether or not Invention 1 violates the enablement requirement and the
support requirement.
(1) Statements concerning the process for producing arginine in Description 1
In addition to the statements in Attachment 12 "Statements in Description 1,"
Description 1 has the following statements on the process for producing arginine.
A. Means for solving problem[0012]
48

The amino acid as the target substance may be any amino acid as long as the gene
involved in biosynthesis and its promoter are known. Specific examples of enzymes
involved in biosynthesis include...
In arginine fermentation, arginine is produced through reactions catalyzed by Nacetylglutamate synthase, N-acetylglutamate kinase, N-acetylglutamylphosphate
reductase,acetylornithine
aminotransferase,
N-acetylornitinase,ornithine
carbamyltransferase, argininosuccinate synthase, and argininosuccinase. These enzymes
are found to be effective. Further, these enzymes are encoded by the respective genes ofargA, argB, argC, argD, argE, argF, argG, and argH in order.
[0019]
Examples of the promoter for argininosuccinate synthase include one that has at least
one kind of DNA sequence selected from the group consisting of TTGCCA, TTGCTA,
and TTGTCA in the -35 region and/or has the TATAAT sequence or said sequence inwhich the ATAAT base is substituted with another base and which does not inhibit thepromotor function in the -10 region. The present invention also provides anargininosuccinate synthase gene having the abovementioned promoter.
In addition, the present invention provides a coryneform arginine-producing
bacterium having the abovementioned gene.
Amino acid can be obtained by culturing a bacterium that produces the coryneform
amino acid of the present invention, preferably L-glutamic acid, in a liquid medium,having the bacterium produce and accumulate the desired amino acid, preferably Lglutamic acid, in the medium, and collecting the amino acid from the medium.
In the present invention, a normal nutrient medium containing a carbon source, anitrogen source, inorganic salts, growth factors and the like is used as a liquid medium
for culturing the abovementioned strain.
As the carbon source, glucose, fructose, sucrose, waste molasses, carbohydrates suchas starch hydrolysate, alcohols such as ethanol and glycerol, and organic acids such as
acetic acid are used. As the nitrogen source, ammonium sulfate, ammonium nitrate,
ammonium chloride, ammonium phosphate, ammonium acetate, ammonia, peptone, meat
extract, yeast extract, corn steep liquor and the like are used. When an auxotrophic mutant
strain is used, it is preferable to add the required substance as a sample or a natural productcontaining the same.
B. Working Example 6 Introduction of a mutation into the promoter region of thearginosuccinate synthase gene of a coryneform arginine-producing bacterium[0077]... 1) Determination of the nucleotide sequence of the upstream region of the argG gene49In order to amplify the argG gene of Brevibacterium flavum by PCR, the nucleotide
sequences of the upstream and downstream regions of the ORF were determined. ...
[0078]
2) Prediction of the promoter site
From the above sequences, a promoter-like sequence upstream of the ORF of the argG
gene was searched using commercially available software (GENETYX). A mutation was
introduced at the site with the highest score (about 120 bp upstream from the first ATG),
and then the activity of the promoter was evaluated.
3) Introduction of a mutation into the promoter sequence and measurement of the activityof the mutant promoter
By performing the first PCR in the site with the highest score, using the chromosomal
DNA of the AJ12092 strain as a template and using the primer for mutagenesis..., andthen performing a PCR again using the same chromosomal DNA as a template, with the
product of the first PCR as the 3' primer, and with ... as the 5' primer, DNA fragments inwhich a mutation was introduced into the target promoter portion were obtained. Next, inorder to measure the activity of this mutant promoter, these DNA fragments were inserted
into the SmaI site of the promoter probe vector pNEOL so as to be in the forward direction
toward the reporter gene lacZ, to obtain plasmids pNEOL-1, pNEOL-2, pNEOL-3,
pNEOL-4 and pNEOL-7. As a control of the activity, plasmid pNEOL-0 was constructedby similarly inserting a DNA fragment, obtained by performing PCR, using chromosomal
DNA of the AJ12092 strain as a template and using ..., upstream of the lacZ gene ofpNEOL.
[0079]
pNEOL-0, pNEOL-1, pNEOL-2, pNEOL-3, pNEOL-4 and pNEOL-7 wereintroduced into the AJ12092 strain. The plasmid was introduced by the electric pulse
method (Unexamined Patent Application Publication No. 1990-207791). ...
These strains were ... β-galactosidase activity was measured.
As shown in Table 20, β-galactosidase activity was detected in AJ12092/pNEOL-0,
indicating that the DNA fragment inserted upstream of the lacZ structural gene functions
as a promoter. In addition, β-galactosidase activity was higher in each plasmid-introduced
strain as compared to AJ12092/pNEOL-0, and it was found that transcriptional activity
was enhanced as shown in Table 20 by introducing a mutation into this promoter-likesequence.
[0080][Table 20]
Relative activity (AJ12092/pNEOL-0=1)
50

AJ12092

nd

AJ12092/pNEOL-0

1.0

AJ12092/pNEOL-1

2.8

AJ12092/pNEOL-2

2.7

AJ12092/pNEOL-3

1.8

AJ12092/pNEOL-4

0.8

AJ12092/pNEOL-7

3.0[0081]
4) Construction of plasmids for mutagenesis
A DNA fragment obtained by performing PCR using the chromosomal DNA of the
AJ12092 strain as a template and using primers 14 and 15 (SEQ ID NOs: 55 and 56) was
inserted into the SmalI site of a multicloning site of cloning vector pHSG398 (produced
by Takara Shuzo Co., Ltd.) to construct plasmid p0. Then, p0 was digested with restriction
enzymes EcoRV and BspHI, and a DNA fragment obtained by similarly digestingpNEOL-3 and pNEOL-7 with restriction enzymes EcoRV and BspHI was ligated toobtain plasmids p3 ... and p7 ... for mutagenesis.
5) Introduction of plasmids for mutagenesis into an Arg-producing bacterium
The abovementioned plasmids were introduced into the Brevibacterium
lactofermentum AJ12092 strain, which is an Arg-producing bacterium. The plasmid wasintroduced by the electric pulse method (Unexamined Patent Application Publication No.
1990-207791). Since these plasmids cannot be replicated autonomously in
Brevibacterium, only strains in which these plasmids have been integrated into thechromosome by homologous recombination can be selected as Cm-resistant strains. The
strain in which the plasmids for mutagenesis have been integrated into the chromosome
was selected as a chloramphenicol resistant strain in a CM2G plate medium containing 5
μg/ml of chloramphenicol (10 g of polypeptone, 10 g of yeast extract, 5 g of glucose, 5 gof NaCl, and 15 g of agar in 1 liter of pure water; pH 7.2).
Next, a strain in which the promoter portion of the argG gene was substituted with thetarget mutant sequence was selected from the strains that had undergone homologous
recombination again and became Cm-sensitive.
As a result, a strain in which the portion was substituted with the sequence of P3(AJ12092-P3) and a strain in which the portion was substituted with the sequence of P7(AJ12092-P7) were obtained.
[0082]
6) Cloning of the argG gene
Based on the nucleotide sequence determined as in 1), oligonucleotides (primers 551and 6) having the nucleotide sequences shown in SEQ ID NOs: 46 and 47 weresynthesized, and PCR was performed using the chromosomal DNA of Brevibacterium
flavum 2247 as a template. ... The obtained DNA fragment was cloned into the SmaI site
in a multiple cloning site of cloning vector pSTV29 (produced by Takara Shuzo Co., Ltd.)to prepare pSTVarG. Further, pargG was prepared in which a fragment containing a
replication origin obtained by treating pSAK4 described in Working Example 1(1) withSalI was inserted into the SalI site of pSTVargG.
7) Introduction of pargG into Brev.
pargG was introduced into the Brevibacterium lactofermentum AJ12092 strain. The
plasmid was introduced by the electric pulse method (Unexamined Patent Application
Publication No. 1990-207791). ...
[0083]
8) ArgG activity of promoter mutant strains
The ArgG activity of the abovementioned two types of ArgG promoter mutant strains
and the strain in which argG was amplified by plasmid (AJ12092/pargG) was measured. ...
Table 21 shows the ArgG activity of the abovementioned two types of ArgG promoter
mutant strains and the strain in which argG was amplified by plasmid (AJ12092/pargG).
As shown in Table 21, by introducing a mutation into the promoter, ArgG activity wasincreased to about twice that of the parent strain in AJ12092-P3 and about three times in
AJ12092-P7. In addition, ArgG activity of AJ12092/pargG was about 4.5 times that ofthe parent strain.
[0084][Table 21]
Relative activity (AJ12092=1)
AJ12092

1.0

AJ12092-P3

2.1

AJ12092-P7

2.9

AJ12092/pargG

4.4[0085]
9) Arg production by promoter mutant strains
ArgG promoter mutant strains were cultured in flasks. As controls, parent strains
AJ12092 and AJ12092/pargG were similarly cultured. ... As shown in Table 22, the Arg
yield was enhanced in the argG promoter mutant strains, and the yield was equivalent tothat of the strain in which argG was amplified by plasmid. In addition, while the culture
time was delayed for the plasmid-amplified strain, the culture time of both promoter
mutant strains AJ12092-P3 and AJ12092-P7 was equivalent to that of the parent strains,52

indicating that their Arg productivity was enhanced as compared to the plasmid-amplified
strain.
[0086][Table 22]
OD

Arg(g/dl)

Culture(h)time Productivity(g/dl/h)

AJ12092

0.502

1.25

48

0.026

AJ12092-P3

0.510

1.47

48

0.031

AJ12092-P7

0.514

1.43

48

0.030

AJ12092/pargG 0.520

1.47

52

0.028

(2) Regarding violation of the enablement requirement and the support
requirement by the process for producing arginine
A. Regarding Invention 1-1(A) The claim of Invention 1-1 is as described in [Claim 1] of Attachment 4-1 "Claims(Patent 1)," and Invention 1-1 is an invention of a process for producing glutamic acid orarginine by using a coryneform bacterium into which a specific nucleotide sequence has
been introduced in the -35 region and/or the -10 region of the promoter sequence of anyof the following: GDH gene, CS gene, ICDH gene, PDH gene, or argininosuccinate
synthase gene.
(B) Description 1 states that, regarding the problem to be solved, Invention 1 provides aprocess for constructing a mutant strain capable of appropriately enhancing andregulating the expression level of an objective gene and having the ability to produce
amino acids in high yield by genetic recombination or mutation, without using a plasmid(paragraphs [0001] and [0006]).
However, statements in Description 1 concerning the process for producing thearginine mentioned in Invention 1-1, from among amino acids, are as described in (1)above. Description 1 has statements on the enzymes relating to fermentation of arginine
and the types of genes encoding them and the fact that argininosuccinate synthase is oneof the enzymes related to fermentation of arginine (paragraphs [0012] and [0019]), andalso describes production of arginine that uses a coryneform arginine-producing
bacterium in which a mutation has been introduced into the promoter region of anargininosuccinate synthase gene as Working Example 6 (paragraphs [0077] through[0086]). However, the GDH gene, CS gene, ICDH gene, and PDH gene are all describedas genes that encode enzymes related to fermentation of glutamic acid (paragraph [0012]),and they are not described to encode enzymes related to fermentation of arginine(paragraph [0012]). Furthermore, there are no statements indicating that these genes are53

related to production of arginine and no working examples of production of arginine using
a bacterium in which a mutation has been introduced into the promoter region of thesegenes.
(C) Accordingly, among the inventions claimed in Invention 1-1, at least the process for
producing arginine by using a coryneform bacterium in which a mutation has been
introduced into the GDH gene, CS gene, ICDH gene, or PDH gene can be considered tofall beyond the scope in which a person skilled in the art can recognize that theabovementioned problem, that is, to construct a mutant strain having the ability to produce
amino acids in high yield by genetic recombination or mutation, can be solved based on
the detailed descriptions of inventions in Description 1. In addition, it cannot be foundthat there was common general technical knowledge based on which a person skilled in
the art could solve the abovementioned problem, apart from the statements in Description
1, with regard to the process for producing arginine by using these genetic mutations.
Consequently, it should be said that the patent relating to Invention 1-1 was granted
in violation of the requirement prescribed in Article 36, paragraph (6), item (i) of the
Patent Act (the support requirement), and should be invalidated by a trial for patent
invalidation under Article 123, paragraph (1), item (iv) of the Patent Act.
B. Regarding Inventions 1-2, 1-3, and 1-4
As described in [Claim 2] through [Claim 4] of Attachment 4-1 "Claims (Patent 1),"Inventions 1-2 and 1-3 are both inventions of a process for producing glutamic acid orarginine by using a coryneform bacterium in which a specific nucleotide sequence has
been introduced in the -35 region and/or the -10 region of the promoter sequence of the
GDH gene. Invention 1-4 is an invention of a process for producing glutamic acid orarginine by using a coryneform bacterium in which a specific nucleotide sequence has
been introduced in the -35 region and/or the -10 region of the promoter sequence of the
CS gene.
Accordingly, due to the same reason as for Invention 1-1, among these inventions, atleast the process for producing arginine can be considered to have fallen beyond the scopein which a person skilled in the art could recognize that the problem, that is, to construct
a mutant strain having the ability to produce amino acids in high yield by genetic
recombination or mutation, can be solved based on Description 1 or the common generaltechnical knowledge at the time.
Consequently, it should be said that the patents relating to Inventions 1-2, 1-3, and 14 were all granted in violation of the requirement prescribed in Article 36, paragraph (6),item (i) of the Patent Act (the support requirement) and should be invalidated by a trial
for patent invalidation under Article 123, paragraph (1), item (iv) of the Patent Act.
54

(3) Summary on the grounds for invalidation regarding Invention 1
According to the above, the patents relating to Invention 1 should all be invalidated
in a trial for patent invalidation without having to determine whether there are any othergrounds for invalidation, so the defense of Article 104-3, paragraph (1) of the Patent Act
is established.
Since the Plaintiff has deleted Claim 3 by Correction 1, and has not asserted redefense of correction for Invention 1-3, the Plaintiff cannot exercise its rights against the
Defendant based on Invention 1-3.
On the other hand, the Plaintiff has asserted re-defense of correction for Inventions 11, 1-2, and 1-4, so with regard to the other grounds for invalidation alleged with regard
to Invention 1-1, 1-2, and 1-4, their grounds for invalidation will be determined based on
Corrected Invention1 after the correction, along with whether the abovementioned groundfor invalidation, which is violation of the support requirement, has been eliminated, when
determining whether re-defense of correction can be established in 6. below.
6. Regarding Issue 5 (Whether re-defense of correction of Patent 1 can be established)(1) Contents of Corrected Invention 1
A. Contents described in Description 1
The statements in the detailed explanation of the invention in Description 1
concerning Corrected Invention 1 are outlined as described in Attachment 12 "Statements
in Description 1."
B. Outline of Corrected Invention 1
According to the claims of Corrected Invention 1 (Attachment 4-2), the statements in
Description 1 referred to in A. above, and the entire import of oral arguments, the outlineof Corrected Invention 1 is found to be as follows.
(A) Technical field/background art
When using genetic recombination as a process for constructing a mutant strain to be
used for producing amino acid by the fermentation method, a plasmid, which can be
replicated autonomously and independently from the chromosome in a cell, has mainly
been used for enhancing the objective gene. However, as the level of enhancement of theobjective gene is decided by the copy number of the plasmid itself, there are often cases
in which the expression level becomes too high due to an excessively high copy number,leading to extremely restrained growth or a decline in the target substance-producing
ability, depending on the type of the objective gene. In addition, as replication of a plasmid
is often unstable, there is a problem that plasmid loss sometimes occurs (paragraphs[0002] and [0003]).
(B) Problem to be solved by Corrected Invention 1
55

The purpose of Corrected Invention 1 is to provide a process for constructing a mutant
strain capable of appropriately enhancing and regulating the expression level of theobjective gene and that has the ability to produce amino acids in high yield by genetic
recombination or mutation, without using a plasmid, and to provide a glutamic acidfermentation method for increasing the yield of glutamic acid and producing glutamic
acid at a lower cost, using a coryneform glutamic acid-producing bacterium (paragraph[0006]).
(C) Means for solving the problem, etc.
In order to solve the abovementioned problem, Corrected Invention 1 adopted a
configuration in which a mutant of a coryneform bacterium is prepared by causing or
introducing by genetic recombination a mutation to the promoter sequence of an aminoacid biosynthetic gene on the chromosome of the coryneform bacterium to make thesequence closer to the consensus sequence, then that mutant is cultured, and as a result,only a mutant that produces a large amount of the target amino acid is collected (paragraph[0007]).
As for examples of enzymes involved in the biosynthesis of amino acid, enzymes suchas glutamate dehydrogenase (GDH) and citrate synthetase (CS) are effective in the caseof glutamic acid fermentation (paragraph [0012]). Meanwhile, the abovementioned
mutation to the promoter sequence may be caused either only for the promoter sequenceof one gene or for the promoter sequences of two or more genes (paragraph [0015]).
In Corrected Invention 1, it is preferable that the DNA sequence in the -35 region of
the promoter of the GDH gene is at least one kind of DNA sequence selected from a groupconsisting of TTGTCA, etc. and/or that the DNA sequence in the -10 region of that
promoter is TATAAT, etc. (paragraph [0017]).
In addition, in Corrected Invention 1, examples of the promoter of the CS gene include
one that has the TTGACA sequence in the -35 region and/or the TATAAT sequence in the-10 region (paragraph [0018]).
Corrected Invention 1 has a significant industrial advantage in that the target aminoacid can be obtained in high yield by introducing a mutation into the promoter region ofan amino acid biosynthetic gene of a coryneform amino acid-producing bacterium andregulating the expression level of the objective gene, and that the target amino acid canbe obtained in high yield in a stable manner without loss as in the case of a plasmid(paragraph [0020]). In addition, Corrected Invention 1 can give a Corynebacterium strain
the ability to produce amino acid, particularly glutamic acid, in high yield without causing
an increase in byproduct aspartic acid and alanine (paragraph [0021]).
(D) Outline of the disclosed working examples
56

a. Working Example 1"ATCC13869/P6-8" is a wild-type strain of a coryneform bacterium (an ATCC13869
strain of Brevibacterium lactofermentum) into which a plasmid of a GDH gene, wherein
the -35 region of the promoter sequence (the TGGTCA sequence in the case of an
ATCC13869 strain) has been modified to the TTGTCA sequence and the -10 region of
the promoter sequence (the CATAAT sequence in the case of an ATCC13869 strain) has
been modified toa TATAAT sequence, has been introduced.
It is indicated that the specific activity of GDH (401.3) of "ATCC13869/p6-8" is about
4.9 times that of "ATCC13869/pGDH," which is a strain into which a plasmid of a GDHgene having the same promoter sequence as that of the parent strain (the ATCC13869
strain) (paragraphs [0022] through [0025], [Table 1]).
b. Working Example 2"FGR2" is Brevibacterium lactofermentum (an AJ13029 strain) to which mutations
have been added, wherein the -35 region of the promoter sequence (the TGGTCA
sequence) of a GDH gene of the AJ13029 strain has been mutated to a TTGTCA sequence,and the -10 region of the promoter sequence (CATAAT sequence) has been mutated to a
TATAAT sequence.
It is indicated that the specific activity of GDH of FGR2 is about 3.4 times that of an
AJ13029 strain, and that its L-glutamic acid production (3.0 g/dl) increased compared to
the AJ13029 strain (2.6 g/dl) (paragraphs [0026], [0029], [0031], and [0032], [Table 5],[Table 6]).
c. Working Example 3"GB02" is a strain into which a mutation whereby the CS gene on the chromosome of
FGR2 referred to in b. above is substituted with a CS gene in which the -10 region of thepromoter sequence has been modified to a TATAAT sequence (the sequence before themutation is TATAGC) has been introduced."GB03" is a strain into which a mutation
whereby the CS gene on the chromosome of FGR2 is substituted with a CS gene in which
the -10 region of the promoter has been modified to a TATAAT sequence and the -35
region of the promoter has been modified to a TTGACA sequence (the sequence before
the mutation is ATGGCT) has been introduced. The -35 region and the -10 region of thepromoter sequence of the GDH gene of GB02 and GB03 are the same as those of FGR2.
It is indicated [i] that the specific activity of citrate synthetase of GB02 is about 1.9
times that of FGR2 and the specific activity of citrate synthetase of GB03 is about 4.0
times that of FGR2, and [ii] that the L-glutamic acid production of GB02 and GB03 (9.4
g/l respectively) increased compared to FGR2 (8.9 g/l).
(Regarding the above, paragraphs [0033] through [0036], paragraphs [0041] through57

[0046], paragraph [0048], [Table 7], [Table 10], [Table 12]).
d. Working Example 7"GA02" is a strain wherein a mutation has been introduced on the chromosome of the
AJ13029 strain referred to in b. above by using the promoter sequence of the GDH geneof FGR2 referred to in b. above, and the -35 region and the -10 region of the promoter
sequence of the GDH gene introduced are the same as those of FGR2.
It is indicated that the specific activity of GDH of GA02 is about 3.5 times that of theparent strain (AJ13029), and that the L-glutamic acid production of GA02 (2.9 g/dl)
increased compared to AJ13029 (2.6 g/dl) (paragraphs [0087] through [0089], [Table 23]).
(2) Whether or not Defendant's Production Processes 1 and 3 fall within the technicalscope of Corrected Invention 1
According to the entire import of oral arguments, it is found that, when Defendant's
Production Processes 1 and 3 are compared with Corrected Invention 1, the results of thecomparison will be as described in Attachment 6-2 "Comparison between Defendant's
Production Processes 1 and 3 and Corrected Invention 1," and therefore Defendant's
Production Process 1 literally falls within the technical scopes of Corrected Invention 11 (corresponding to Invention 1-1), Corrected Invention 1-2 (corresponding to Invention1-2), and Corrected Invention 1-3 (corresponding to Invention 1-4), whereas Defendant's
Production Process 3 literally falls within the technical scope of Corrected Invention 1-1
but does not fall within the technical scope of Corrected Invention 1-3.
(3) Regarding Exhibit Otsu 6 Invention
A. Contents described in Exhibit Otsu 6 Document
The contents described in Exhibit Otsu 6 Document include the following (Exhibit
Otsu 6, the entire import of oral arguments; for Figures 1 and 5 and Table 2, see
Attachment 14 "Drawings of Cited Documents").
(A) Abstract (Lines 1 through 18 on page 1297)"Relatively limited information about promoter structures in Corynebacterium
glutamicum has been available until now. ... A comparative analysis of the newly
characterized promoter sequences together with published promoters from C. glutamicum
revealed conserved sequences centred about 35 bp (ttGcca) and 10 bp (TA.aaT) upstreamof the TS [transcriptional start] site. The position of these motifs and the motifs
themselves are comparable to the -35 and -10 promoter consensus sequences of otherGram-positive and Gram-negative bacteria, indicating that they represent transcription
initiation signals in C. glutamicum."(B) Figure 1 (page 1301)"Fig. 1. Nucleotide sequences of C. glutamicum promoters aligned according to their58

TS sites. Putative -35 and -10 region identified by the promscan program are underlined.
The promoter sequences of hom, thrC, fda, lysA, ask, gdh, glt, gap, pgk and trp were takenfrom references cited in the text."(C) Discussion
a. Line 11 in the right column on page 1305 through line 2 in the left column on page
1306
"All 18 fragments tested also drove transcription of cat in E. coli, indicating that theisolated promoters were active in this organism too. This result was not unexpected since
many of the genes cloned from C. glutamicum were expressed in E. coli as indicated byheterologous complementation of appropriate auxotrophs ... Conversely, several E. coli
genes were efficiently expressed in C. glutamicum ... and the E. colitac, lacUV5, and trp
promoters have been shown to be functional in corynebacteria ... All these results indicatethat the general structure of promoters from C. glutamicum is similar to that of promotersfrom E. coli. However, there were also reports on corynebacteria-specific promoterswhich were obviously not functional in E. coli ..."
b. Lines 3 through 38 in the left column on page 1306
"The similarity of the primary structure of promoters from C. glutamicum with thatof promoters from other bacteria was substantiated by comparative computer analysis.
Both programs applied in this study showed that in our set of C. glutamicum promotersthe most correlated region is located around 10 bp upstream of the TS site and comprises
the hexamer TA.AAT. This or a similar motif can be found in all promoters. Anotherconserved region was detected about 35 bp upstream of the TS site. The consensus
hexamer TTGCCA found in this region differs from the E. coli consensus TTGACA onlyin the fourth position. The role of the TTGCCA motif is not that obvious since it is clearlyseen (> 3 bp matching) in only 14 promoters out of the 33. In several others the motif was
not easily discernible, indicating a lower conservation in C. glutamicum promoters.
However, from the position relative to the TS site, from the spacing (a mean of 17‘35bp) and from the sequence of the two conserved hexamer motifs, it is obvious that thesesignals in C. glutamicum promoters are comparable to the -10 and -35 promoter consensus
sequences of E. coli ... and other eubacteria, e.g. Bacillus ..., Eactobacillus ... andStreptococcus ... The significance of the C. glutamicum consensus motifs found by our
analyses is corroborated by the fact that the tac promoter (11 out of 12 positions identicalto consensus) was found to be very efficient in C. glutamicum ... and that an alteration of
the -10 hexamer in the E. coli lac promoter from TATGTT to TATATT, thus increasing
the similarity to the consensus sequence, led to higher efficiency of the promoter in C.
glutamicum ..."
59

c. Lines 18 through 42 in the right column on page 1306
"In some Gram-positive bacteria, e.g. Bacillus and Streptococcus, most of the -10 and-35 promoter regions match their consensus sequences TATAAT and TTGACA,
respectively, nearly perfectly ... In contrast, this does not hold for the set of C. glutamicumpromoters studied here. Several of the bases in the C. glutamicum promoter consensus
motifs are only moderately conserved (see Fig. 5), indicating only relatively moderateconservation of the motif as a whole. Since the conservation level of promoter consensus
sequences probably reflects the requirements of the major RNA polymerase σ-factor in agiven organism ..., it was interesting to compare the degree of consensus conservation per
position in our study with that in compilations of promoters from other bacteria. As shownin Table 2, nearly all bases in the -35 and -10 consensus hexamers in the Bacillus,
Eactobacillus and Streptococcus promoters are much more and those in the E. coli
promoters are considerably more conserved than in the set of C. glutamicum promoters.
These data suggest that the recognition specificity of the major RNA polymerase in theorganisms listed decreases in the order Bacillus/Lactobacillus/Streptococcus > E. coli >C. glutamicum."d. Table 2 (page 1307)"Table 2. Degree of consensus conservation in the -35 and -10 motifs in compilationsof promoters from different organisms
Promoter compilations for the different organisms were taken from the following
references: C. glutamicum, 33 promoters (this work); E. coli, 263 promoters ...; Bacillus
subtilis, 237 promoters ...; Eactobacillus, 30 promoters ...; Streptococcus, 17
promoters ..."
e. Line 2 from the bottom in the right column on page 1306 through line 6 in the left
column on page 1307
"Since the activity of promoters in E. coli can be correlated to a major extent with
their similarity to the -35 and -10 consensus hexamers ..., we assumed that the observed
CAT activity roughly correlates with the identity score of a given promoter with thepredicted consensus promoter sequence. However, such a correlation could not berecognized."
f. Lines 4 through 10 in the right column on page 1307
"It becomes clear from the data presented here that further work, e.g. stringent
mutational and biochemical analysis of selected promoters, is necessary to gain detailed
information on the structure-function relationship of promoters (or promoter classes) inC. glutamicum and thus a better understanding of gene expression in this industrially
important organism."60

B. Outline of Exhibit Otsu 6 Invention
As mentioned in A(B) above, Figure 1 of Exhibit Otsu 6 Document shows nucleic
acid sequences of the promoters of Corynebacterium glutamicum genes, including the
GDH gene and the CS gene. The figure indicates nucleic acid sequences of the promotersof Corynebacterium glutamicum genes which have a "TGGTCA" sequence in the -35
region and a "CATAAT" sequence in the -10 region of the promoter sequence of the GDHgene ("p-gdh"), and a "TGGCTA" sequence in the -35 region and a "TAGCGT" sequencein the -10 region of the promoter sequence of the CS gene ("p-glt").
Consequently, Exhibit Otsu 6 Document is found to disclose Exhibit Otsu 6 Invention
having the following configuration as a configuration to be compared with Corrected
Invention 1.
"A coryneform bacterium which has a TGGTCA sequence in the -35 region and a
CATAAT sequence in the -10 region of the promoter sequence of the GDH gene, and a
TGGCTA sequence in the -35 region and a TAGCGT DNA sequence in the -10 region of
the promoter sequence of the CS gene on the chromosome of the coryneform bacterium"
C. Other matters disclosed in Exhibit Otsu 6 Document
According to the matters described in Exhibit Otsu 6 Document mentioned in A.
above, Exhibit Otsu 6 Document is found to disclose the following, in addition to Exhibit
Otsu 6 Invention referred to in B. above.
(A) As a result of analyzing promoters of Corynebacterium glutamicum genes, conserved
sequences are ttGcca.a in the -35 region and ggTA.aaT in the -10 region, and the positionof these motifs and the motifs themselves correspond to consensus sequences in the -35
region and the -10 region of promoters of other Gram-positive and Gram-negative
bacteria (A. (A) above, Figure 5).
(B) Between Corynebacterium glutamicum and E. coli, there are examples where thepromoter sequence of either is functional in the other, suggesting similarity in the general
structure, etc. of promoter regions of Corynebacterium glutamicum genes and promoter
regions of E. coli, but there were also multiple reports on corynebacteria-specificpromoters which were obviously not functional in E. coli (A. (C) a., b. above).
(C) Several of the bases in the consensus motifs of Corynebacterium glutamicum are only
moderately conserved, and the conservation levels of most bases were lower than thoseof Gram-positive bacteria, such as Bacillus and Streptococcus, and E. coli (A. (C) c., d.
above, Figure 5, Table 2).
(D) Whereas the activity of promoters in E. coli can be correlated to a major extent with
their similarity to the consensus sequences in the -35 region and the -10 region, theactivity of promoters in Corynebacterium glutamicum was not recognized to correlate61with their similarity to the predicted consensus sequence (A. (C) e. above).
(4) Regarding Exhibit Otsu 9 Invention
A. Contents described in Exhibit Otsu 9 Document
The contents described in Exhibit Otsu 9 Document include the following (Exhibit
Otsu 9; for Table 9, see Attachment 14 "Drawings of Cited Documents")(A) Claims (Lines 2 through 8 on page 55)"1. a coryneform L-glutamic acid-producing bacterium deficient in α-ketoglutarate
dehydrogenase activity due to occurrence of substitution, deletion, insertion, addition, orinversion of one or more nucleotides in a nucleotide sequence of a gene coding for an
enzyme having α-KGDH activity or a promoter thereof existing on chromosome;
2. a method of producing L-glutamic acid comprising the steps of cultivating thecoryneform L-glutamic acid-producing bacterium described in Claim 1 in a liquidmedium, to allow L-glutamic acid to be produced and accumulated in a culture liquid,and collecting it"(B) Description
a. Technical field (Lines 4 through 11 on page 1)"The present invention relates to breeding and utilization of coryneform bacteria used
for fermentative production of L-glutamic acid... In particular, the present invention
relates to coryneform L-glutamic acid-producing bacteria deficient in α-ketoglutarate
dehydrogenase (α-KGDH), a method of producing L-glutamic acid by using thebacteria..."b. Background art (Line 13 on page 1 through line 5 on page 2)"L-Glutamic acid has been hitherto industrially produced by a fermentative method
using coryneform bacteria belonging to the genus Brevibacterium or Corynebacterium.
Recently, it has been revealed that a mutant strain of Escherichia coli, in which the αKGDH activity is deficient or lowered, and the glutamic acid-decomposing activity islowered, has high L-glutamic acid productivity...
On the contrary, it was reported that a mutant strain having lowered α-KGDH activity
had approximately the same L-glutamic acid productivity as that of its parent strain in thecase of a bacterium belonging to the genus Brevibacterium... Therefore, it has been
believed that the level of α-KGDH activity is not important for production of L-glutamic
acid in coryneform bacteria.
On the other hand, it was found that a mutant strain of a L-glutamic acid-producing
bacterium belonging to the genus Brevibacterium having lowered α-KGDH activity
produces L-glutamic acid at high efficiency ... when the bacterium is cultivated in a
medium which contains a material containing an excessive amount of biotin as a carbon
62source without addition of materials which suppress an effect of biotin such as penicillins
and surface-active agents... However, since it has been believed that the level of α-KGDH
activity is not important for production of L-glutamic acid in the coryneform bacteria as
described above, there has been no example in which an α-KGDH gene of a coryneform
L-glutamic acid-producing bacterium is cloned and analyzed. Further, mutant strains ofcoryneform bacteria being completely deficient in α-KGDH have been unknown."c. Disclosure of the invention(a) "An object of the present invention is to obtain an α-KGDH gene originating fromcoryneform L-glutamic acid-producing bacteria, prepare recombinant DNA containing
the gene, clarify the influence of the level of α-KGDH activity on fermentative productionof L-glutamic acid by using microorganisms transformed with the recombinant DNA, andthus provide a new methodology in breeding of coryneform L-glutamic acid-producing
bacteria. More specifically, an object of the present invention is to obtain a coryneform
L-glutamic acid-producing bacterium deficient in α-KGDH activity by destroying an αKGDH gene existing on chromosomal DNA, and provide a method of producing Lglutamic acid by using the bacterium. ...
The present inventors have obtained an α-KGDH gene originating from a coryneform
L-glutamic acid-producing bacterium, clarified its structure, transformed a coryneform
L-glutamic acid-producing bacterium by using a plasmid into which the gene isincorporated, and investigated the level of α-KGDH activity and L-glutamic acidproductivity of obtained transformants. As a result, it has been found that the α-KGDH
activity remarkably affects production of L-glutamic acid. Further, the present inventors
have found that a strain, in which the α-KGDH activity is deleted by destroying an αKGDH gene existing on chromosome of a coryneform L-glutamic acid-producing
bacterium, produces and accumulates a considerable amount of L-glutamic acid when it
is cultivated in a medium containing an excessive amount of biotin without adding any
substance for suppressing the action of biotin such as surfactant and penicillin." (Lines 7
through 27 on page 2)(b) "Namely, the present invention provides:(1) a coryneform L-glutamic acid-producing bacterium deficient in α-KGDH activity
due to occurrence of substitution, deletion, insertion, addition, or inversion of one or more
nucleotides in a nucleotide sequence of a gene coding for an enzyme having α-KGDH
activity or a promoter thereof existing on chromosome;(2) a method of producing L-glutamic acid comprising the steps of cultivating thecoryneform L-glutamic acid-producing bacterium described in the aforementioned item
(1) in a liquid medium, to allow L-glutamic acid to be produced and accumulated in a63culture liquid, and collecting it; ..." (Lines 3 through 18 on page 3)(c) "Utilization of the gene of the present invention includes preparation of α-KGDH
activity-deficient strains by insertion of a drug-relevant gene or the like, preparation ofstrains with weak activity in vitro mutation, preparation of expression lowered strains bymodification of a promoter and so on, which makes it possible to efficiently breed abacterial strain in which the L-glutamic acid productivity is further improved as comparedwith conventional coryneform L-glutamic acid-producing bacteria.
A strain deficient in α-KGDH activity can be obtained either by a method which uses
a chemical reagent to induce mutation, or by a method which resides in genetic
recombination. However, in the case of the method for introducing mutation by using achemical reagent, it is relatively easy to obtain a strain in which the α-KGDH activity islowered, but it is difficult to obtain a strain in which the activity is completely deficient.
In order to obtain the latter strain, it is advantageous to use a method in which an α-KGDH
gene existing on chromosome is modified or destroyed by means of a genetic homologous
recombination method on the basis of the structure of the α-KGDH gene having beenclarified as described above.
...
Specifically, substitution, deletion, insertion, addition or inversion of one or aplurality of nucleotides is caused in a nucleotide sequence in a coding region or apromoter region of the α-KGDH gene by means of a site-directed mutagenesis method ...
or a treatment with a chemical reagent such as sodium hyposulfite and hydroxylamine ...
The gene thus modified or destroyed is used to substitute a normal gene on chromosome.
It is thereby possible to delete the activity of α-KGDH as a gene product, or extinguishtranscription of the α-KGDH gene." (Line 20 on page 8 through line 10 on page 9)"The method for substituting a normal gene on chromosome of a coryneform Lglutamic acid-producing bacterium with the gene thus obtained by introduction of
mutation to give modification or destruction includes a method which utilizes
homologous recombination ... Selection of such bacterial strains makes it possible toobtain a bacterial strain in which a normal gene on the chromosome is substituted with agene into which nucleotide substitution, deletion, insertion, addition or inversion is
introduced to provide modification or destruction." (Line 29 on page 9 through line 13 on
page 10)"A coryneform L-glutamic acid-producing bacterium deficient in α-KGDH activity
thus obtained is remarkably more excellent in L-glutamic acid productivity especially ina medium containing an excessive amount of biotin than strains having partially lowered
α-KGDH activity." (Lines 14 through 16 on page 10)64(d) "In order to improve the L-glutamic acid productivity, it is advantageous to enhanceglutamic acid biosynthetic genes. Examples of enhancement of the glutamic acidbiosynthesis system genes include phosphofructokinase in the glycolytic pathway(PFK ...), phosphoenolpyruvate carboxylase in the anaplerotic pathway (PEPC ...), citratesynthase in the TCA cycle (CS ...), aconitate hydratase (ACO ...), isocitrate
dehydrogenase (ICDH ...), glutamate dehydrogenase for amination reaction (GDH ...),and so on." (Lines 11 through 20 on page 11)"In order to obtain the genes described above, the following methods may be available.
(1) As a mutant strain in which mutation arises in an objective gene and acharacteristic character is presented, a mutant strain is obtained wherein the character
disappears by introducing the objective gene. A gene which complements the characterof the mutant strain is obtained from chromosome of a coryneform bacterium.
(2) When an objective gene has been already obtained from another organism, and its
nucleotide sequence has been clarified, the objective gene is obtained by a technique of
hybridization using DNA in a region having high homology as a probe.
(3) When a nucleotide sequence of an objective gene is fairly clarified in detail, a genefragment containing the objective gene is obtained by means of a PCR method ... using
chromosome of a coryneform bacterium as a template.
...
When the gene is obtained in accordance with the methods of (2) and (3) describedabove, if an objective gene has no original promoter, the objective gene can be expressedby inserting a DNA fragment having promoter activity in coryneform bacteria into a
position upstream from the objective gene. In order to enhance expression of the objective
gene, it may be available to ligate the objective gene at a position downstream from astrong promoter. Strong promoters, which function in cells of coryneform bacteria,
include lac promoter, tac promoter, trp promoter, etc. from Escherichia coli ... In addition,
trp promoter from a bacterium belonging to the genus Corynebacterium is also a
preferable promoter ... In Examples of the present invention, trp promoter from acoryneform bacterium has been used for expression of the PEPC gene." (Line 21 on page
11 through line 16 on page 12)
d. Description of preferred embodiments(a) "Example 3: Preparation of α-KGDH Gene-Deficient Strain
According to the fact that the production of L-glutamic acid was suppressed byamplification of the α-KGDH gene, it was expected, on the contrary, that the yield ofglutamic acid could be improved by destroying the α-KGDH gene. A gene-destroyed
strain was obtained by a homologous recombination method using a temperature65

sensitive plasmid described in Japanese Patent Laid-open No. 1993-7491." (Lines 18
through 22 on page 23)"A nucleotide sequence of the α-KGDH gene on chromosome was investigated byusing the sensitive strain, and it was confirmed that the α-KGDH gene was substituted
into the deficient type. The strain was designated as ΔS strain." (Lines 21 through 23 on
page 24)(b) "Example 4: Preparation of Plasmids for Amplifying gdh, gltA and icd Genes" (Line
26 on page 24)"Next, a promoter of tryptophan operon known as a promoter to function in
Brevibacterium lactofermentum (...) was inserted at a position upstream from the ppc geneon pHSG-ppc'. ... Thus a plasmid pHSG-ppc, in which one copy of the promoter of thetryptophan operon was inserted at a position upstream from the ppc gene, was obtained."(Lines 2 through 15 on page 26)(c) "Example 7: Confirmation of Expression of Each Gene on pGDH, pGLTA, pPPC,
pICD, pGDH+GLTA+ICD, and pGDH+GLTA+PPC
It was confirmed whether or not each of the genes on pGDH, pGLTA, pPPC, pICD,
pGDH+GLTA+ICD, and pGDH+GLTA+PPC was expressed in cells of Brevibacterium
lactofermentum, and these plasmids functioned for gene amplification. Specifically, eachof the plasmids was introduced into Brevibacterium lactofermentum ATCC13869 bymeans of an electric pulse method (...)." (Lines 3 through 8 on page 28)(d) "Example 8: L-glutamic Acid Production by ΔS Strain, and ΔS Strains with Amplified
gdh, gltA, ppc and icd Genes"(Lines 13 through 14 on page 30)"(1) ... It was confirmed that the ΔS strain produced and accumulated L-glutamic acidat a high yield although it was cultivated in the medium containing an excessive amountof biotin." (Lines 10 and 11 on page 31)"(2) ... pGDH, pGLTA, pPPC, pICD, pGDH+GLTA+ICD, or pGDH+GLTA+PPC
prepared as described above was introduced into the ΔS strain to evaluate L-glutamic acidproductivity of transformants in which each of the plasmids was introduced. Introductionof the plasmids into cells of Brevibacterium lactofermentum was performed in accordancewith an electric pulse method (...)." (Lines 12 through 18 on page 31)"Evaluation of L-glutamic acid productivity of the ΔS strain and the obtainedtransformants was performed as described in the aforementioned item (1). ... Results areshown in Table 9." (Lines 22 through 24 on page 31)
e. Industrial applicability"It has been revealed that the level of α-KGDH activity of coryneform L-glutamic
acid-producing bacteria affects fermentative production of L-glutamic acid. Therefore, it
66becomes possible to efficiently breed bacterial strains having further improved Lglutamic acid productivity as compared with conventional coryneform L-glutamic acidproducing bacteria, by preparing α-KGDH gene activity-deficient strains by insertion of
drug-relevant genes and so on, by preparing activity-leaky strains by in vitro mutation,and by preparing strains with lowered expression by modification of promoters and soon." (Lines 13 through 18 on page 33)
B. Outline of Exhibit Otsu 9 Invention(A) According to A. above, Exhibit Otsu 9 Document is found to disclose the following.
a. In a coryneform L-glutamic acid-producing bacterium, the activity of the α-KGDH
gene remarkably affects production of L-glutamic acid, and a coryneform L-glutamic
acid-producing bacterium in which the α-KGDH activity is deleted produces andaccumulates a considerable amount of L-glutamic acid (A. (B) c. (a) above). One methodof deleting the activity of the α-KGDH gene is to modify or destroy the α-KGDH geneby causing occurrence of substitution, deletion, insertion, addition, or inversion of one ormore nucleotides in a nucleotide sequence of a gene coding for an enzyme having αKGDH activity or a promoter thereof existing on the chromosome (A. (B) c. (b), (c)above).
b. Besides deletion of the activity of the α-KGDH gene, there is a method that improves
the glutamic acid productivity by enhancing the glutamic acid biosynthetic genes. As amethod to enhance genes, it may be available to ligate the objective gene for the purposeof enhancing expression of the objective gene. Strong promoters, which function in cellsof coryneform bacteria, include lac promoter, tac promoter, trp promoter, etc. from
Escherichia coli, and trp promoter from a bacterium belonging to the genus
Corynebacterium (A. (B) c. (d) above).
c. A working example in which the activity of the α-KGDH gene on the chromosome ofa coryneform bacterium is deleted, and then glutamic acid biosynthetic genes, such as the
GDH gene and the CS gene (gltA), are enhanced by introducing plasmids (vectors)
containing these genes (A. (B) d. above).
(B) Exhibit Otsu 9 Invention
According to (A) above, Exhibit Otsu 9 Document is found to disclose Exhibit Otsu
9 Invention having the following configuration as a configuration to be compared with
Corrected Invention 1.
"A method of producing L-glutamic acid by the fermentation method comprising thesteps of cultivating, in a medium, a coryneform bacterium, which is deficient in αketoglutarate dehydrogenase activity due to occurrence of substitution, deletion, insertion,
addition, or inversion of one or more nucleotides in a nucleotide sequence of a gene67

coding for an enzyme having α-KGDH activity or a promoter thereof existing on thechromosome, and into which a vector wherein a glutamic acid biosynthetic gene, such as
the GDH gene or the CS gene, is ligated at a position downstream from a strong promoter
has been introduced, to allow L-glutamic acid to be produced and accumulated in themedium, and collecting it from the medium."(C) Regarding the Defendant's allegations
The Defendant alleges that Exhibit Otsu 9 Document discloses an invention of "amethod of producing L-glutamic acid by the fermentation method comprising the stepsof cultivating, in a medium, a coryneform bacterium in which a mutation has been
introduced into the promoter sequence of the glutamate dehydrogenase (GDH) gene or
citrate synthetase (CS) gene on the chromosome of the coryneform bacterium, to allow
L-glutamic acid to be produced and accumulated in the medium, and collecting it fromthe medium."As mentioned in A. (B) c. (c) above, Exhibit Otsu 9 Document describes a method of
introducing a mutation into a gene or the promoter sequence thereof on a chromosome byutilizing homologous recombination, but this description has been described as a meansfor causing defects in the activity of the α-KGDH gene, and not as a means for enhancing
the GDH gene, etc. on a chromosome. In addition, as mentioned in (A) b. above, Exhibit
Otsu 9 Document discloses a method of ligating the objective gene at a position
downstream from a strong promoter as a method to enhance glutamic acid biosynthetic
genes. However, the only specific method for enhancing the GDH gene or the CS genedescribed in Exhibit Otsu 9 Document is the method of introducing vectors (plasmids) ofthese genes referred to in A. (B) d. above, and it does not describe a method of modifying
the promoter sequence of the GDH gene or the CS gene on a chromosome.
In this way, it cannot be said that Exhibit Otsu 9 Document discloses introduction ofa mutation into the promoter sequence of the GDH gene or the CS gene on thechromosome of a coryneform bacterium. Therefore, the Defendant's abovementioned
allegation cannot be adopted, and it is reasonable to find Exhibit Otsu 9 Invention to be
as mentioned in (B) above.
(5) Regarding matters described in publicly known documents other than Exhibit Otsu 6
Document and Exhibit Otsu 9 Document
A. Regarding Exhibit Otsu 8 Document(A) Matters described in Exhibit Otsu 8 Document
The contents described in Exhibit Otsu 8 Document include the following (Exhibit
Otsu 8, Exhibit Ko 25).
a. Introduction
68

"Citrate synthase (EC4.1.3.7) catalyses a crucial step at the entry of the citric acidcycle, i.e. the condensation of acetyl-CoA and oxaloacetate to form citrate and CoA. The
key position of this enzyme within central metabolism has given rise to much interest in
its structural, kinetic, regulatory and molecular characteristics and, therefore, it has beenstudied in great detail from a variety of different organisms (...)." (Lines 1 through 9 in
the left column on page 1818)"The same authors reported that a classically obtained mutant of C. glutamicum ssp.
flavum with reduced citrate synthase activity was able to produce significant amounts ofaspartate and lysine (...). This and the key position of the enzyme in the carbon flow fromcarbohydrates to the citric acid cycle, and thus also to the amino acids derived therefrom,suggest that citrate synthase may be an important target in the genetic construction ofdefined amino-acid-producing C. glutamicum strains.
We describe here the citrate synthase of C. glutamicum with respect to its regulation,
the isolation of the citrate synthase gene (gltA), its nucleotide sequence, the homologous
and heterologous expression and its transcriptional organization." (Lines 27 through 40
in the left column on page 1818)
b. Results and discussion(a) "It is noteworthy that the gltA-overexpressing C. glutamicum strains showed slower
growth on all media tested (e.g. on LB medium a doubling time of 120 min instead of 80min), indicating a slight impairment of the cells by the enhanced level of citrate synthase."(Lines 32 through 36 in the right column on page 1820)(b) "To test the effect of increased citrate synthase activity on glutamate secretion,
standard glutamate fermentations (Hoischen & Krämer, 1989) were performed with C.
glutamicum WT and WT (pJC-gltA3A). In these experiments identical glutamate
secretion rates of about 17 μmol min−1 (g dry weight)−1 were found for both strains. Thus,
the capacity of C. glutamicum to secrete glutamate cannot be enhanced by simply
elevating the citrate synthase enzyme level." (Lines 17 through 26 in the left column on
page 1821)(c) "The nucleotide sequence obtained and the deduced amino acid sequence of the C.
glutamicum citrate synthase are shown in Fig. 2." (Lines 10 through 12 in the right column
on page 1821)"Fig. 2. Nucleotide sequence of the 3007 bp SalI-HindIII fragment and the deduced
citrate synthase amino acid sequence. The transcriptional initiation site (↦), the putative 10 region (—) ... are shown." (Figure 2 on pages 1822 through 1823; a "TATAGC"
sequence is shown in the putative -10 region in Figure 2)(B) According to the matters described in Exhibit Otsu 8 Document mentioned in (A)69above and the entire import of oral arguments, Exhibit Otsu 8 Document discloses thefollowing: citrate synthetase (CS) catalyzes a crucial reaction of the citric acid cycle; theproduction of aspartate and lysine increased in Corynebacterium glutamicum with
reduced CS activity; overexpression of CS slows the growth of Corynebacterium
glutamicum; and in Corynebacterium glutamicum in which CS activity was increased byintroducing plasmids, the glutamic acid productivity was not improved compared to astrain to which no mutation was added. It also discloses that the sequence in the -10 regionof the promoter of the CS gene of wild-type Corynebacterium glutamicum was deduced
as being TATAGC.
B. Regarding Exhibit Otsu 10 Document(A) Matters described in Exhibit Otsu 10 Document
Exhibit Otsu 10 Document has the following description concerning the promoter
sequence of E. coli (Exhibit Otsu 10).
"Six mutants with a 21-fold increase in promoter strength compared with the wildtype were mutated in the -35 promoter region from TTGTCA to the consensus sequence
TTGACA. The -10 region sequence TACAAT was mutated to the consensus sequence
TATAAT in three mutants exhibiting an ampC promoter seven times stronger than thewild-type." (Lines 4 through 10 of Abstract in the left column on page 875)
"The very conserved TTG sequence of the -35 region is followed downstream by threeless stringently conserved nucleotides." (Lines 18 through 20 of Introduction in the left
column on page 875)
(B) According to the matters described in Exhibit Otsu 10 Document mentioned in (A)above, Exhibit Otsu 10 Document discloses that, in E. coli, mutants in which the
TTGTCA sequence in the -35 region was mutated to the consensus sequence TTGACA
showed a 21-times increase in promoter strength, that mutants in which the TACAAT
sequence in the -10 region was mutated to the consensus sequence TATAAT showed aseven-time increase in promoter strength, and that the TTG sequence is conserved in the-35 region.
C. Regarding Exhibit Otsu 11 Document
Exhibit Otsu 11 Document discloses that, in E. coli, the -35 region and the -10 regionof the tac promoter sequence are TTGACA and TATAAT, respectively, and the -35 region
and the -10 region of the lacUV5 promoter are TTTACA and TATAAT, respectively (Fig.
1b on page 3540) (Exhibit Otsu 11, the entire import of oral arguments).
D. Regarding Exhibit Otsu 12 Document(A) Matters described in Exhibit Otsu 12 Document
The contents described in Exhibit Otsu 12 Document include the following (Exhibit70

Otsu 12, the entire import of oral arguments).
a. Claims"1. A DNA fragment (c) having a nucleotide sequence (a) represented by TAGACA
and a nucleotide sequence (b) represented by TATAAT at a position 15 to 20 nucleotide
pairs downstream of said nucleotide sequence (a), and capable of functioning as apromoter in a coryneform bacterium cell." (Lines 5 through 9 in the left column on page1)
b. Detailed explanation of the invention(a) Working Example 2"Introduction of a synthetic promoter into pPR3:
The promoter was synthesized by using a DNA synthesizer ... (making it a fragmentwith BamHI ends at both ends). The nucleotide sequence of that DNA fragment is shown
below." (Lines 13 through 18 in the upper right column on page 6; for the cited nucleotide
sequence, see Attachment 14 "Drawings of Cited Documents")"By reacting 0.5 μg of pPR3, the plasmid prepared in Working Example 1, withrestriction enzyme BamHI (5 units) at 37℃ for one hour, the plasmid DNA wascompletely degraded.
The abovementioned synthetic promoter DNA and the plasmid DNA degradation
product were mixed, ... E. coli HB101 Competent Cells (produced by Takara Shuzo) weretransformed by using this solution.
The transformed strains were cultured in ..., and were grown. The plasmids of thesegrown strains were extracted by the alkaline SDS method ...
The obtained plasmids were transformed into plasmid-cured Brevibacterium flavum
MJ-233 strains (FERM BP-1497) according to the method described in item (E) of
Working Example 1, and a plasmid was extracted by using the method described in item(A) of Working Example 1.
It was confirmed that the synthetic DNA was integrated into pPR3 based on thecleavage patterns of restriction enzymes, including BamHI, KpnI, and SacI, and thisplasmid was named "pPR3BT2." (Line 1 in the lower left column through line 11 in thelower right column on page 6.)(b) Working Example 3"Measurement of the strength of the synthetic promoter:
The strength of the promoter inserted into pPR3 in Working Example 2 was checkedby measuring the activity of chloramphenicol acetyltransferase (CAT).
A Brevibacterium flavum MJ-233 strain carrying pPR3 and a Brevibacterium flavum
MJ-233 strain carrying pPR3BT2 were each precultured overnight in a test tube
71containing 10 ml medium prepared by adding 50 μg/ml kanamycin to semisynthetic
medium A described in item (A) of Working Example 1. Then, the culture solution was
cultured for about six hours in a conical flask containing 100 ml of the abovementioned
medium, after which bacterial cells were harvested and used for activity measurement.
The CAT activity was measured by a method of W. V. Shaw et al. ... As a result, the MJ233 strain carrying pPR3BT2 had about 14 times higher CAT activity compared to the
MJ-233 strain carrying pPR3 without the insertion of the promoter." (Line 12 in the lower
right column on page 6 through line 11 in the upper left column on page 7)(B) According to the matters described in (A) above, Exhibit Otsu 12 Document discloses
that a promoter having a TAGACA sequence in the -35 region and a TATAAT sequencein the -10 region functioned in a coryneform bacterium, and enhanced the expression of
the objective gene (the CAT gene) to 14 times compared to the level of a strain without
the insertion of the promoter.
E. Regarding Exhibit Otsu 35 Document(A) Matters described in Exhibit Otsu 35 Document
The contents described in Exhibit Otsu 35 Document include the following (For
Tables 2, 10, 11, and 13, see Attachment 14 "Drawings of Cited Documents").
a. Prior art"L-glutamic acid has a wide range of uses as a seasoning, and it is industrially
produced by a fermentation method in which a glutamic acid-producing coryneformbacterium is cultured to cause it to produce L-glutamic acid, and the produced L-glutamic
acid is separated from the culture of the bacterium." (Lines 10 through 14 in the lower
left column on page 2)"... attempts have been made to increase the production rate of L-glutamic acid andalso efficiently produce L-glutamic acid by enhancing the activity of enzymes involved
in glutamic acid biosynthesis, and the cloning of various enzyme genes involved in thebiosynthetic pathway of glutamic acid is under way in order to achieve this objective."(Lines 13 through 19 in the lower right column on page 2)
b. Problem to be solved by the present invention"However, no case has been reported so far regarding a strain capable of sufficiently
improving the L-glutamic acid productivity in L-glutamic acid fermentation." (Lines 5through 7 in the upper right column on page 3)
c. Means and actions for solving the problem"As a result of intensive studies to solve the abovementioned problem, the present
inventors succeeded in producing various strains wherein at least two or more types of
enzyme genes, among the enzyme genes involved in the glutamic acid biosynthetic72pathway, were simultaneously enhanced by using recombinant DNA technology. Whenthey performed glutamic acid fermentation by using these strains, they found that, in thecase of performing glutamic acid fermentation by using a strain transformed with arecombinant DNA containing at least two or more types of enzyme genes derived from a
glutamic acid-producing coryneform bacterium involved in the glutamic acidbiosynthetic pathway, at least including the glutamate dehydrogenase (... GDH) gene andthe isocitrate dehydrogenase (... ICDH) gene, not only the level of L-glutamic acidaccumulated in the medium, but also the yield based on sugar improved remarkably
compared to the case of using the parent strain, leading to the completion of the present
invention." (Line 9 in the upper right column through line 6 in the lower left column on
page 3)"Recombinant plasmids to be used as materials for producing a multi-enhanced strain
include pAG1001 containing the GDH gene, pAG3001 containing the ICDH gene,
pA5001 containing the AH gene, and pAG4003 containing the CS gene. ... they are in theform of the vector plasmid pAG50 of a glutamic acid-producing coryneform bacterium
into which a GDH gene-containing fragment, ICDH gene-containing fragment, AH genecontaining fragment, or CS gene-containing fragment is incorporated." (Lines 8 through18 in the lower right column on page 6)"Examples of the recombinant DNA carried by the glutamic acid-producing
coryneform bacterium of the present invention include plasmids pIG101, pAIG321,
pCIG231, pCAIG4 and the like mentioned below. pIG101 is a plasmid wherein the GDHgene and the ICDH gene derived from a glutamic acid-producing coryneform bacterium
are simultaneously incorporated into pAG50. pAIG321 is a plasmid wherein the AH gene,
the ICDH gene, and the GDH gene are simultaneously incorporated into pAG50. Also,pCIG231 is a plasmid wherein three enzymes, CS, ICDH, and GDH, are simultaneously
incorporated into pAG50. Furthermore, pCAIG4 is a plasmid wherein four enzymes, CS,
AH, ICDH, and GDH, have been simultaneously incorporated into pAG50." (Lines 7
through 19 in the upper left column on page 7)
d. Working Example 1"This working example shows an example of producing a double-enhanced strain inwhich the activities of GDH and ICDH are simultaneously enhanced by using a glutamic
acid-producing coryneform bacterium.
As recombinant plasmids containing the GDH gene and the ICDH gene, pAG1001
and pAG3001 were used respectively." (Lines 3 through 8 in the upper right column on
page 8)"Corynebacterium melassecola (...) carrying plasmid pAG1001 (…) was cultured in ...
73

The GDH activity was obtained by measuring ... The results are shown in Table 2."(Lines 19 through 18 in the upper left column on page 16)
e. Working Example 3"The working example shows an example of producing a triple-enhanced strain inwhich CS + ICDH + GDH are enhanced. It also shows an example of producing a doubleenhanced strain in which CS + ICDH are enhanced and a double-enhanced strain in which
CS + GDH are enhanced.
As materials for producing a recombinant plasmid, the abovementioned pAG1001
and pAG3001 as well as recombinant plasmid pAG4003 containing the CS gene wereused." (Lines 1 through 8 in the lower left column on page 35)"(6) Measurement of the CS activity of plasmid pAG4001-carrying strain
Corynebacterium melassecola (...) 801 carrying plasmid pAG4001 was ... cultured. ...
The CS activity was measured ... As a result, as shown in Table 10, the plasmid pAG4001carrying strain showed higher CS specific activity than the vector pAG50-carrying strain
and the strain carrying no plasmid." (Line 7 in the upper left column through line 4 in theupper right column on page 41)"pCI31 and pCG5 are a recombinant plasmid simultaneously containing CS and
ICDH and a recombinant plasmid simultaneously containing CS and GDH, respectively.
The recombinant plasmid of CS + ICDH + GDH can be obtained by integrating a GDH
fragment at the EcoRI breakpoint of pCI31, and more detailed analysis was performed
regarding plasmid pCIG231 in the present invention." (Lines 12 through 18 in the lower
right column on page 42)"Cell extracts were prepared from host strains respectively carrying the pCI31, pCG5,
and pCIG231 plasmids (Corynebacterium melassecola 801), and the enzyme activities ofCS, ICDH, and GDH were compared with the case of using a cell extract of a hostbacterium that does not carry any plasmids. ...
Based on the results in Table 11, it was confirmed that all target enzyme activities
were enhanced in the pCI31-carrying strain, the pCG5-carrying strain, and the pCIG231carrying strain." (Line 9 in the upper left column through line 15 in the upper right column
on page 43)
f. Working Example 5"This working example shows an example of glutamic acid fermentation using themultiple-enhanced strains obtained in Working Examples 1 through 4...
... The results obtained are shown in Table 13. ...
As is clear from Table 13, at least the strains in which both GDH and ICDH enzymes
were simultaneously enhanced showed higher results in the amount of glutamic acid74

accumulated and the yield based on sugar compared to those of the other strains." (Line
17 in the upper left column on page 44 through line 5 in the upper left column on page
45)(B) According to the matters described in (A) above, Exhibit Otsu 35 Document discloses
glutamic acid-producing coryneform bacteria in which, among the enzyme genes
involved in the glutamic acid biosynthetic pathway, the GDH gene, the CS gene, theICDH gene, etc. are enhanced by introducing plasmids carrying these genes, and discloses
that, among these, glutamic acid-producing coryneform bacteria in which at least the
GDH gene and the ICDH gene were simultaneously enhanced showed improvement inglutamic acid-producing ability. On the other hand, Table 13 of the document discloses
that the glutamic acid productivity of strains in which the GDH gene or the CS gene wasindividually enhanced (9.2 g/dl) did not substantially differ from the productivity of theirparent strain (9.1 g/dl).
(6) Whether or not Corrected Invention 1 lacks an inventive step based on Exhibit Otsu 6
Invention as the primary cited document
A. Regarding involvement of an inventive step in Corrected Invention 1-1(A) Comparison between Exhibit Otsu 6 Invention and Corrected Invention 1-1
The following are found as a result of comparing Exhibit Otsu 6 Invention referred toin (3) B. above with Corrected Invention 1-1.
a. Common feature
They are inventions relating to a coryneform bacterium having specific nucleotide
sequences in the -35 region and the -10 region of the promoter sequence of the GDH gene
and/or having a specific nucleotide sequence in the -10 region of the promoter sequenceof the CS gene on the chromosome of the coryneform bacterium.
b. Differences
Difference 1: The coryneform bacterium in Corrected Invention 1-1 has a TTGTCA
sequence introduced into the -35 region and a TATAAT sequence introduced into the -10
region of the promoter sequence of the GDH gene (1-A'-1), whereas the coryneformbacterium in Exhibit Otsu 6 Invention has a TGGTCA sequence in the -35 region and a
CATAAT sequence in the -10 region of the promoter sequence of the GDH gene.
Difference 2: The coryneform bacterium in Corrected Invention 1-1 has a TATAAT
sequence introduced into the -10 region of the promoter sequence of the CS gene (1-A'2), whereas the coryneform bacterium in Exhibit Otsu 6 Invention has a TAGCGT
sequence in the -10 region of the promoter sequence of the CS gene.
Difference 3: Corrected Invention 1-1 is a method of producing glutamic acid by thefermentation method comprising the steps of cultivating a coryneform bacterium,75

allowing glutamic acid to be accumulated in a medium, and collecting it from the medium(1-A-3, 1-B'), whereas Exhibit Otsu 6 Invention is an invention of a coryneformbacterium.
Meanwhile, with regard to Constituent Features 1-A'-1 and 1-A'-2 from among theconstituent features of Corrected Invention 1-1, it suffices for either one to be fulfilled.
Consequently, it can be said that if the configuration relating to either Difference 1 orDifference 2 could have been easily conceived of by a person skilled in the art, Corrected
Invention 1-1 includes a configuration which could have been easily conceived of basedon Exhibit Otsu 6 Invention.
(B) Regarding the ease in conceiving of Difference 1 (to adopt a TTGTCA sequence inthe -35 region and a TATAAT sequence in the -10 region of the GDH gene)a. Regarding suggestions in Exhibit Otsu 6 Document(a) As mentioned in (3) B. above, Figure 1 of Exhibit Otsu 6 Document discloses nucleic
acid sequences of the promoters of various genes of wild-type Corynebacterium
glutamicum, and in these sequences, it discloses the respective promoter sequences of the
GDH gene and the CS gene of the wild-type strain relating to Exhibit Otsu 6 Invention (a"TGGTCA" sequence in the -35 region and a "CATAAT" sequence in the -10 region of
the promoter sequence of the GDH gene, and a "TGGCTA" sequence in the -35 region
and a "TAGCGT" sequence in the -10 region of the promoter sequence of the CS gene).
In addition, Exhibit Otsu 6 Document describes that in relation to consensus
sequences of the promoters of Corynebacterium glutamicum genes, in the promoter
sequences of Corynebacterium glutamicum genes, conserved sequences are ttGcca.a in
the -35 region and ggTA.aaT in the -10 region, and their positions, etc. correspond toconsensus sequences in the -35 region and the -10 region of promoters of other Grampositive and Gram-negative bacteria ((3) C. (A) above).
On the other hand, Exhibit Otsu 6 Document has no description suggesting that
expression of the GDH gene and the CS gene, which are glutamic acid biosynthetic genes
and specific genes on the chromosome of a coryneform bacterium, should be enhanced
in order to improve the glutamic acid-producing ability in a method of producing glutamic
acid by a fermentation method using a coryneform bacterium, or that, as a specific method
therefor, the -35 region and the -10 region of the promoter sequences of these genes
should be modified to make them closer to the consensus sequences of coryneformbacteria.
Consequently, as there is no motivation for a person skilled in the art to change the 35 region of the promoter of the GDH gene on the chromosome of the coryneformbacterium in Exhibit Otsu 6 Invention to a TTGTCA sequence and its -10 region to a76

TATAAT sequence based on suggestions in Exhibit Otsu 6 Document, it is not found thata person skilled in the art could have easily conceived of the configuration relating to
Difference 1.
(b) Regarding the Defendant's allegation
The Defendant alleges that matters including the following were known as common
general technical knowledge as of Priority Date 1: [i] enhancement of glutamic acidbiosynthetic genes, particularly enhancement of the GDH activity or the CS activity, wasadvantageous for improving the L-glutamic acid productivity of coryneform bacteria; and[ii] in E. coli and Corynebacterium glutamicum, expression of the objective gene waspromoted by making the sequences in the -35 region and the -10 region of the promoterof the gene closer to the consensus sequences. On this basis, the Defendant alleges that it
would have been easy for a person skilled in the art who read Exhibit Otsu 6 Document
to conceive of changing the sequences of the -35 region and the -10 region of the promoterof the GDH gene on the chromosome of the coryneform bacterium in Exhibit Otsu 6
Invention to the consensus sequences of Corynebacterium glutamicum described in
Exhibit Otsu 6 Document (a "TTGCCA" sequence in the -35 region and a "TAT.AT"
sequence in the -10 region) or to sequences close to them in order to improve the glutamic
acid-producing ability, and hence it would have been easy to conceive of adopting a
TTGTCA sequence in the -35 region and a TATAAT sequence in the -10 region of the
GDH gene.
However, even if the general actions of GDH and CS as enzymes involved in glutamic
acid biosynthesis referred to in (8) D. (B) below, and, as described in Exhibit Otsu 9
Document ((4) B. (A) above), the fact that it is desirable to enhance the expression ofgenes such as the GDH gene and the CS gene involved in glutamic acid biosynthesis inorder to improve the glutamic acid-producing ability of coryneform bacteria were known
as of Priority Date 1, there is no sufficient evidence to find that a method to introduce
specific mutations into specific regions of the promoters of these genes on thechromosome was known as a specific method for such enhancement.
In addition, even if it was known as of Priority Date 1 that there were cases where, in
E. coli, expression of the objective gene was promoted by making the sequences in the 35 region and the -10 region of the promoter of the gene closer to the consensus sequencesof the gene, as described in Exhibit Otsu 10 Document ((5) B. (B) above), there is no
sufficient evidence to find that similar knowledge existed with regard to coryneformbacteria. In this regard, Exhibit Otsu 6 Document describes that there are cases where
promoters of E. coli genes functioned in coryneform bacteria and suggests similarity
between promoter regions of Corynebacterium glutamicum genes and promoter regions77

of E. coli genes ((3) C. (B) above), but it also describes that there were multiple reports
on coryneform bacteria-specific promoters which were obviously not functional in E. coli((3) C. (B) above) and that the conservation levels of the abovementioned consensus
motifs of Corynebacterium glutamicum were lower than those of E. coli ((3) C. (C)above), indicating that there are differences in the characteristics of the sequences in the
promoter regions of genes between Corynebacterium glutamicum and E. coli. Moreover,as mentioned in (3) C. (D) above, Exhibit Otsu 6 Document describes, with regard to therelation between the activity of gene promoters and the process of making the sequencesin their -35 region and -10 region similar to the consensus sequences, that the activity ofpromoters of E. coli genes can be correlated to a major extent with their similarity to theconsensus sequences, but that no correlation could be recognized between the activity ofpromoters of Corynebacterium glutamicum genes and their similarity to the consensus
sequences. Therefore, it cannot be said that the statements in Exhibit Otsu 6 Document
concerning consensus sequences of Corynebacterium glutamicum and those concerning
E. coli promoters, etc. suggest that, in order to enhance the objective gene of thecoryneform bacterium, nucleotide sequences in the -35 region and the -10 region of thepromoters of genes on the chromosome of the coryneform bacterium should be modified
to the consensus sequences of coryneform bacteria or to sequences close to them.
Meanwhile, as mentioned in (3) A. (C) a. above, Exhibit Otsu 6 Document indicates
the tac promoter and the lacUV5 promoter as E. coli promoters functional in coryneformbacteria, and as mentioned in (5) C. above, according to statements in Exhibit Otsu 11
Document, the sequence in the -10 region of these promoters is found to be a "TATAAT"
sequence, while according to the statements in Exhibit Otsu 10 Document referred to in(5) B. above, this sequence is found to be the consensus sequence of E. coli. However,Exhibit Otsu 6 Document does not mention the impact of the tac promoter, etc. on theactivity of the GDH gene or the CS gene of coryneform bacteria, and there is no sufficientevidence either to find the existence of common general technical knowledge that the
GDH gene or the CS gene of coryneform bacteria could be enhanced by using thesepromoters (statements in Exhibit Otsu 64 Document, which the Defendant points out tobe in relation to the functions of these promoters in coryneform bacteria, are statements
relating to the activity of the CAT gene). By also taking into consideration theabovementioned statement in Exhibit Otsu 6 Document indicating that there aredifferences in the characteristics of the consensus sequences between E. coli and
Corynebacterium glutamicum, it cannot be said that Exhibit Otsu 6 Document suggested
introduction of a "TATAAT" sequence in the -10 region of the promoter of the GDH geneor the CS gene according to the sequence in the -10 region of the tac promoter, etc. or the78

consensus sequence of E. coli.
Consequently, the Defendant's abovementioned allegation cannot be adopted.
b. Regarding combination with matters described in publicly known documents other thanExhibit Otsu 6 Document
While taking into account the suggestions in Exhibit Otsu 6 Document examined ina. above, an examination will be made as to whether it was easy for a person skilled in
the art to conceive of the configuration relating to Difference 1 by combining mattersdescribed in other publicly known documents.
(a) Regarding matters described in Exhibit Otsu 9 Document
As mentioned in (4) B. (A) above, Exhibit Otsu 9 Document provides a descriptionthat the enhancement of glutamic acid biosynthetic genes is advantageous for improving
the L-glutamic acid productivity, and provides an example in which the GDH gene andthe CS gene are enhanced as an example of enhancement of glutamic acid biosynthetic
genes.
On the other hand, the only specific disclosure of a method to enhance objective genes
in Exhibit Otsu 9 Document is a method to ligate the objective genes at a position
downstream from a strong promoter and introduce them as vectors, and the document
does not disclose an example to add mutations into the promoter sequences of theobjective genes on the chromosome ((4) B. (A), (B) above).
In this respect, with regard to introduction of a mutation into the promoter sequenceof a gene on a chromosome, Exhibit Otsu 9 Document describes a method to introduce amutation into the promoter sequence on a chromosome by utilizing homologous
recombination as a method to obtain a coryneform bacterium deficient in α-KGDH
activity ((4) A. (B) c. (c) above). However, it is merely described as a means for causing
defects in α-KGDH activity, and not as a means for enhancing the glutamic acidbiosynthetic enzyme activity of the GDH gene, etc. on the chromosome of a coryneformbacterium.
Apart from this, Exhibit Otsu 9 Document has statements on strong promoters thatfunction in coryneform bacteria ((4) A. (B) c. (d) above), but it has no statements
concerning the relation between sequences of these promoters and the consensus
sequences of the promoters of genes of coryneform bacteria.
Consequently, it should be said that even if a person skilled in the art who came across
Exhibit Otsu 6 Invention recognized the contents described in Exhibit Otsu 9 Document,
motivation to introduce the sequence mentioned in Corrected Invention 1-1 into thepromoter region of the GDH gene on the chromosome of the coryneform bacterium in
Exhibit Otsu 6 Invention cannot be found.
79

Meanwhile, although the strong promoters that function in coryneform bacteria
described in Exhibit Otsu 9 Invention include those of which -10 region is a "TATAAT"
sequence (of those mentioned in (4) A. (B) c. (d) above, the sequence of the tac promoter
described in Exhibit Otsu 11 Document referred to in (5) C. above), this point does notoverturn the abovementioned conclusion, considering that Exhibit Otsu 9 Document does
not describe introduction of a mutation into the promoter sequence of the objective geneon the chromosome as a method to enhance the objective gene as mentioned above.
(b) Regarding matters described in Exhibit Otsu 10 Document
Matters disclosed in Exhibit Otsu 10 Document are as mentioned in (5) B. (B) above.
It discloses that, in E. coli, the promoter strength was enhanced in a mutant in which thesequence in the -35 region or the -10 region of the promoter was mutated to the consensus
sequence and that the TTG sequence was conserved in the -35 region, but it makes nodisclosure concerning sequences of the promoters of coryneform bacteria.
According to the results of the examination made in a. (b) above, even if a person
skilled in the art who read Exhibit Otsu 6 Document recognized the contents described in
Exhibit Otsu 10 Document concerning the promoter region of E. coli, motivation to
introduce the sequence mentioned in Corrected Invention 1-1 into the promoter region of
the GDH gene on the chromosome of the coryneform bacterium in Exhibit Otsu 6
Invention in order to enhance the objective gene of the coryneform bacterium cannot befound.
(c) Regarding matters described in Exhibit Otsu 11 Document
Matters disclosed in Exhibit Otsu 11 Document are as mentioned in (5) C. above. It
discloses that, in E. coli, the -35 region and the -10 region of the tac promoter sequence
are TTGACA and TATAAT, respectively, and that the -35 region and the -10 region of thelacUV5 promoter sequence are TTTACA and TATAAT, respectively, but the disclosure isneither related to the sequences of the promoters of coryneform bacteria nor related to theconsensus sequences of the promoters of genes of coryneform bacteria.
As mentioned in (3) A. (C) a. above, even if the mention of the tac promoter and thelacUV5promoter in Exhibit Otsu 6 Document is taken into consideration, according to
the results of the examination made in a. (b) above, motivation cannot be found for a
person skilled in the art to introduce the sequence mentioned in Corrected Invention 1-1
into the promoter region of the GDH gene on the chromosome of the coryneformbacterium in Exhibit Otsu 6 Invention in order to enhance the objective gene of thecoryneform bacterium based on the contents described in Exhibit Otsu 6 Document andExhibit Otsu 11 Document.
(d) Regarding matters described in Exhibit Otsu 12 Document
80Matters disclosed in Exhibit Otsu 12 Document are as mentioned in (5) D. (B) above.
It discloses that a promoter having a TAGACA sequence in the -35 region and a TATAAT
sequence in the -10 region functioned in a coryneform bacterium, and enhanced theexpression of the objective gene (the CAT gene) to 14 times compared to the level of astrain without the insertion of the promoter.
On the other hand, Exhibit Otsu 12 Document merely describes the results of theexamination on the relation between a promoter having a specific sequence and theactivity of a specific gene (the CAT gene), and it neither discloses nor suggests theconsensus sequences in the -35 region and the -10 region of the promoters of coryneformbacteria and also does not disclose a strain in which a mutation has been introduced into
the promoter sequence of a gene on the chromosome of a coryneform bacterium.
Consequently, even if a person skilled in the art who read Exhibit Otsu 6 Document
recognized the contents described in Exhibit Otsu 12 Document, motivation to introduce
the sequence mentioned in Corrected Invention 1-1 into the promoter region of the GDHgene on the chromosome of the coryneform bacterium in Exhibit Otsu 6 Invention inorder to enhance the objective gene of the coryneform bacterium cannot be found.
(e) Regarding matters described in Exhibit Otsu 35 Document
According to (5) E. (A) above, Exhibit Otsu 35 Document discloses an example inwhich glutamic acid biosynthetic genes, including the GDH gene, the CS gene, and the
ICDH gene, are enhanced as a method to improve the glutamic acid productivity.
On the other hand, in Exhibit Otsu 35 Document, the method specifically disclosed
as a method to enhance these objective genes is a method of introducing plasmids(vectors) carrying these genes, and there is no statement about adding mutations to thepromoter sequences of genes on the chromosome ((5) E. (A), (B) above).
Moreover, Exhibit Otsu 35 Document also discloses that the glutamic acid-producing
ability improved in strains in which at least the GDH gene and the ICDH gene have been
simultaneously enhanced, and that the glutamic acid-producing ability of strains in which
the GDH gene or the CS gene was individually enhanced did not substantially differ fromthat of their parent strain. Hence, it cannot be said that Exhibit Otsu 35 Document suggests
a method of individually or simultaneously enhancing the GDH gene and the CS gene asa method to improve the glutamic acid-producing ability.
Consequently, even if a person skilled in the art who read Exhibit Otsu 6 Document
recognized the contents described in Exhibit Otsu 35 Document, motivation to introduce
the sequence mentioned in Corrected Invention 1-1 into the promoter region of the GDHgene on the chromosome of the coryneform bacterium in Exhibit Otsu 6 Invention inorder to enhance the objective gene of the coryneform bacterium cannot be found.
81

c. According to the results of the examination made in a. and b. above, a person skilled in
the art is not found to have been able to easily conceive of the configuration relating to
Difference 1 based on Exhibit Otsu 6 Document as of Priority Date 1, by combining thecontents of the document with the contents described in the publicly known documents
referred to in b. above and other common general technical knowledge.
(C) Regarding the ease in conceiving of Difference 2 (to adopt a TATAAT sequence inthe -10 region of the CS gene)
Similarly to (B) a. above, there is no motivation for a person skilled in the art tochange the -10 region of the promoter of the CS gene on the chromosome of
Corynebacterium glutamicum in Exhibit Otsu 6 Invention to a TATAAT sequence basedon suggestions in Exhibit Otsu 6 Document. In addition, according to the contents
described in the respective documents referred to in (B) b. above, even if a person skilledin the art who read Exhibit Otsu 6 Document recognized these contents, there is also no
motivation for the person to introduce the abovementioned mutation into the coryneformbacterium in Exhibit Otsu 6 Invention.
Meanwhile, as mentioned in (5) A. (B) above, Exhibit Otsu 8 Document discloses thatthe sequence in the -10 region of the promoter of the CS gene of wild-type
Corynebacterium glutamicum was deduced as being TATAGC, unlike in Exhibit Otsu 6
Invention. However, even if a person skilled in the art who read Exhibit Otsu 6 Document
recognized this statement, there is no motivation for the person to change the sequence inthe -10 region of the promoter of the CS gene on the chromosome of the coryneformbacterium in Exhibit Otsu 6 Invention to a TATAAT sequence, similar to the above.
Consequently, a person skilled in the art is not found to have been able to easily
conceive of the configuration relating to Difference 2 based on Exhibit Otsu 6 Document
as of Priority Date 1, by combining the contents of the document with the contents
described in the publicly known documents referred to in (5) above and other common
general technical knowledge.
(D) As mentioned in (B) and (C) above, neither the configuration relating to Difference 1
nor that relating to Difference 2 could have been easily conceived of based on Exhibit
Otsu 6 Invention. Consequently, without having to make determinations on other points,
it cannot be said that a person skilled in the art could have easily conceived of Corrected
Invention 1-1 based on Exhibit Otsu 6 Invention as the primary cited document, and hencea ground for invalidation, i.e. lack of an inventive step, (Article 123, paragraph (1), item(ii) and Article 29, paragraph (2) of the Patent Act) based on Exhibit Otsu 6 Invention as
the primary cited document is not found for Corrected Invention 1-1.
B. Regarding involvement of an inventive step in Corrected Invention 1-2
82

The configuration of Corrected Invention 1-2 is included in the configuration of
Corrected Invention 1-1, and when Exhibit Otsu 6 Invention referred to in (3) B. above
and Corrected Invention 1-2 are compared, they differ in respect to Difference 1 andDifference 3 referred to in A. (A) b. above.
In addition, similarly to A. (B) above, a person skilled in the art is not found to havebeen able to easily conceive of the configuration relating to Difference 1 based on Exhibit
Otsu 6 Document as of Priority Date 1, by combining the contents of the document withthe contents described in the publicly known documents referred to in A. (B) b. above andother common general technical knowledge.
Consequently, without having to make determinations on other points, it cannot be
said that a person skilled in the art could have easily conceived of Corrected Invention 12 based on Exhibit Otsu 6 Invention as the primary cited document, and hence a groundfor invalidation, i.e. lack of an inventive step, (Article 123, paragraph (1), item (ii) and
Article 29, paragraph (2) of the Patent Act) based on Exhibit Otsu 6 Invention as theprimary cited document is not found for Corrected Invention 1-2.
C. Regarding involvement of an inventive step in Corrected Invention 1-3
The configuration of Corrected Invention 1-3 is included in the configuration of
Corrected Invention 1-1, and when Exhibit Otsu 6 Invention referred to in (3) B. above
and Corrected Invention 1-2 are compared, they differ at least in respect to Difference 1,
Difference 2, and Difference 3 referred to in A. (A) b. above.
In addition, similarly to A. (B) and (C) above, a person skilled in the art is not foundto have been able to easily conceive of either the configuration relating to Difference 1 orthat relating to Difference 2 based on Exhibit Otsu 6 Document as of Priority Date 1, by
combining the contents of the document with the contents described in the publicly known
documents referred to in A. (B) b. and A. (C) above and other common general technicalknowledge.
Consequently, without having to make determinations on other points, it cannot be
said that a person skilled in the art could have easily conceived of Corrected Invention 13 based on Exhibit Otsu 6 Invention as the primary cited document, and hence a groundfor invalidation, i.e. lack of an inventive step, (Article 123, paragraph (1), item (ii) and
Article 29, paragraph (2) of the Patent Act) based on Exhibit Otsu 6 Invention as theprimary cited document is not found for Corrected Invention 1-3.
(7) Whether or not Corrected Invention 1 lacks an inventive step based on Exhibit Otsu 9
Invention as the primary cited document
A. Regarding involvement of an inventive step in Corrected Invention 1-1(A) Comparison between Exhibit Otsu 9 Invention and Corrected Invention 1-1
83

The following are found as a result of comparing Exhibit Otsu 9 Invention referred toin (4) B. (B) above with Corrected Invention 1-1.
a. Common feature
They are methods of producing L-glutamic acid by the fermentation method
comprising the steps of cultivating, in a medium, a coryneform bacterium in which amutation has been introduced into the nucleotide sequence of the promoter of a gene on
the chromosome, to allow L-glutamic acid to be produced and accumulated in the medium,and collecting it from the medium.
b. Differences
Difference 1: The coryneform bacterium in Corrected Invention 1-1 has a TTGTCA
sequence introduced into the -35 region and a TATAAT sequence introduced into the -10
region of the promoter sequence of the GDH gene on the chromosome (1-A'-1), whereas
in the coryneform bacterium in Exhibit Otsu 9 Invention, no such specification is made,and a vector wherein the GDH gene is ligated at a position downstream from a strong
promoter has been introduced into the bacterium.
Difference 2: The coryneform bacterium in Corrected Invention 1-1 has a TATAAT
sequence introduced into the -10 region of the promoter sequence of the CS gene on thechromosome (1-A'-2), whereas in the coryneform bacterium in Exhibit Otsu 9 Invention,no such specification is made, and a vector wherein the CS gene is ligated at a position
downstream from a strong promoter has been introduced into the bacterium.
Meanwhile, with regard to Constituent Features 1-A'-1 and 1-A'-2 from among theconstituent features of Corrected Invention 1-1, it suffices for either one to be fulfilled.
Consequently, it can be said that if the configuration relating to either Difference 1 orDifference 2 could have been easily conceived of, Corrected Invention 1-1 includes aconfiguration which could have been easily conceived of by a person skilled in the art
based on Exhibit Otsu 9 Invention.
(B) Regarding the ease in conceiving of Difference 1 (to introduce a TTGTCA sequencein the -35 region and a TATAAT sequence in the -10 region of the promoter sequence of
the GDH gene on the chromosome)a. According to the results of the examination made in (6) A. (B) b. (a) above, Exhibit
Otsu 9 Document provides a description that enhancement of glutamic acid biosynthetic
genes is advantageous for improving the L-glutamic acid productivity, and provides anexample in which the GDH gene and the CS gene are enhanced as an example ofenhancement of glutamic acid biosynthetic genes. However, the only specific disclosureof a method to enhance the GDH gene and the CS gene, etc. in Exhibit Otsu 9 Document
is a method to ligate these genes at a position downstream from a strong promoter and84

introduce them as vectors, and the document does not disclose an example to add
mutations into the promoter sequences of the objective genes on the chromosome. The
document contains no statements that would motivate modification of the nucleotide
sequences in the -35 region and the -10 region of the promoter sequences of the GDHgene and the CS gene, which are glutamic acid biosynthetic genes and specific genes on
the chromosome of a coryneform bacterium, to make them closer to the consensus
sequences of coryneform bacteria.
Consequently, even if a person skilled in the art who read Exhibit Otsu 9 Invention
recognized the contents described in Exhibit Otsu 6 Document, Exhibit Otsu 10
Document, Exhibit Otsu 11 Document, Exhibit Otsu 12 Document, or Exhibit Otsu 35
Document examined in (3), (5), and (6) A. (B) above, there is no motivation in Exhibit
Otsu 9 Invention for a person skilled in the art to pay attention to the sequences in the 35 region and the -10 region of the promoter sequence of the GDH gene and the extent ofenhancement of the expression level of the objective gene as well as their relevance toimproving the glutamic acid-producing ability, or to introduce the sequences mentionedin Corrected Invention 1-1 into the -35 region and the -10 region of the promoter sequenceof the GDH gene on the chromosome of the coryneform bacterium in order to obtain amutant strain having the ability to produce glutamic acid in high yield, and it is not foundthat a person skilled in the art could have easily conceived of the configuration relating
to Difference 1.
b. The Defendant alleges that the process of enhancing the expression of the GDH geneor the CS gene, not on a vector, but on a chromosome by using a promoter having a
specific sequence in order to solve problems that occur due to the use of a vector is merely
a matter that could have been easily conceived of by a person skilled in the art. However,as common general technical knowledge as of Priority Date 1, it was known that themethod of enhancing the objective gene by using a vector involved the problems
described in paragraphs [0002] and [0003] of Description 1, including the facts that there
are often cases in which the expression level of the objective gene becomes too high,leading to extremely restrained growth or a decline in the target substance-producing
ability, and that loss of the plasmid itself occurs. Therefore, even if a method to introduce
a mutated gene on a chromosome by utilizing homologous recombination was known(however, as mentioned in (4) A. (B) c. (c) above, Exhibit Otsu 9 Document described it
as a means for causing defects in α-KGDH activity, and not as a means for enhancing agene), in light of the abovementioned contents described in Exhibit Otsu 9 Document andthe publicly known documents indicated by the Defendant, it should be said that there is
no motivation for a person skilled in the art who came across Exhibit Otsu 9 Invention to85

modify the sequences at specific positions of the promoters of the GDH gene and the CS
gene of a coryneform bacterium to make them closer to the consensus sequences ofcoryneform bacteria, and hence this does not overturn the conclusion mentioned in a.
above.
(C) Regarding the ease in conceiving of Difference 2 (to introduce a TATAAT sequencein the -10 region of the promoter sequence of the CS gene on the chromosome)
Due to the same reason as in (B) above, even if a person skilled in the art who cameacross Exhibit Otsu 9 Invention recognized the contents described in Exhibit Otsu 6
Document, Exhibit Otsu 8 Document, Exhibit Otsu 10 Document, Exhibit Otsu 11
Document, Exhibit Otsu 12 Document, or Exhibit Otsu 35 Document examined in (3),(5), and (6) A. (B) and (C) above, there is no motivation in Exhibit Otsu 9 Invention fora person skilled in the art to pay attention to the sequence in the -10 region of the promoterof the CS gene and the extent of enhancement of the expression level of the objective
gene as well as their relevance to improving the glutamic acid-producing ability, or to
introduce the sequence mentioned in Corrected Invention 1-1 into the -10 region of thepromoter of the CS gene on the chromosome in order to obtain a mutant strain having theability to produce glutamic acid in high yield, and it is not found that a person skilled in
the art could have easily conceived of the configuration relating to Difference 2.
(D) As mentioned in (B) and (C) above, neither the configuration relating to Difference 1
nor that relating to Difference 2 could have been easily conceived of based on Exhibit
Otsu 9 Invention. Consequently, without having to make determinations on other points,
it cannot be said that a person skilled in the art could have easily conceived of Corrected
Invention 1-1 based on Exhibit Otsu 9 Invention as the primary cited document, and hencea ground for invalidation, i.e. lack of an inventive step, (Article 123, paragraph (1), item(ii) and Article 29, paragraph (2) of the Patent Act) based on Exhibit Otsu 9 Invention as
the primary cited document is not found for Corrected Invention 1-1.
B. Regarding involvement of an inventive step in Corrected Invention 1-2
The configuration of Corrected Invention 1-2 is included in the configuration of
Corrected Invention 1-1, and when Exhibit Otsu 9 Invention referred to in (4) B. (B)above and Corrected Invention 1-2 are compared, they differ at least in respect toDifference 1 referred to in A. (A) b. above.
In addition, similarly to A. (B) above, a person skilled in the art is not found to havebeen able to easily conceive of the configuration relating to Difference 1 based on Exhibit
Otsu 9 Document as of Priority Date 1, by combining the contents of the document withthe contents described in the publicly known documents referred to in (3), (5), and (6) A.
(B) above and other common general technical knowledge.
86Consequently, without having to make determination on other points, it cannot be said
that a person skilled in the art could have easily conceived of Corrected Invention 1-2
based on Exhibit Otsu 9 Invention as the primary cited document, and hence a ground forinvalidation, i.e. lack of an inventive step, (Article 123, paragraph (1), item (ii) and Article
29, paragraph (2) of the Patent Act) based on Exhibit Otsu 9 Invention as the primarycited document is not found for Corrected Invention 1-2.
C. Regarding involvement of an inventive step in Corrected Invention 1-3
The configuration of Corrected Invention 1-3 is included in the configuration of
Corrected Invention 1-1, and when Exhibit Otsu 9 Invention referred to in (4) B. (B)above and Corrected Invention 1-3 are compared, they differ at least in respect toDifference 1 and Difference 2 referred to in A. (A) b. above.
In addition, similarly to A. (B) and (C) above, a person skilled in the art is not foundto have been able to easily conceive of either the configuration relating to Difference 1 orthat relating to Difference 2 based on Exhibit Otsu 9 Document as of Priority Date 1, by
combining the contents of the document with the contents described in the publicly known
documents referred to in (3), (5), and (6) A. (B) and (C) above and other common generaltechnical knowledge.
Consequently, without having to make determinations on other points, it cannot be
said that a person skilled in the art could have easily conceived of Corrected Invention 13 based on Exhibit Otsu 9 Invention as the primary cited document, and hence a groundfor invalidation, i.e. lack of an inventive step, (Article 123, paragraph (1), item (ii) and
Article 29, paragraph (2) of the Patent Act) based on Exhibit Otsu 9 Invention as theprimary cited document is not found for Corrected Invention 1-3.
(8) Whether or not Corrected Invention 1 violates the enablement requirement or the
support requirement
A. Problem to be solved by Corrected Invention 1
The problem to be solved by Corrected Invention 1 is, as mentioned in (1) B. (B)above, to provide a process for constructing a mutant strain capable of appropriately
enhancing and regulating the expression level of the objective gene and that has the ability
to produce amino acids in high yield by genetic recombination or mutation, without usinga plasmid, and to provide a glutamic acid fermentation method for increasing the yield ofglutamic acid and producing glutamic acid at a lower cost, using a coryneform glutamic
acid-producing bacterium (paragraph [0006]).
B. Regarding the process for producing L-arginine
As Corrected Inventions 1-1, 1-2, and 1-3 all no longer include the process for
producing L-arginine due to Correction 1, the violation of the support requirement87regarding the process for producing L-arginine mentioned in 5. (2) above is found to havebeen eliminated by Correction 1 with regard to these inventions.
Similarly, Corrected Invention 1-1, 1-2, and 1-3 are not found to violate theenablement requirement in relation to the process for producing L-arginine either.
C. Regarding introduction of a mutation into the promoter of the ICDH gene, the PDH
gene, or the argininosuccinate synthase gene
Although Invention 1-1 before correction included inventions using a coryneformbacterium in which a specific nucleotide sequence has been introduced into the ICDH
gene, the PDH gene, or the argininosuccinate synthase gene, Corrected Invention 1-1 nolonger includes these inventions due to Correction 1.
Consequently, Corrected Invention 1-1 is not found to violate either the support
requirement or the enablement requirement in relation to introduction of a mutation into
the promoter of the ICDH gene, the PDH gene, or the argininosuccinate synthase gene.
D. Regarding introduction of a mutation into the promoter of the GDH gene(A) Statements in Description 1
As mentioned in (1) B. above, the detailed explanation of the invention in Description
1 states the following: [i] in order to solve the problem mentioned in A. above, aconfiguration can be adopted in which a mutant of a coryneform bacterium is preparedby causing or introducing by genetic recombination a mutation to the promoter sequenceof an amino acid biosynthetic gene on the chromosome of the coryneform bacterium tomake the sequence closer to the consensus sequence, then that mutant is cultured, and as
a result, only a mutant that produces a large amount of the target amino acid is collected(paragraph [0007]), while for examples of enzymes involved in the biosynthesis of aminoacid, enzymes such as GDH and CS are effective in the case of glutamic acid fermentation(paragraph [0012]), and the abovementioned mutation may be caused either only for thepromoter sequence of one gene or for the promoter sequences of two or more genes(paragraph [0015]); and [ii] as an example of configuration of such mutation, it ispreferable that, in the promoter sequence of the GDH gene, the -35 region is TTGTCA or
the -10 region is TATAAT (paragraph [0017]).
As mentioned in (1) B. above, Description 1 discloses the following: [iii] Working
Example 2 describes a method to produce a strain wherein the -35 region of the promoter
sequence of a GDH gene of the parent strain (AJ13029) has been mutated to a TTGTCA
sequence, and the -10 region of the promoter sequence has been mutated to a TATAAT
sequence, as "FGR2," and the fact that the specific activity of GDH of FGR2 is about 3.4
times that of the parent strain, while its L-glutamic acid-producing ability also increased
compared to the parent strain (paragraphs [0026], [0029], [0031], and [0032], [Table 5],88[Table 6]); and [iv]Working Example 7 also describes a method to produce a strain
wherein the same mutation as that in "FGR2" has been introduced into the GDH gene on
the chromosome ("GA02"), and the fact that the specific activity of GDH of GA02 isabout 3.5 times that of the parent strain, while its L-glutamic acid-producing ability
increased compared to the parent strain (paragraphs [0087] through [0089], [Table 23]).
(B) Common general technical knowledge concerning actions of GDH and CS in theglutamic acid production process
According to Exhibit Otsu 17 (Iwanami Seibutsugaku Jiten [Iwanami biology
dictionary], 4th ed., published on July 12, 1996) and the entire import of oral arguments,
the following common general technical knowledge is found to have existed as of thetime of the filing of the application for Patent Right 1 with regard to involvement of GDH
and CS in glutamic acid biosynthesis.
a. The citric acid cycle (TCA cycle) is involved in glutamic acid biosynthesis by way of
supplying materials for synthesis of glutamic acid.
b. The TCA cycle is an oxidation process which completely degrades acetyl CoA
produced by glycolysis and other catabolic action into water and carbon dioxide. It
consists of eight steps and repeats the cycle where acetyl CoA is condensed withoxaloacetic acid to form citric acid, which is then converted into isocitric acid, αketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, L-malic acid, andoxaloacetic acid in order, and citric acid is produced again.
c. The citric acid-producing enzyme (CS) is an enzyme which condenses oxaloacetic acidwith acetyl CoA, and catalyzes a reaction to synthesize citric acid in the TCA cycle.
d. L-glutamic acid is produced from α-ketoglutaric acid, which is an intermediate productin the TCA cycle. GDH is an enzyme which reductively fixes ammonia to α-ketoglutaricacid and catalyzes a reaction to produce L-glutamic acid.
(C) According to the statements in Description 1 mentioned in (A) above and thecommon general technical knowledge mentioned in (B) above, it is found that a person
skilled in the art who read Description 1 could have recognized that the problem
mentioned in A. above, which is to provide a process for constructing a mutant strain
capable of appropriately enhancing and regulating the expression level of the objective
gene and that has the ability to produce amino acids in high yield, without using a plasmid,
could be solved by the method of producing L-glutamic acid wherein a mutation has been
introduced into the promoter of the GDH gene in Corrected Invention 1, that is, a method
to introduce a TTGTCA sequence in the -35 region and a TATAAT sequence in the -10
region of the promoter sequence of the GDH gene on the chromosome of a coryneformbacterium used for producing glutamic acid.
89

In addition, according to the statements in Description 1 mentioned above, it is foundthat, based on these statements, a person skilled in the art could have been able to obtaina strain in which the abovementioned sequences are respectively introduced into the -35
region and the -10 region of the promoter sequence of the GDH gene on the chromosomeof a coryneform bacterium and work Corrected Invention 1 without undergoing excessive
trial and error.
Consequently, Corrected Invention 1 is not found to violate either the support
requirement or the enablement requirement in relation to the introduction of a mutation
into the promoter of the GDH gene mentioned above.
E. Regarding introduction of a mutation into the promoter of the CS gene(A) Statements in Description 1
As mentioned in (1) B. above and D. above, the detailed explanation of the invention
in Description 1 states, in addition to the statements mentioned in [i] in D. above, the factthat examples of the configuration of a mutation that could be adopted for solving the
problem mentioned in A. above include one that has the TTGACA sequence in the -35
region and/or the TATAAT sequence in the -10 region of the promoter sequence of the CS
gene (paragraph [0018]). Then, after describing Working Example 2 relating to "FGR2"
wherein the glutamic acid-producing ability has been improved by introducing mutations
into the promoter sequence of the GDH gene mentioned in [iii] in D. above, it describes,
in Working Example 3, a method of producing "GB02" wherein the promoter sequenceof the GDH gene on the chromosome of the abovementioned "FGR2" in Working
Example 2 remains the same, but a mutation has been introduced to make the -10 regionof the promoter sequence of the CS gene a TATAAT sequence, and a method of producing"GB03" wherein mutations have been introduced to make the -35 region of the promoter
sequence a TTGACA sequence and the -10 region of the promoter sequence a TATAAT
sequence, as well as the fact that the specific activity of CS of GB02 is about 1.9 times
that of FGR2 and the specific activity of CS of GB03 is about 4.0 times that of FGR2,
and that their L-glutamic acid-producing ability both further improved compared to FGR2.
(B) Regarding Corrected Invention 1-1
a. Comprehensively considering the statements in Description 1 mentioned in (A) above
and the common general technical knowledge mentioned in D. (B) above, it is found thata person skilled in the art who read Description 1 could have recognized that the problem
mentioned in A. above, which is to provide a process for constructing, without using aplasmid, a mutant strain capable of appropriately enhancing and regulating the expression
level of the objective gene and that has the ability to produce amino acids in high yield,
could be solved by the method of producing L-glutamic acid wherein a mutation has been90

introduced into the promoter of the CS gene in Corrected Invention 1-1, that is, a method
to introduce a TATAAT sequence in the -10 region of the promoter sequence of the CS
gene on the chromosome of a coryneform bacterium used for producing glutamic acid. In
addition, according to the statements in Description 1 mentioned in (A) above, it is foundthat, based on these statements, a person skilled in the art could have been able to obtaina strain in which the abovementioned sequence is introduced into the -10 region of thepromoter sequence of the CS gene on the chromosome of a coryneform bacterium andwork Corrected Invention 1-1 without undergoing excessive trial and error.
b. Regarding the Defendant's allegations(a) The Defendant alleges, with regard to a configuration included in Corrected Invention
1-1 which uses a strain wherein no mutation is introduced into the promoter sequence of
the GDH gene and a mutation is only introduced into the promoter sequence of the CS
gene (a configuration that fulfills Constituent Feature 1-A'-2, but not Constituent Feature
1-A'-1), that Description 1 contains no working example wherein a mutation is only
introduced into the promoter sequence of the CS gene, and it was known in documents
published before the filing of the application (Exhibit Otsu 8 Document, Exhibit Otsu 35
Document) that the glutamic acid productivity would not improve by enhancing the CS
activity without enhancing the GDH activity in a coryneform bacterium, and hence a
person skilled in the art could not have recognized that the glutamic acid productivity
would improve based on the statements in Description 1 and the common generaltechnical knowledge as of the time of the filing of the application.
(b) Indeed, as mentioned in (A) above, GB02 and GB03, wherein a mutation has been
introduced into the promoter of the CS gene on the chromosome, in Working Example 3,
were strains produced by introducing a further mutation into FGR2 in Working Example
2, and therefore, the mutation mentioned in Working Example 2 has been introduced not
only into the promoter of the CS gene, but also into the promoter sequence of the GDH
gene, which means that Description 1 contains no working example relating to a strain
wherein a mutation is only introduced into the promoter sequence of the CS gene.
However, as mentioned in (A) above, Working Example 3 shows the results of comparing
FGR2 with GB02 and GB03, wherein the promoter sequence of the GDH gene of FGR2
remains the same as in Working Example 2 and a mutation is further introduced to makethe -10 region of the promoter sequence of the CS gene a TATAAT sequence. Therefore,
it can be said that Working Example 3 discloses the impact which the method of
introducing the abovementioned sequence into the promoter sequence of the CS gene on
the chromosome has on the glutamic acid-producing ability.
(c) Moreover, the Defendant alleges that it was known since before the filing of the91

application that the glutamic acid productivity would not improve by enhancing the CS
activity without enhancing the GDH activity in a coryneform bacterium.
While the contents described in Exhibit Otsu 8 Document indicated by the Defendant
are as mentioned in (5) A. above, the document discloses that overexpression of the CS
gene slows the growth of Corynebacterium glutamicum and that in Corynebacterium
glutamicum in which CS activity was increased by introducing plasmids the glutamic acidproductivity was not improved compared to a strain to which no mutation was added. In
addition, Exhibit Otsu 35 Document discloses, as mentioned in (5) E. above, that withregard to glutamic acid-producing coryneform bacteria in which the GDH gene, the CS
gene, the ICDH gene, etc. are enhanced by introducing plasmids carrying these genes, theglutamic acid-producing ability of strains in which the GDH gene or the CS gene wasindividually enhanced did not substantially differ from that of their parent strain.
However, in abovementioned Exhibit Otsu 8 Document and Exhibit Otsu 35
Document, the examples where the glutamic acid-producing ability did not improve byenhancing the CS gene individually were all examples in which the CS gene wasenhanced by introducing plasmids, and the method of enhancement in these examples
differs from that employed in Corrected Invention 1-1, which is to change the promoter
sequence of the objective gene on the chromosome. Moreover, regarding enhancement ofgenes by introducing plasmids, Exhibit Otsu 35 Document shows a result that theglutamic acid-producing ability also did not improve compared to the parent strain in thecase of enhancing the GDH gene individually, and this also differs from the experimentresults of FGR2, where the GDH gene was individually enhanced by changing thesequence of the promoter of the objective gene on the chromosome, as shown in Working
Example 2 in Description 1. According to statements in paragraphs [0002] and [0003] of
Description 1 and the entire import of oral arguments, with regard to enhancement of theobjective gene by plasmids, it is found to have been known since before the filing of theapplication that there are cases in which the expression level of the objective gene
becomes too high, leading to extremely restrained growth or a decline in the target
substance-producing ability. When this point is also taken into consideration, even when
the same objective gene is enhanced in a coryneform bacterium, the impact on
improvement of the glutamic acid-producing ability could differ depending on theenhancement method. Thus, it cannot be said, based on the statements in Exhibit Otsu 8
Document and Exhibit Otsu 35 Document, that the glutamic acid-producing ability does
not improve by enhancing the CS gene individually, when the method of introducing
plasmids is not adopted, and there is no other evidence sufficient to find that there wassuch common general technical knowledge as of the time of the filing of the application.
92

(d) According to (a) through (c) above, the conclusion mentioned in a. above cannot be
overturned even by taking into consideration the Defendant's allegations.
(C) Regarding Corrected Invention 1-3
Similarly to (B) above, it is found that, based on the statements in Description 1
mentioned in (A) above and the common general technical knowledge mentioned in D.
(B) above, a person skilled in the art who read Description 1 could have recognized thatthe problem mentioned in A. above, which is to provide a process for constructing amutant strain capable of appropriately enhancing and regulating the expression level ofthe objective gene and that has the ability to produce amino acids in high yield, without
using a plasmid, could be solved by the method of producing L-glutamic acid wherein amutation has been introduced into the promoter of the CS gene in Corrected Invention 13, that is, a method to introduce a TATAAT sequence in the -10 region of the promoter
sequence of the CS gene on the chromosome of a coryneform bacterium used for
producing glutamic acid or a method to introduce a TTGACA sequence in the -35 region
and a TATAAT sequence in the -10 region of the promoter sequence. In addition,according to the statements in Description 1 mentioned in (A) above, it is found that,based on these statements, a person skilled in the art could have been able to obtain astrain in which the abovementioned sequences are introduced into the promoter sequenceof the CS gene on the chromosome of a coryneform bacterium and work Corrected
Invention 1-3 without undergoing excessive trial and error.
F. Regarding the promoter sequence of Corynebacterium glutamicum wild-type strain
ATCC13869
The Defendant indicates that the sequences in regions surrounding the -35 region andthe -10 region of the promoter sequence of SEQ ID NO: 1 in paragraph [0025] of
Description 1 in Working Example 1 differ from the sequences of the wild-type strain
ATCC13869 described in Exhibit Ko 22 at two positions, and alleges that the experimentin Description 1 was performed based not on wild-type strain ATCC13869 but a different
strain, and therefore it cannot be said that Description 1 has demonstrated a change inactivity caused by mutating the promoter sequence of a wild-type strain, at least withregard to the GDH gene.
As mentioned in (1) B. (D) a. above, however, in Working Example 1, a plasmid
wherein a mutation has been introduced into the promoter sequence of the GDH gene has
been produced, but a mutation has not been introduced into the promoter sequence of the
GDH gene on the chromosome of a coryneform bacterium, and as mentioned in D. (A)above, the improvement of the glutamic acid-producing ability in a strain wherein thesequences prescribed in Corrected Invention 1 have been introduced into the promoter
93

sequence of the GDH gene on the chromosome was confirmed in Working Example 2and Working Example 7.
The nucleotide sequences of the promoter region of the GDH gene of the AJ13029
strain and of FGR1 and FGR2, which are AJ13029 strains into which mutations have been
introduced, used in Working Example 2 are described in [Table 6] of Description 1, but
the Defendant has not made any particular indication about these sequences, and similarly,
the Defendant has not made any particular indication about the strain into which amutation has been introduced in Working Example 7.
If so, it cannot be said that the Defendant's abovementioned indications about thestatements on the promoter sequence in Working Example 1 affect the conclusion
mentioned in D. above to the effect that Corrected Invention 1 fulfills the support
requirement and the enablement requirement in relation to the introduction of a mutation
into the promoter of the GDH gene.
With regard to the statements on the promoter sequence of ATCC13869 in Working
Example 1, the Defendant has not made specific allegations regarding violation of the
support requirement and the enablement requirement, apart from making theabovementioned allegations in relation to introduction of a mutation into the GDH gene.
Consequently, Corrected Invention 1 is not found to violate the support requirement or
the enablement requirement based on the abovementioned statements on the sequence.
G. Regarding regions surrounding the -35 region and the -10 region of the promoter
The Defendant alleges that, as Corrected Invention 1 does not specify the sequencesin regions surrounding the -35 region and the -10 region of the GDH gene and the CS
gene, a person skilled in the art cannot recognize that the glutamic acid productivity would
improve by making the regions surrounding the -35 region and the -10 region arbitrary
sequences or lengths, even by referring to the working examples and other statements in
Description 1 as well as common general technical knowledge, and hence the support
requirement is not fulfilled, and due to the same reason, the statements in the detailed
explanation of the invention in Description 1 do not fulfill the enablement requirement.
As mentioned in (1) B. (D) b. above, "FGR2" in Working Example 2 is a strain
wherein mutations are introduced to mutate the -35 region and the -10 region of thepromoter sequence of the GDH gene of the parent strain, AJ13029, to the sequences
mentioned in Corrected Invention 1. Meanwhile, [Table 6] of Description 1 shows thatthe sequence in the region between the -35 region and the -10 region of the promoter
sequence of the GDH gene of "FGR2" is the same as that of the parent strain, AJ13029,
except for deletion of one nucleotide, and apart from this, Description 1 contains nostatements to the effect that a mutation has been added to the sequence of the parent strain94

with regard to the sequences in the regions surrounding the -35 region and the -10 regionof the promoter of GDH of FGR2.
In addition, also with regard to the "GB02" and "GB03" strains in Working Example
3 and the "GA02" strain in Working Example 7, as examined in D. and E. above,
Description 1 contains no statements to the effect that a mutation has been added to thesequences in the regions surrounding the -35 region and the -10 region of the promoter of
the CS gene or the GDH gene when producing the respective strains.
According to the above, it should be said that, even if the fact that the sequences andlengths of the regions surrounding the -35 region and the -10 region affect the promoter
activity was publicly known, a person skilled in the art who read Description 1 could haveunderstood that, in Corrected Invention 1 for which the problem to be solved is tointroduce on the chromosome a mutation to make the -35 region or the -10 region of thepromoter sequence of the GDH gene or the CS gene a specific sequence, the regions
surrounding the -35 region and the -10 region of the promoter sequence, which are notspecified in the invention, can be the same as those in a non-modified strain, as allegedby the Defendant.
Consequently, it cannot be said, based on the fact that the sequences in the regions
surrounding the -35 region and the -10 region have not been specified, that Corrected
Invention 1 falls beyond the scope in which a person skilled in the art can recognize thatthe problem to be solved by the invention can be solved in light of the statements in thedetailed explanation of the invention and the common general technical knowledge as of
the time of the filing of the application. Therefore, the Defendant's allegation of aviolation of the support requirement relating to this point is groundless. Also, for the same
reason, it can be said that a person skilled in the art can work Corrected Invention 1
without undergoing excessive trial and error based on the statements in Description 1,
even if the sequences in the regions surrounding the -35 region and the -10 region of thepromoter sequence have not been specified, and hence, the Defendant's allegation of aviolation of the enablement requirement relating to this point is also groundless.
(9) Summary on the defense of invalidity concerning Patent Right 1
A. Regarding whether or not the requirements for re-defense of correction are fulfilled
While Correction 1 satisfies the requirements for correction as mentioned in No. 2, 2.
(4) above, Defendant's Production Process 1 literally falls within the technical scopes ofCorrected Invention 1-1 (corresponding to Invention 1-1), Corrected Invention 1-2(corresponding to Invention 1-2), and Corrected Invention 1-3 (corresponding toInvention 1-4), whereas Defendant's Production Process 3 literally falls within thetechnical scope of Corrected Invention 1-1 but does not fall within the technical scope of95

Corrected Invention 1-3, as mentioned in (2) above.
Among the grounds for invalidation of Invention 1, the violations of the support
requirement referred to in 5. above have all been eliminated in Corrected Invention 1, as
mentioned in (8) B. above. In addition, with regard to the other grounds for invalidation
alleged by the Defendant in relation to Invention 1 and Corrected Invention 1, no groundfor invalidation is found to exist at least in Corrected Invention 1 after the correction.
B. Regarding claims concerning Defendant's Production Process 1
According to A. above, without having to make determinations on other points, inregard to the claims based on Patent Right 1 concerning Defendant's Production Process
1, the requirements for re-defense of correction are fulfilled for Corrected Invention 1-1(corresponding to Invention 1-1), Corrected Invention 1-2 (corresponding to Invention 12), Corrected Invention 1-3 (corresponding to Invention 1-4), and hence the defense ofinvalidity based on Article 104-3, paragraph (1) of the Patent Act is groundless for all theparts for which the re-defense of correction is allowed.
On the other hand, according to 5. (3) above, as the Plaintiff has not asserted redefense of correction with regard to the claims based on Invention 1-3, the Plaintiff cannot
exercise its rights against the Defendant based on the abovementioned defense ofinvalidity.
According to the above, the claims for which the Plaintiff can exercise its rights
against the Defendant based on Patent Right 1 in relation to Defendant's Production
Process 1, without being restricted by the defense of invalidity, are the claims based on
Invention 1-1 (corresponding to Corrected Invention 1-1), Invention 1-2 (corresponding
to Corrected Invention 1-2), and Invention 1-4 (corresponding to Corrected Invention 13).
C. Regarding claims concerning Defendant's Production Process 3
According to A. above, without having to make determinations on other points, inregard to the claims based on Patent Right 1 concerning Defendant's Production Process
3, the requirements for re-defense of correction are fulfilled for Corrected Invention 1-1,
and hence the defense of invalidity based on Article 104-3, paragraph (1) of the Patent
Act is groundless for the part for which the re-defense of correction is allowed.
On the other hand, as mentioned in A. above, because Defendant's Production Process
3 does not fall within the technical scope of Corrected Invention 1-3 (corresponding toInvention 1-4), re-defense of correction cannot be found to be established for the claims
based on Invention 1-4. Accordingly, the Plaintiff cannot exercise its rights against the
Defendant based on the abovementioned defense of invalidity with regard to the claims
based on Invention 1-4 (corresponding to Corrected Invention 1-3).
96

According to the above, the claims for which the Plaintiff can exercise its rights
against the Defendant based on Patent Right 1 in relation to Defendant's Production
Process 3, without being restricted by the defense of invalidity, are the claims based on
Invention 1-1 (corresponding to Corrected Invention 1-1).
7. Regarding Issue 6 (whether re-defense of correction of Patent 2 (Correction 2) can beestablished)In light of the case, the ground for invalidation of the invention claimed in Patent 2
will be determined based on Issue 6.
(1) Whether Correction 2 satisfies the requirements for correction
A. Regarding correction concerning the number of amino acids (regarding Claim 1)
In Correction 2, the number of amino acids to be substituted, etc. in (ii) of Claim 1
was corrected from "one or several" before correction to "one to five," but it can be said
that this correction is intended for restriction of the claims (Article 134-2, paragraph (1),item (i) of the Patent Act).
The Defendant asserts a violation of the requirements for correction (a violation of
Article 126, paragraph (5) of the Patent Act) on the basis that Description 2 contains nostatement on a numerical scope to make the number of amino acids to be substituted, etc.
"one to five," but paragraph [0034] of Description 2 attached to the written application
states a numerical scope as follows: "The yggB gene may be a gene having an amino acidsequence in which one or several amino acids are substituted, deleted, inserted, or addedin an amino acid sequence of SEQ ID NO: 6, 62, 68, or 84 so far as the yggB geneenhances L-glutamic acid-producing ability of the coryneform bacterium. The number of'several' as referred to herein means, for example, 2 to 20, preferably 2 to 10, and more
preferably 2 to 5." Therefore, the abovementioned correction is a correction within thescope of the matters disclosed in the description, claims, or drawings attached to a written
application, and complies with the requirement under Article 126, paragraph (5) of the
Patent Act as applied mutatis mutandis pursuant to Article 134-2, paragraph (9) of thatAct.
Furthermore, as this correction limits the number of amino acids to be substituted, etc.
and does not substantially enlarge or alter the scope covered by the invention of Claim 1
before correction, it also complies with the requirement under Article 126, paragraph (6)of the Patent Act as applied mutatis mutandis pursuant to Article 134-2, paragraph (9) ofthat Act, and hence it satisfies all of the requirements for correction.
B. Regarding correction of the strain name (regarding paragraph [0033] of Description 2)
While Correction 2 makes a correction, in the part "The gene having a sequence of
nucleotide numbers 1437 to 3035 of SEQ ID NO: 5 is a yggB gene of Corynebacterium97

glutamicum ATCC13032 strain" in the third sentence in paragraph [0033] of Description
2 attached to the written application, to change "ATCC13032 strain" to "ATCC13869
strain" (Exhibits Ko 51 and 94), the Defendant alleges that this correction is not acorrection of an error and thus impermissible.
First, in paragraph [0033] of Description 2 before correction, the nucleotide sequenceof SEQ ID NO: 5 and the nucleotide sequence of SEQ ID NO: 83 are both indicated as
being sequences derived from the ATCC13032 strain, and the sequences in the parts ofthese nucleotide sequences that correspond to the yggB gene partially do not coincidewith each other (Exhibit Ko 4-2), so either of them is apparently an error.
In addition, while paragraph [0033] before correction has a statement "The gene
having a sequence of nucleotide numbers 501 to 2099 of SEQ ID NO: 83 is encoded tonucleotide numbers 1336092 to 1337693 in the genome sequence registered as GenBank
Accession No. NC_003450 of Corynebacterium glutamicum ATCC13032, and isregistered as NCgl 1221 (NP_600492. Reports small-conductance ...[gi:19552490]),"according to evidence (Exhibit Ko 94) and the entire import of oral arguments, it is foundto be possible to confirm that SEQ ID NO: 83 is a sequence of the ATCC13032 strain bycomparing information on the sequence of NCgl1221, which had been published as of thetime of the filing of the application, and SEQ ID NO: 83.
Moreover, considering that paragraph [0103] before correction contains a statement
indicating that the yggB gene of SEQ ID NO: 5 was derived from the "ATCC13869
strain," it is found that the statement in paragraph [0033] indicating that SEQ ID NO: 5
is derived from the "ATCC13032 strain" is an error, and correcting this to the"ATCC13869 strain" is found to constitute correction of an error or incorrect translation(Article 134-2, paragraph (1), item (ii) of the Patent Act).
In addition, in light of such contents of correction, this correction also complies withthe requirements under Article 126, paragraphs (5) and (6) of the Patent Act as appliedmutatis mutandis pursuant to Article 134-2, paragraph (9) of that Act, and satisfies all of
the requirements for correction.
C. The Defendant has not specifically indicated a violation of the requirements forcorrection concerning Correction 2 apart from the corrections referred to in A. and B.
above, and according to evidence (Exhibits Ko 51-1 and 94) and the entire import of oral
arguments, all corrections in Correction 2 were made within the scope of the matters
indicated in the description, claims, or drawings attached to the written application forthe purpose of restriction of the claims (Article 134-2, paragraph (1), item (i) of the Patent
Act), correction of errors or incorrect translations (item (ii) of that paragraph), or
correction of a statement of claims which cites another statement of claims to a statement98which does not cite that other statement of claims (item (iv) of that paragraph), and they
do not substantially enlarge or alter the claims; therefore, they satisfy the requirements
for correction (Article 134-2, paragraph (9) and Article 126, paragraphs (5) and (6) of the
Patent Act).
(2) Regarding whether the Defendant's Production Processes fall within the technicalscope of Corrected Invention 2
A. Regarding Defendant's Production Processes 1 through 3
As examined in 3. above, the bacteria used in Defendant's Production Processes 1
through 3 are all Corynebacterium glutamicum into which a mutant-type yggB gene,
wherein an A100T mutation has been added to the yggB gene having the amino acidsequence of SEQ ID NO: 6 and the amino acid sequence after the mutation is SEQ ID
NO: 22, has been introduced, indicating an improvement in the glutamic acid-producing
ability compared to a non-modified strain.
Consequently, when comparing Defendant's Production Processes 1 through 3 andCorrected Invention 2, the result is found to be as shown in Attachment 7-2 "Comparison
between Defendant's Production Processes 1 through 3 and Corrected Invention 2."
Accordingly, the bacteria used in Defendant's Production Processes 1 through 3 all fallwithin the technical scopes of Corrected Inventions 2-1, 2-2, 2-3, and 2-4, andDefendant's Production Processes 1 through 3, which use those bacteria, fall within thetechnical scope of Corrected Invention 2-5.
B. Regarding Defendant's Production Process 4(A) As mentioned in 4. (5) A. above, Strains [xii] and [xiii] used in Defendant's Production
Process 4 are both Corynebacterium glutamicum into which a mutant-type yggB gene,
wherein an A98T mutation and a V241I mutation have been introduced into a yggB genederived from Corynebacterium callunae (DSM20147 strain), has been introduced,
indicating an improvement in the glutamic acid-producing ability compared to a nonmodified strain in the presence of an excess of biotin.
Consequently, Strains [xii] and [xiii] used in Defendant's Production Process 4 fulfill
Constituent Features 2-E'-1 and 2-E'-2 of Corrected Invention 2-2, but do not fulfill 2-D',
and do not fulfill any of the constituent features of Corrected Invention 2-3. In addition,
Defendant's Production Process 4 literally fulfills Constituent Feature 2-H' of Corrected
Invention 2-5, except for the part citing Claims 1, 2, and 4 through 10, and fulfills
Constituent Features 2-I' and 2-J.
(B) The configuration using the 19-type mutation is a configuration that is included inCorrected Invention 2-5 even after Correction 2, and it is a method of producing theglutamic acid in Claim 11 using a coryneform bacterium into which the mutant-type yggB99

gene mentioned in (e) of Claim 6 citing Claim 4 after Correction 2, has been introduced.
(C) The differences between Defendant's Production Process 4 and the configuration
using the 19-type mutation comprised in Defendant's Invention 2-5 lie in the parts relating
to Constituent Features 2-D', 2-F-1, 2-F'-2, 2-F-3, and 2-H' (literally non-fulfilling parts
mentioned in (A) above), and their specific contents are the same as Differences 1 through3 mentioned in 4. (5) B. above.
Meanwhile, the circumstances concerning the first through fifth requirements of thedoctrine of equivalents with regard to Defendant's Production Process 4 using Strains [xii]and [xiii] and the configuration using the 19-type mutation comprised in Corrected
Invention 2-5 are the same as those mentioned in 4. (6) through (10) above, and nocircumstances that would overturn the conclusions regarding the respective requirements
are found in the evidence for the present case.
Consequently, it is reasonable to construe that Defendant's Production Process 4 using
Strains [xii] and [xiii] falls within the technical scope of Corrected Invention 2-5 as being
an equivalent to the configuration using the 19-type mutation indicated in the claims.
(3) Whether or not Corrected Invention 2 lacks novelty based on Exhibit Otsu 42
Invention
A. Statements in Exhibit Otsu 42 Document
The contents described in Exhibit Otsu 42 Document include the following (Exhibit
Otsu 42, the entire import of oral arguments).
(A) Paragraph [0003]"For example, Corynebacterium glutamicum is a Gram-positive bacterium identified
as a glutamic acid-producing bacterium, and many amino acids are produced by mutants
thereof. For example, 1,000,000 ton/year of L-glutamic acid which is useful as aseasoning for umami (delicious taste), 250,000 ton/year of L-lysine which is a valuableadditive for livestock feeds and the like, and several hundred ton/year or more of otheramino acids, such as L-arginine, L-proline, L-glutamine, L-tryptophan, and the like, havebeen produced in the world (Nikkei Bio Yearbook 99, published by Nikkei BP (1998))."(B) Paragraph [0009]"An object of the present invention is to provide a polynucleotide and a polypeptide
derived from a microorganism of coryneform bacteria which are industrially useful,sequence information of the polynucleotide and the polypeptide, a method for analyzing
the microorganism, an apparatus and a system for use in the analysis, and a method forbreeding the microorganism."(C) Paragraph [0010]"The present invention provides a polynucleotide and an oligonucleotide derived from100

a microorganism belonging to coryneform bacteria, ... a polypeptide encoded by thepolynucleotide ..."(D) Table 1 (Lines 1 through 2 on page 110)
The following sequence: SEQ NO (DNA) "1401": SEQ NO (a. a.) "4901"; Initial (nt)"1337567"; Terminal (nt) "1336095"; ORF (bp) "1473"; db Match "sp: YILV＿CORGL";
Homologous gene "Corynebacterium glutamicum ATCC13032 yilV"; Identity (%)"100.0"; Similarity (%) "100.0"; Matched length (a. a.) "62"; Function "hypotheticalmembrane protein."
B. According to evidence (Exhibit Ko 4-2, Exhibits Otsu 42 and 43) and the entire
import of oral arguments, the sequence mentioned in A. (D) above, which is described in
Exhibit Otsu 42 Document, is the sequence of the polypeptide of SEQ ID NO: 4901
encoded by the gene of SEQ ID NO: 1401, and this polypeptide sequence is found tocorrespond to the amino acid sequence of SEQ ID NO: 84 consisting of 533 amino acids
mentioned in Description 2, which is a sequence of a yggB gene derived from
Corynebacterium glutamicum, excluding the amino acids at positions 1 through 42.
C. The Defendant alleges that the sequence of SEQ ID NO: 4901 in Exhibit Otsu 42
Document discloses the amino acid sequence described in (ii) of Claim 1 of Patent 2 (thestatement after Correction 2 is as follows: "(ii) substitution, deletion, or insertion of one
to five amino acids in a region selected from the group consisting of amino acid numbers
1 to 23, 86 to 108, and 110 to 132 of an amino acid sequence of SEQ ID NO: 6, 62, 68,
84, or 85.").
However, as mentioned in B. above, the sequence of SEQ ID NO: 4901 in Exhibit
Otsu 42 Document has excluded all amino acids at positions 1 through 42 from the aminoacid sequence of SEQ ID NO: 84 in Description 2, and therefore it does not constitute theabovementioned amino acid sequence in (ii) of Claim 1 after Correction 2.
Consequently, without having to make determinations on other points, it cannot be
said that Corrected Invention 2-5 citing Claim 1 or Claim 10 includes the same invention
as Exhibit Otsu 42 Invention, and hence a ground for invalidation, i.e. lack of novelty,(Article 123, paragraph (1), item (ii) and Article 29, paragraph (1), item (iii) of the Patent
Act) based on Exhibit Otsu 42 Invention does not exist with regard to Corrected Invention
2-5.
(4) Whether or not Corrected Invention 2 lacks an inventive step based on Exhibit Otsu
24 Invention as the primary cited document
A. Regarding Exhibit Otsu 24 Invention
The contents described in Exhibit Otsu 24 Document are as mentioned in 4. (2) D.
(A) above, and as examined in 4. (2) D. (B) above, although Exhibit Otsu 24 Document101

suggests that Corynebacterium glutamicum has osmoregulated channels having similar
characteristics as mechanosensitive channels of E. coli, it does not show that theosmoregulated channels contribute to efflux of glutamic acid. Rather, it states that thechannels were unrelated to efflux of glutamic acid, and derived a conclusion that effluxof glutamic acid is mediated not by osmoregulated channels, but by a carrier system as
had been indicated before.
Consequently, it is reasonable to find that Exhibit Otsu 24 Document discloses aninvention of "Corynebacterium glutamicum having osmoregulated channels for whichsimilarity with mechanosensitive channels of E. coli is suggested" (Exhibit Otsu 24
Invention), and the Defendant's allegation that Exhibit Otsu 24 Invention discloses"Corynebacterium glutamicum having osmoregulated channels, related to efflux ofglutamic acid" cannot be adopted.
B. Regarding involvement of an inventive step in Corrected Invention 2-5 citing Claim 1(A) Comparison between Exhibit Otsu 24 Invention and Corrected Invention 2-5 citing
Claim 1
The following are found as a result of comparing Exhibit Otsu 24 Invention with
Corrected Invention 2-5 citing Claim 1.
a. Common feature
The bacteria mentioned in Exhibit Otsu 24 Invention and Claim 1 are both coryneformbacteria having osmoregulated channels.
b. Differences
Exhibit Otsu 24 Invention and Corrected Invention 2-5 citing Claim 1 differ in thefollowing respects.
Difference 1: Corrected Invention 2-5 is an invention of a method of producing
glutamic acid comprising the steps of cultivating a coryneform bacterium in a medium,to allow glutamic acid to be produced and accumulated in the medium or within thebacterial cell, and collecting it from the medium or the bacterial cell, whereas Exhibit
Otsu 24 Invention is an invention of a coryneform bacterium.
Difference 2: In Corrected Invention 2-5, a mutant-type yggB gene is introduced into
the coryneform bacterium, and this mutant-type yggB gene is one into which any of themutations mentioned in (i), (i'), (i''), or (ii) of Claim 1 has been introduced, indicating an
improvement in the glutamic acid-producing ability compared to a non-modified strain,
whereas in the coryneform bacterium of Exhibit Otsu 24 Invention, the yggB gene is not
specified, and thus the glutamic acid-producing ability is not specified.
(B) Ease in conceiving of Difference 1
There is no dispute between the parties concerning the fact that a person skilled in the102

art could have easily conceived of Difference 1 as of Priority Date 2.
(C) Ease in conceiving of Difference 2
According to the results of the examination made in 4. (2) D. above, the fact that
osmoregulated channels contribute to efflux of glutamic acid in coryneform bacteria
including Corynebacterium glutamicum is not found to have been well-known to persons
skilled in the art as of Priority Date 2, and Exhibit Otsu 24 Document also does not statethat osmoregulated channels contribute to efflux of glutamic acid. Therefore, it had beenconcluded that efflux of glutamic acid is mediated not by osmoregulated channels, but by
a carrier system.
The Defendant alleges that a person skilled in the art could have easily conceived ofadopting the configuration relating to Difference 2 by applying Exhibit Otsu 26
Document, Exhibit Otsu 29 Document, Exhibit Otsu 30 Invention, Exhibit Otsu 31
Invention, and Exhibit Otsu 41 Document as well as common general technicalknowledge to Exhibit Otsu 24 Invention. However, the abovementioned documents andinventions contain no statements that suggest efflux of glutamic acid from osmoregulatedchannels in coryneform bacteria (Exhibits Otsu 26, 29 through 31, and 41), and as
mentioned above, such well-known art or common general technical knowledge is notfound to have existed as of Priority Date 2.
If so, it should be said that there is no motivation in Exhibit Otsu 24 Invention for a
person skilled in the art to pay attention to the yggB gene that encodes osmoregulatedchannels in coryneform bacteria by applying the abovementioned documents andinventions as well as common general technical knowledge; thus, a person skilled in theart could not have easily conceived of the configuration relating to Difference 2 as ofPriority Date 2.
(C) Accordingly, it cannot be said that a person skilled in the art could have easily
conceived of Corrected Invention 2-5 citing Claim 1 based on Exhibit Otsu 24 Invention
as the primary cited document.
C. Regarding involvement of an inventive step in Corrected Invention 2-5 citing Claim 4,
Claim 6, or Claim 10
Claim 4 after Correction 2 is an invention of a coryneform bacterium into which amutant-type yggB gene having a mutation that is included in the mutation mentioned in(ii) of Claim 1 has been introduced, while Claim 6 and Claim 10 after Correction 2 areinventions of coryneform bacteria that have further restricted Claim 1. Therefore,
according to the results of the examination made in A. above, similar to the case of
Corrected Invention 2 citing Claim 1, it cannot be said that a person skilled in the art
could have easily conceived of any of Corrected Invention 2-5 citing Claim 4, Claim 6,
103

or Claim 10 based on Exhibit Otsu 24 Invention as the primary cited document.
D. Consequently, a ground for invalidation, i.e. lack of an inventive step, (Article 123,
paragraph (1), item (ii) and Article 29, paragraph (2) of the Patent Act) based on Exhibit
Otsu 24 Invention as the primary cited document is not found for Corrected Invention 25 citing Claim 1, Claim 4, Claim 6, or Claim 10.
(5) Whether or not Corrected Invention 2 violates the enablement requirement or the
support requirement
A. Regarding culture conditions(A) Regarding Working Example 8 and Working Example 10
As mentioned in 4. (3) B. (B) above, Working Example 8 indicates that under acondition with an excess of biotin (a non-inducing condition), the glutamic acidproduction increases as compared to a non-modified strain due to the configuration using
the 19-type mutation included in Corrected Invention 2-5, and the experiment in Exhibit
Otsu 44 performed by the Defendant does not overturn the credibility of the experimentresults in Working Example 8.
In addition, as mentioned in 4. (3) B. (A) above, although Working Example 10
indicates that the glutamic acid production increases under an inducing condition due tothe configuration using the 19-type mutation, it does not indicate that such effect is only
observed under an inducing condition (paragraphs [0125] through [0128]).
(B) Regarding a violation of the support requirement
The problem to be solved by Corrected Invention 2-5 is "to provide novel art toimprove the L-glutamic acid-producing ability of a coryneform bacterium when Lglutamic acid is produced by using the coryneform bacterium" (paragraph [0007]).
As mentioned in (A) above, it can be said that Working Examples 8 and 10 disclosethat the configuration using the 19-type mutation included in Corrected Invention 2-5
improves the glutamic acid-producing ability not only under an inducing condition, butalso under a non-inducing condition, and there is no other evidence sufficient to find thatthe abovementioned problem can be solved only under an inducing condition.
Consequently, it cannot be said, based on the fact that use of an inducing condition as
the culture condition is not required, that Corrected Invention 2-5 falls beyond the scopein which a person skilled in the art can recognize that the problem to be solved by theinvention can be solved in light of the statements in the detailed explanation of theinvention and the common general technical knowledge as of the time of the filing of theapplication. Therefore, the Defendant's allegation of a violation of the support
requirement relating to this point is groundless.
(C) Regarding a violation of the enablement requirement104According to the results of the examination made in 4. (3) B. above, it can be said thata person skilled in the art can work the invention relating to the configuration using the
19-type mutation without undergoing excessive trial and error based on the statements on
culture conditions in Description 2 and common general technical knowledge as of thetime of the filing of the application, not only under an inducing condition, but also undera non-inducing condition.
Also with regard to other configurations included in Corrected Invention 2-5, nocircumstances are found in terms of culture conditions which suggest that a person skilledin the art cannot achieve those configurations under a non-inducing condition based on
the statements on culture conditions in Description 2 and common general technicalknowledge as of the time of the filing of the application, and hence the Defendant's
allegation of a violation of the enablement requirement relating to this point is groundless.
B. Regarding the mutations mentioned in (i) and (i') of Claim 1(A) According to the results of the examination made in 4. (2) above, the technical ideaof Corrected Invention 2 is found to be to promote efflux of glutamic acid and improve
the glutamic acid-producing ability by focusing on the fact that the YggB protein, which
is an osmoregulated channel encoded by the yggB gene of a coryneform bacterium, isinvolved in efflux of glutamic acid, and by modifying the YggB protein by using amutant-type yggB gene into which a C-terminal side mutation or mutations intransmembrane regions are introduced. Meanwhile, the mutations mentioned in (i) and(i') of Claim 1 introduce a mutation into the part of the mutant-type yggB gene that
encodes the C-terminal side region (paragraph [0070]).
(B) Description 2 provides an explanation on the C-terminal side mutation in general inparagraph [0070], and as its example, it describes in paragraph [0071] a 2A-1-type
mutation wherein the amino acid sequence in a region positioned downstream fromposition 419 in the C-terminal side of the amino acid sequence (positions 419 through533 of the amino acid sequence of SEQ ID NO: 6) is substituted with a sequence
consisting of five amino acids which is derived from a short insertion sequence, and statesthat the 2A-1-type mutation includes a mutation wherein the region positioned
downstream from position 419 in the C-terminal side of SEQ ID NOs: 6, 62, 68, 84 and
85 is deleted or substituted. Further, Working Examples 5 and 6 in Description 2 state that
it was confirmed that a glutamic acid-producing ability was improved in the 2A-1-type
mutation wherein the region positioned downstream from position 419 in the C-terminalside is substituted with five amino acids (paragraphs [0107] through [0113]).
(C) Based on the basic technical idea mentioned in (A) above, it is found that a person
skilled in the art who read the statements in Description 2 referred to in (B) above would
105

recognize that, with regard to the mutations mentioned in (i) and (i') of Claim 1, the threedimensional structure of the YggB protein would be modified by introduction of amutation such as deletion or substitution, in a region positioned downstream fromposition 419 in the C-terminal side, to thereby improve the glutamic acid-producing
ability.
Although the Defendant alleges that Description 2 contains no statements on acoryneform bacterium into which the mutation mentioned in (i) of Claim 1 was introduced,
as mentioned above, the 2A-1-type mutation that was introduced in Working Examples 5
and 6 is a mutation wherein 115 amino acids at positions 419 through 533 in the Cterminal side region have been substituted with five amino acids, and in view of theembodiment of the mutation, it can be said that a person skilled in the art can recognizethat even if the region positioned downstream from position 419 in the C-terminal side
region is deleted, the three-dimensional structure of the YggB protein in the part encodedby amino acids in the C-terminal side region would be similarly modified, and theglutamic acid-producing ability would be improved.
In addition, the Defendant alleges that it is not possible to understand that the
mutations in (i') of Claim 1 other than the 2A-1-type mutation mentioned in Working
Examples 5 and 6 also have the same effect. However, as mentioned above, it can be said
that a person skilled in the art can recognize, based on the statements in Description 2,
the basic principle that the glutamic acid-producing ability is improved due to a change
in the three-dimensional structure of the YggB protein caused by, for example, insertionof an insertion sequence to the region positioned downstream from position 419 in the Cterminal side.
Consequently, with regard to coryneform bacteria of which glutamic acid-producing
ability has been improved by introduction of the mutations mentioned in (i) and (i') of
Claim 1, it cannot be said that Corrected Invention 2-5 citing Claim 1 falls beyond thescope in which a person skilled in the art can recognize that the problem to be solved by
the invention can be solved in light of the statements in the detailed explanation of theinvention. Therefore, the Defendant's allegation of a violation of the support requirement
relating to this point is groundless. In addition, it can be said that a person skilled in theart can obtain the abovementioned coryneform bacteria of which glutamic acid-producing
ability has been improved and work Corrected Invention 2-5 without undergoingexcessive trial and error based on the abovementioned statements in Description 2, andhence, it cannot be said that the invention fails to fulfill the enablement requirement.
C. Regarding the mutation mentioned in (i'') of Claim 1(A) Paragraph [0072] of Description 2 discloses 12 substitutable prolines in the region106

positioned downstream from position 419 in the C-terminal side of the yggB gene, andthat it is considered that these prolines play an important role in maintaining the threedimensional structure of the YggB protein. In addition, Working Examples 12 and 13 in
Description 2 state that a mutant strain was produced wherein proline at position 424 in
the amino acid sequence of SEQ ID NO: 68 was substituted with leucine (66-type
mutation), and proline at position 437 in the amino acid sequence of SEQ ID NO: 6 wassubstituted with serine (22-type mutation), and that the 22-type mutant strain mentionedin Working Example 13 actually improved the glutamic acid-producing ability(paragraphs [0130] through [0138]).
Moreover, according to evidence (Exhibits Ko 44 through 46) and the entire importof oral arguments, it is found to have been well-known to persons skilled in the art thatproline is generally an amino acid having a special property which is involved in theformation of the three-dimensional structure of a protein.
(B) It can be said that, based on the statements in Description 2 mentioned in (A) above
and common general technical knowledge concerning the property of proline, as well as
the basic technical idea of Corrected Invention 2 mentioned in B. (A) above, a person
skilled in the art can recognize that, by substituting the proline in the region positioned
downstream from position 419 in the C-terminal side with another amino acid, the threedimensional structure of the YggB protein in the part encoded by amino acids in that
region would be modified, and the glutamic acid-producing ability would be improved.
Consequently, with regard to coryneform bacteria of which glutamic acid-producing
ability has been improved by introduction of the mutation mentioned in (i'') of Claim 1,
it cannot be said that Corrected Invention 2-5 citing Claim 1 falls beyond the scope inwhich a person skilled in the art can recognize that the problem to be solved by theinvention can be solved in light of the statements in the detailed explanation of theinvention. In addition, it can be said that a person skilled in the art can obtain theabovementioned coryneform bacteria of which glutamic acid-producing ability has been
improved and work Corrected Invention 2-5 without undergoing excessive trial and error
based on the abovementioned statements in Description 2. Therefore, the invention is notfound to violate the support requirement or the enablement requirement in relation to thispoint.
The Defendant indicates among other matters that while many prolines could be
subject to substitution in the mutation mentioned in (i'') of Claim 1, the glutamic acidproductivity has been actually confirmed only for the 22-type mutation mentioned in
Working Example 13, and that the result could differ if proline at a different position was
substituted from among the abovementioned prolines. However, given that the basic
107

principle has been clarified and that the substitutable prolines are specifically disclosed
in Description 2 as mentioned above, even by taking the Defendant's allegation into
consideration, the invention is not found to violate the support requirement or the
enablement requirement, as mentioned above.
D. Regarding the mutation mentioned in (ii) of Claim 1 (a mutation in the transmembraneregion)(A) The mutation mentioned in (ii) of Claim 1 is a mutation to be introduced into theyggB gene wherein one to five amino acids are substituted, deleted, or inserted in thetransmembrane region.
Paragraph [0073] of Description 2 states, as generalities concerning mutations in thetransmembrane region, that the YggB protein is presumed to have five transmembraneregions, that these transmembrane regions correspond to amino acid numbers 1 to 23 (thefirst transmembrane region), amino acid numbers 86 to 108 (the fourth transmembraneregion), and amino acid numbers 110 to 132 (the fifth transmembrane region) in the aminoacid sequences of SEQ ID NOs: 6, 62, 68, 84, and 85, which are stated in (ii) of Claim 1
as regions into which mutations should be introduced, as well as the details of themutations to be introduced into the transmembrane regions.
As specific examples of the mutations, Description 2 mentions the A1-type mutation
as a mutation in the first transmembrane region, the 19-type mutation as a mutation in the
fourth transmembrane region, and the L30-type mutation and the 8-type mutation as
examples of mutations in the fifth transmembrane region (paragraphs [0074] through[0076]). Moreover, Description 2 states the following: as Working Example 7, a methodof producing a strain into which the A1-type mutation has been introduced and the factthat the glutamic acid-producing ability has been improved in this strain (paragraphs[0114] through [0118]); as Working Examples 8 and 10, a method of producing a strain
into which the 19-type mutation has been introduced and the fact that the glutamic acidproducing ability has been improved in this strain (paragraphs [0119] through [0121] and[0125] through [0128]); as Working Example 9, a method of producing a strain into which
the L30-type mutation has been introduced and the fact that the glutamic acid-producing
ability has been improved in this strain (paragraphs [0122] through [0124]); and as
Working Example 11, a method of producing a strain into which the 8-type mutation has
been introduced (paragraph [0129]).
(B) It can be said that, based on the statements in Description 2 mentioned in (A) above
and the basic technical idea of Corrected Invention 2 mentioned in B. (A) above, a person
skilled in the art can recognize the basic principle for solving the problem, which is adding
modification to the transmembrane region of the YggB protein which forms the108osmoregulated channel relating to efflux of glutamic acid to promote efflux of glutamic
acid and improve the glutamic acid-producing ability.
Considering that Description 2 discloses working examples in which the glutamic
acid-producing ability was improved for three transmembrane regions, and that (ii) of
Claim 1 restricts the regions into which the mutation should be introduced and also
restricts the number of amino acids to be substituted, deleted, or inserted to one to five,with regard to coryneform bacteria of which glutamic acid-producing ability has been
improved by introduction of the mutation mentioned in (ii) of Claim 1, it cannot be said
that Corrected Invention 2-5 citing Claim 1 falls beyond the scope in which a person
skilled in the art can recognize that the problem to be solved by the invention can besolved in light of the statements in the detailed explanation of the invention. In addition,
it can be said that a person skilled in the art can obtain the abovementioned coryneformbacteria of which glutamic acid-producing ability has been improved and work Corrected
Invention 2-5 without undergoing excessive trial and error based on the abovementioned
statements in Description 2. Therefore, the invention is not found to violate the support
requirement or the enablement requirement in relation to this point.
E. Regarding SEQ ID NO: 85(A) The mutations mentioned in (i), (i'), (i''), or (ii) of Claim 1 include not only those that
use the amino acid sequences of SEQ ID NOs: 6, 62, 68, and 84, but also those that use
the amino acid sequence of SEQ ID NO: 85 of the yggB gene before mutation.
Paragraph [0034] of Description 2 states that the amino acid sequence of the yggB
gene into which a mutation is to be introduced may be a gene having an amino acidsequence in which one or several amino acids are substituted, deleted, etc. in an aminoacid sequence of SEQ ID NO: 6, 62, 68, or 84, and that, in the case of substitution, it
should preferably be a conservative substitution, while showing specific examples ofconservative substitution of the respective types of amino acids. Then, paragraph [0035]
discloses SEQ ID NO: 85 as a sequence wherein 14 locations of amino acids in the aminoacid sequence of SEQ ID NO: 6 that may be substituted or deleted are indicated by Xaa.
In addition, Description 2 also has statements on conservative substitution in paragraphs[0078] and [0079].
Furthermore, according to evidence (Exhibit Ko 48), it is found to have been wellknown to persons skilled in the art as of the time of the filing of the application that, as aproperty of protein, the functions of the protein are maintained even if substitution
between amino acids having similar size and similar polarity (conservative substitution)
occurs at a site other than an active center of a protein or a site of functional importancein the protein.
109

(B) In light of the statements in Description 2 mentioned in (A) above and common
general technical knowledge as of the time of the filing of the application, a person skilledin the art can recognize that the locations indicated by Xaa in SEQ ID NO: 85 are sitesthat are not of functional importance in the protein and that may be substituted or deleted
as mentioned above, and recognize a certain scope of substitutions that are permissible,
based on the abovementioned significance of conservative substitution. If so, it can besaid that even if Description 2 contains no working example in which a mutation has been
added to the amino acid sequence of SEQ ID NO: 85, given the points examined in B.
through D. above, a person skilled in the art can recognize that the glutamic acidproducing ability can also be improved by introducing the respective mutations
mentioned in Claim 1 into the amino acid sequence of SEQ ID NO: 85, similar to the caseof the amino acid sequences of the other SEQ ID Nos listed in Claim 1.
Considering that the amino acids that can be substituted, etc. from SEQ ID NO: 6(Xaa) are concretely specified for SEQ ID NO: 85, and details of desirable substitutions
are disclosed in Description 2 as mentioned in (A) above, with regard to coryneformbacteria of which glutamic acid-producing ability has been improved by introduction of
the respective mutations mentioned in Claim 1 into the amino acid sequence of SEQ ID
NO: 85 as well, it cannot be said that Corrected Invention 2-5 citing Claim 1 falls beyond
the scope in which a person skilled in the art can recognize that the problem to be solvedby the invention can be solved in light of the statements in the detailed explanation of theinvention and common general technical knowledge as of Priority Date 2. In addition, it
can be said that a person skilled in the art can obtain the abovementioned coryneformbacteria of which glutamic acid-producing ability has been improved and work Corrected
Invention 2-5 without undergoing excessive trial and error based on the abovementioned
statements in Description 2 and common general technical knowledge as of the time of
the filing of the application. Therefore, the invention is not found to violate the support
requirement or the enablement requirement in relation to this point.
F. Regarding the mutation mentioned in Claim 4
The mutations to be introduced in Claim 4 after Correction 2 are included in themutations mentioned in (ii) of Claim 1.
In addition to the point examined in D. above, with regard to Claim 4, Correction 2
further restricts the amino acids to be substituted from alanine at position 100 or 111.
Given that the 19-type mutation mentioned in Working Examples 8 and 10 is disclosedwith regard to a mutation that substitutes alanine at position 100 with threonine, the 8type mutation mentioned in Working Example 11 is disclosed with regard to a mutationthat substitutes alanine at position 111 with threonine, and the L30-type mutation
110

mentioned in Working Example 9 with regard to a mutation that substitutes alanine atposition 111 with valine, all of which are mutations included in Claim 4, Corrected
Invention 2-5 citing Claim 4 is also not found to violate the support requirement or the
enablement requirement. According to the results of the examination made in E. above,the same applies to the part of the mutation mentioned in Claim 4 which introduces amutation into the amino acid sequence of SEQ ID NO: 85.
G. Regarding the mutation mentioned in Claim 6(A) The amino acid sequences of SEQ ID NOs: 8, 20, 22, 24, 64, 70, and 74 mentionedin (a), (c), (e), (g), (i), (k), and (m) of Claim 6 respectively correspond to the amino acidsequences encoded by the yggB gene after the 2A-1-type mutation mentioned in Working
Example 6, the A1-type mutation mentioned in Working Example 7, the 19-type mutation
mentioned in Working Examples 8 and 10, the L30-type mutation mentioned in Working
Example 9, the 8-type mutation mentioned in Working Example 11, the 66-type mutation
mentioned in Working Example 12, and the 22-type mutation mentioned in Working
Example 13.
The Defendant alleges that, with regard to the mutant-type yggB genes mentioned in(b), (d), (f), (h), (j), (l), and (n) of Claim 6 wherein deletion, etc. of one to five aminoacids has been further added to the respective amino acid sequences after these mutations,
it is not possible to understand what kind of mutant protein would improve the glutamic
acid productivity in a medium containing an excess of biotin as a result of adding
substitution, etc. to one to five amino acids at arbitrary positions.
However, as mentioned in E. (A) above, it is found to have been well-known topersons skilled in the art as of the time of the filing of the application that, as a property
of protein, the functions of the protein are maintained even if substitution between aminoacids having similar size and similar polarity (conservative substitution) occurs at a siteother than an active center of a protein or a site of functional importance in the protein.
In addition, paragraphs [0078] and [0079] of Description 2 state that a mutation that may
be added to the amino acid sequence of SEQ ID NO: 8, 20, 22, 24, 64, 70, or 74 ispreferably conservative substitution, while showing specific examples of conservative
substitution that may be added to these amino acid sequences.
(B) Considering the statements in Description 2 mentioned in (A) above and common
general technical knowledge, the points examined in B. through D. above, as well as thefact that, regarding the mutant-type yggB genes mentioned in (b), (d), (f), (h), (j), (l), and(n) of Claim 6, the number of amino acids that may be substituted from the amino acidsequence of SEQ ID NO: 8, 20, 22, 24, 64, 70, or 74 are restricted to one to five, withregard to coryneform bacteria of which glutamic acid-producing ability in the case of111

being cultured in a medium containing an excess of biotin has been improved byintroduction of the respective mutant-type yggB genes mentioned above, it cannot be said
that Corrected Invention 2-5 citing Claim 6 falls beyond the scope in which a person
skilled in the art can recognize that the problem to be solved by the invention can besolved in light of the statements in the detailed explanation of the invention and common
general technical knowledge as of the time of the filing of the application. In addition, it
can be said that a person skilled in the art can obtain the abovementioned coryneformbacteria of which glutamic acid-producing ability in the case of being cultured in a
medium containing an excess of biotin has been improved and work Corrected Invention
2-5 without undergoing excessive trial and error based on the abovementioned statements
in Description 2 and common general technical knowledge as of Priority Date 2.
Therefore, the invention is not found to violate the support requirement or the enablement
requirement in relation to this point.
H. Regarding the mutation mentioned in Claim 10
Claim 10 adds a restriction so that the coryneform bacteria into which the mutanttype yggB genes mentioned in Claims 1, 4, and 6 are to be introduced are limited tobacteria belonging to the genus Corynebacterium or the genus Brevibacterium.
Paragraphs [0012] and [0013] of Description 2 state that the coryneform bacteria
covered by the invention include, in addition to a bacterium belonging to the genus
Corynebacterium, a bacterium which has been conventionally classified into the genus
Brevibacterium but is currently classified into the genus Corynebacterium, and also
Brevibacterium bacteria which are very closely related to the genus Corynebacterium
bacteria, and indicate specific examples of these bacteria. In addition, the working
examples also contain examples in which a mutant-type yggB gene has been introduced
into Corynebacterium glutamicum and Brevibacterium flavum, both of which are
currently classified into the genus Corynebacterium (paragraph [0119] of Working
Example 8, paragraph [0129] of Working Example 11, etc.).
According to these statements in Description 2 and the results of the examination
made in B. through G. above, Corrected Invention 2-5 citing Claim 10 is also not foundto violate the support requirement or the enablement requirement.
I. Regarding the method of producing and collecting glutamic acid (Claim 11)
The Defendant alleges, with regard to Invention 2-5 before correction, that
Description 2 contains no statements about allowing glutamic acid to be produced in themedium and about collecting glutamic acid from the bacterial cell, and that the invention
therefore violates the enablement requirement and the support requirement in relation tothis point. However, the Defendant has not asserted a ground for invalidation in relation112

to this point regarding Corrected Invention 2-5.
In Claim 11 after Correction 2, the statement about collecting glutamic acid from thebacterial cell has been deleted, so violation of the enablement requirement and the support
requirement in relation to this point does not present a problem with regard to Corrected
Invention 2-5. Meanwhile, as to the point to cultivate a coryneform bacterium in a
medium and "to allow L-glutamic acid to be produced and accumulated in the medium"(Claim 11 after Correction 2), according to the statements in paragraphs [0085] through[0090] of Description 2 and evidence (Exhibits Ko 8 and 9, Exhibit Otsu 2) as well as theentire import of oral arguments, it is found to have been a well-known matter for persons
skilled in the art as of Priority Date 2.
Consequently, Corrected Invention 2-5 is not found to violate the enablement
requirement or the support requirement with regard to the method of producing andcollecting glutamic acid.
(6) Whether or not Corrected Invention 2 violates the clarity requirement
The Defendant alleges, with regard to "an excess of biotin" mentioned in Claim 6 ofPatent 2, that the amount intended by the phrase "an excess" is unknown.
However, paragraph [0032] of Description 2 states "'a condition with an excess of
biotin' means, for example, a condition in which the medium contains biotin in an amountof 30 μg/L or more, preferably 40 μg/L, and more preferably 50 μg/L." As it is possible
to understand the meaning and details of "an excess of biotin" from the statements in
Description 2, the statements on this point do not violate the clarity requirement (Article
36, paragraph (6), item (ii) of the Patent Act).
Consequently, Corrected Invention 2-5 citing Claim 6 does not contain the ground forinvalidation, i.e. a violation of the clarity requirement.
(7) Summary on the defense of invalidity concerning Patent Right 2
As mentioned above, Correction 2 satisfies the requirements for correction, and the
Defendant's Production Processes all fall within the technical scope of Corrected
Invention 2-5.
Moreover, with regard to the respective grounds for invalidation asserted by the
Defendant with regard to Invention 2-5 and Corrected Invention 2-5, no ground forinvalidation is found to exist at least for Corrected Invention 2-5 after correction. Thus,
all of the requirements for re-defense of correction are satisfied, and therefore, without
having to make determinations on other points, all of the Defendant's defenses ofinvalidity based on Article 104-3, paragraph (1) of the Patent Act against claims based on
Patent Right 2 are groundless.
8. Regarding Issue 8 (occurrence or non-occurrence and the value of damages)
113

(1) Organization of the periods of use of the Defendant's Production Processes and thecorrespondence with the Patent Rights
A. As mentioned in 1. (4) above, with regard to use or non-use of the Defendant's
Production Processes in each period during the subject period, Defendant's Production
Process 1 was used in the periods of use of Strains [ii] and [x] described in Attachment 9"Comparison Table of Allegations concerning Processes for Producing the MSG in
Question," and the processes used in the other periods are as described in the "Plaintiff's
alternative claims" column in that Attachment.
Consequently, the periods of use of the Defendant's Production Processes can beorganized as follows.
(A) Defendant's Production Process 1
From January 2011 through May 2014
From August 2015 through June 2016
(B) Defendant's Production Process 2
From April through June, 2014, and November and December of that year
June 2015
From May through August, 2016
(C) Defendant's Production Process 3
From June 2014 through November 2015
(D) Defendant's Production Process 4
From July 2016 through December 2017
B. According to 2., 5. (3), and 6. (9) above, the Defendant's Production Processes against
which claims can be filed based on Patent Right 1 are Defendant's Production Processes
1 and 3, whereas according to 3., 4. (11), and 7. (7) above, claims based on Patent Right
2 can be filed against all of the Defendant's Production Processes.
If so, the relation of the Patent Rights with the Defendant's Production Processes
against which rights can be exercised based thereon and with the Defendant's Products
produced by those processes can be organized in the order of the time of production as
follows. The MSG in question produced during the period from 2011 through 2017
(hereinafter referred to as the "period of use of the Defendant's Production Processes")
represents products produced by any of the Defendant's Production Processes (theDefendant's Products), and rights can be exercised against them based on Patent Right 1
or 2.
(A) January 2011 through August 22, 2013
Defendant's Production Process 1 was used during this period. As this was before
registration of establishment of Patent Right 2, rights can be exercised based on Patent
114

Right 1 against Defendant's Product 1.
(B) August 23, 2013 (the date of registration of establishment of Patent Right 2) throughJune 2016
Defendant's Production Process 1 or 3 was used throughout this period. Rights can be
exercised based on Patent Rights 1 and 2 against Defendant's Product 1 or 3 produced byeither of these processes.
Defendant's Production Process 2 was additionally used during a part of this period.
Rights can be exercised based on Patent Right 2 against Defendant's Product 2 producedby this process.
(C) July 2016 through December 2017
Defendant's Production Process 2 or 4 was used throughout this period. Rights can be
exercised based on Patent Right 2 against Defendant's Product 2 or 4 produced by eitherof these processes.
(2) Scope of working of inventions for which the Defendant is liable for tort
A. Finding of facts
According to the basic facts, etc. in No. 2, 2 above, the evidence shown below, andthe entire import of oral arguments, the following facts are found with regard to
production of the MSG in question and the sale thereof to Japanese customers during theperiod of use of the Defendant's Production Processes.
(A) Relationship of the CJ GroupThe Defendant and CJ Indonesia belong to the CJ Group, CJ Indonesia is a wholly
owned subsidiary company of CJCJ, the central company of the CJ Group, and the
Defendant is a wholly owned subsidiary company of CJ Company, a holding company,
holding 44% of CJCJ's shares (No. 2, 2. (1) B. above).
In the CJ Group, the group companies share roles in producing and selling CJ-brandand other products in countries around the world. According to the website of the bio
section of the CJ Group, the section's production bases are located in Asia, North America,and South America, and the factory of CJ Indonesia in Indonesia is introduced as one of
the section's production bases, but the section has no production base in Japan. The
website indicates that the section's sales bases are located in Asia, Europe, North America,
South America, the Middle East, and North Africa and that CJCJ in South Korea is theheadquarters, and introduces CJ Indonesia in Jakarta and the Defendant in Tokyo as someof the section's sales bases in Asia (Exhibit Ko 97).
CJCJ has a research and development section, and the strain used for the productionof MSG in question in CJ Indonesia's factory in Indonesia during the period of use of the
Defendant's Production Processes was a strain that had been developed by CJCJ and sent115

to CJ Indonesia (Exhibits Otsu 1, 2, and 5).
(B) Portion sold by the Defendant
During the period of use of the Defendant's Production Processes, the Defendantpurchased, from CJ Indonesia, the MSG in question that had been produced in Indonesia
by CJ Indonesia, imported it into Japan from Indonesia, and both offered to transfer it andtransferred it in Japan (the portion sold by the Defendant in No. 2, 2. (7) A. above;
Exhibits Otsu 101, 104, 111, and 113).
(C) Portion sold by CJ Indonesia
During the period of use of the Defendant's Production Processes, CJ Indonesia soldthe MSG in question which it had produced by itself in Indonesia to Japanese customers
as a seller, separately from the portion sold by the Defendant (the portion sold by CJ
Indonesia in No. 2, 2. (7) A. above; Exhibit Otsu 101). The specific mode of thistransaction was as follows.
a. Upon conclusion of sales contracts for the portion to be sold by CJ Indonesia, the orderforms from Japanese customers contained a statement that the seller of the MSG inquestion was CJ Indonesia (Exhibits Otsu 102-1 and 103-1).
However, an order form to be sent to CJ Indonesia was sometimes sent to the
Defendant by a Japanese customer, and in such case, the Defendant sent that order form
to CJ Indonesia (Exhibit Otsu 103-1).
b. With regard to the portion to be sold by CJ Indonesia, the MSG in question was shipped
in Indonesia and transported to Japan with CJ Indonesia as the shipper. The invoices andbills of lading, etc. relating to that transportation also stated that the shipper was CJ
Indonesia.
Upon transportation of the MSG in question to Japan, the invoices relating to thetransportation stated that the terms and conditions between CJ Indonesia and Japanesecustomers were to be cost, insurance, and freight (CIF) or cost and freight (CFR) tradeterms. Generally, the terms and conditions of CIF require the seller to bear the cost oftransporting the products to the port of discharge and the insurance cost, whereas thoseof CFR require the seller to bear only the cost of transporting the products to the port ofdischarge. In both cases, once the seller completes the export and customs clearance
procedures and shipping at the port of export, the products are transferred to the buyer,
and at that time, the risk burden is also transferred from the seller to the buyer (Exhibits
Ko 104 and 105, Exhibits Otsu 45, 46, 102, 103, and 106).
c. Between the Defendant and CJ Indonesia, there was a commission contract under which
CJ Indonesia was to pay the Defendant an amount equivalent to ●(omitted)●% or ●(omitted)●% of the sales amount of the portion sold by CJ Indonesia as a commission116

(hereinafter the contract is referred to as the "Commission Contract" and the commission
under this contract is referred to as the "Commission"), during the period of use of the
Defendant's Production Processes. The total amount of the Commission paid from 2011
through 2017, which is the period of use of the Defendant's Production Processes, is ●(omitted)● yen (Exhibit Otsu 101).
d. As work related to transactions of the portion sold by CJ Indonesia, the Defendant atleast engaged in delivery of samples and recovery of defective products of the MSG inquestion at various times, in addition to the forwarding of order forms mentioned in a.
above, in Japan (Exhibit Otsu 101).
(D) The Defendant's accounting of the selling, general, and administrative expenses forthe portion sold by the Defendant, etc.
a. During the period of use of the Defendant's Production Processes, the Defendant
recorded, in accounting, expenses relating to the portion sold by the Defendant as well as
expenses relating to the portion sold by CJ Indonesia under the expense items "freight
expenses," "warehouse expenses," "personnel expenses," "service expenses," and"others," as selling, general, and administrative expenses concerning the sale of the MSG
in question. When comparing the total amounts of the abovementioned selling, general,and administrative expenses that were recorded during the period of use of the
Defendant's Production Processes, the ratio between the selling, general, andadministrative expenses relating to the portion sold by the Defendant and those relating
to the portion sold by CJ Indonesia was roughly 4:3 (Exhibit Otsu 101).
b. "Freight expenses" and "warehouse expenses"
In the Defendant's accounting, the standards based on which the "freight expenses"
and the "warehouse expenses" concerning the sale of the MSG in question were to be
distributed between the portion sold by the Defendant and the portion sold by CJ
Indonesia were instructed by CJCJ.
Specifically, with regard to "freight expenses," all transportation expenses concerning
the MSG in question were proportionally distributed according to the sales amount of the
portion sold by the Defendant and the total amount of Commission until the middle of2013. Subsequently, the transportation expenses were recorded as expenses for the portion
sold by the Defendant, in principle, and only the expenses corresponding to indirect
expenses for delivery of samples and recovery of defective products, etc. were
proportionally distributed according to the sales amount of the portion sold by the
Defendant and the total amount of Commission. Meanwhile, with regard to warehouse
expenses, all warehouse expenses relating to the MSG in question were proportionally
distributed according to the sales amount of the portion sold by the Defendant and the117total amount of Commission until 2016, and in 2017, all warehouse expenses were
proportionally distributed according to the trading amount of the portion sold by the
Defendant and the trading amount of the portion sold by CJ Indonesia (Exhibits Otsu 99
and 101).
c. "Personnel expenses," "service expenses," and "others"
In accounting, the Defendant recorded the selling, general, and administrative
expenses concerning the sale of the MSG in question that are categorized under the
expense items "personnel expenses," "service expenses," and "others" by the following
procedure.
First, expenses such as "personnel expenses" that arose in the Defendant'sorganization overall were distributed among the respective departments according to thesize of their trading amounts, etc. Then, of the expenses thus distributed to the "bio"department handling the MSG in question, expenses concerning the MSG in question
were calculated according to the proportion of the sales of the MSG in question in thedepartment's total sales. Further, those expenses concerning the MSG in question were
distributed between the portion sold by the Defendant and the portion sold by CJ
Indonesia according to their respective trading amounts. The standards for such
distribution of expenses had been decided by agreement between the Defendant and CJCJ
(Exhibits Otsu 99 and 101).
(E) Regarding the sales values and sales volumes of the Defendant's Products during theperiod of use of the Defendant's Production Processes
The sales values of the Defendant's Products relating to the portion sold by the
Defendant and the portion sold by CJ Indonesia during the period of use of the
Defendant's Production Processes (2011 through 2017) are as shown in 1. of Attachment
10 "List of Sales Values/Volumes," and the sales volumes thereof are as shown in 2. ofthat Attachment (an undisputed fact).
(F) Sales situation of the MSG in question to Japanese customers since 2018
Since 2018, when selling the MSG in question produced by CJ Indonesia to Japan,
CJCJ has acted as the seller instead of CJ Indonesia, and CJCJ has directly sold the
products to Japanese buyers, or has sold the products to the Defendant and then the
Defendant sold them to Japanese buyers (Exhibits Otsu 99, 101, and 112).
B. Regarding working of the inventions in Japan with regard to the portion sold by the
Defendant
While the MSG in question produced during the period of use of the Defendant's
Production Processes represents products produced by any of the Defendant's Production
Processes (the Defendant's Products) as mentioned in (1) above, the Defendant has
118

imported, transferred, and offered to transfer the MSG in question with regard to the
portion sold by the Defendant during that period as mentioned in A. (B) above, and thus
the Defendant is found to have worked Invention 1 or Invention 2 (Article 2, paragraph(3), item (iii) of the Patent Act).
C. Regarding working of the inventions in Japan with regard to the portion sold by CJ
Indonesia(A) Regarding assertion about an allegation or evidence presented after its time
The Defendant asserts that the allegation on the offer to transfer made by the
Defendant, etc. with regard to the portion sold by CJ Indonesia should be dismissed
without prejudice as an allegation or evidence presented after its time.
However, the allegation on the offer to transfer the MSG in question had been madefrom the stage of filing the complaint, as allegation on one of the acts of working theinventions conducted by the Defendant, and the Defendant's assertion that there is aportion sold by CJ Indonesia apart from the portion sold by the Defendant had not
explicitly been made before the submission of Defendant's Brief 17 dated February 20,
2019 and the Defendant's Brief 18 dated April 18, 2019. Considering these facts, it cannot
be said that the Plaintiff's act of alleging the offer to transfer as an act of working theinventions in relation to the portion sold by CJ Indonesia in Plaintiff's Brief 18 dated
October 11, 2019 constitutes an act of additionally presenting an allegation after the time
for doing so.
Consequently, the Defendant's abovementioned petition seeking dismissal of that
allegation without prejudice as being an allegation or evidence presented after its time is
to be dismissed without prejudice.
(B) Regarding import and transfer
a. According to the facts found in A. (C) above, the sales contracts for the portion sold by
CJ Indonesia were concluded between CJ Indonesia and Japanese customers. Based on
the CIF or CFR trade terms stated in the invoices upon transportation of the MSG inquestion to Japan (A. (C) b. above), it is reasonable to find that the transfer of the MSG
in question to the buyers was conducted at the time of the shipping in Indonesia, and there
is no sufficient evidence to find that, on the contrary, the MSG in question had beentransferred to buyers after the discharge at the port of discharge or that the procedure forimport to Japan had been conducted by CJ Indonesia, which is the seller.
If so, it is reasonable to find that the transfer of the MSG in question relating to the
portion sold by CJ Indonesia had been conducted outside Japan, and also no fact can befound that the Defendant, etc. was importing them into Japan. It should be said that theimport of the MSG in question relating to the portion sold by CJ Indonesia into Japan had
119been conducted by the buyers to whom the MSG in question had been transferred.
b. Regarding the Plaintiff's allegations
The Plaintiff alleges that the portion sold by CJ Indonesia should actually be evaluatedto have been imported into Japan and transferred in Japan by the Defendant, etc. However,even by taking into consideration the relationship between the Defendant and CJ
Indonesia in the CJ Group mentioned in A. (A) above, the fact that order forms to be sent
to CJ Indonesia were sometimes sent to the Defendant as mentioned in A. (C) a. above,
the involvement of the Defendant in the portion sold by CJ Indonesia mentioned in A. (C)c. and d. above, and the processing in the Defendant's accounting mentioned in A. (D)above, they are not sufficient to overturn the determination mentioned in a. above that
conclusion of the sales contract and the transfer of the MSG in question relating to the
portion sold by CJ Indonesia are not found to have been conducted in Japan, and hence
the Plaintiff's abovementioned allegation cannot be adopted.
In addition, the Plaintiff alleges as follows: [i] even if CJ Indonesia and Japaneseclients concluded contracts and Japanese clients directly withdraw the MSG in question
as cargo from the container yard, an act of bringing cargo into a bonded area should beconstrued to constitute an act of import with regard to goods that infringe patent rights,
so CJ Indonesia's act constitutes an import; [ii] the act of transfer to Japanese clients by ashipping company, to which transportation has been entrusted by CJ Indonesia and which
is deemed to act at the instruction of CJ Indonesia, is conducted in Japan, and therefore
there was transfer in Japan by the Defendant, etc. However, given that the transfer of the
MSG in question to the buyers is found to have been conducted at the time of the shippingin Indonesia, and that the MSG in question is not found to have been transferred to buyers
after the discharge at the port of discharge as mentioned in a. above, with regard to thePlaintiff's allegation [i] above, it cannot be said that there was an act of import by the
Defendant or CJ Indonesia, irrespective of whether or not an act of bringing cargo into abonded area constitutes the act of import under Article 2, paragraph (3), item (iii) of the
Patent Act, and for the same reason, the Plaintiff's allegation [ii] also cannot be adopted.
(C) Regarding the offer to transfer
a. As mentioned in (B) above, the transfer of the MSG in question itself is not found tohave been conducted in Japan with regard to the portion sold by CJ Indonesia. However,
it is reasonable to find that the Defendant was conducting sales activities relating to the
portion sold by CJ Indonesia, jointly with CJ Indonesia, with regard to the portion soldby CJ Indonesia during the period of use of the Defendant's Production Processes, andthe Defendant is found to have made an offer to transfer, in light of the following: therelationship between the Defendant and CJ Indonesia in the CJ Group mentioned in A.
120

(A) above; the fact that order forms for the portion sold by CJ Indonesia were sometimes
submitted to the Defendant and were sent to CJ Indonesia via the Defendant as mentionedin A. (C) a. above; the fact that a Commission Contract was concluded between the
Defendant and CJ Indonesia to the effect that CJ Indonesia would pay a part of the sales
amount of the portion sold by CJ Indonesia to the Defendant as mentioned in A. (C) c.
above; the fact that the Defendant engaged in delivery of samples and recovery ofdefective products of the MSG in question in Japan with regard to the portion sold by CJ
Indonesia as mentioned in A. (C) d. above; and the fact that expenses relating to the
portion sold by CJ Indonesia were recorded in the Defendant's accounting based on
instructions by or based on an agreement with CJCJ as mentioned in A. (D) above.
In addition, since the MSG in question produced during the period of use of the
Defendant's Production Processes represents products produced by any of the Defendant's
Production Processes (the Defendant's Products) as mentioned in B. above, theDefendant's offer to transfer the MSG in question during that period constitutes theworking of Invention 1 or Invention 2 (Article 2, paragraph (3), item (iii) of the Patent
Act) by the Defendant.
b. Regarding the Defendant's allegations(a) The Defendant alleges that an "offer to transfer" is an act to be conducted by the seller,
which is the future transferor, and that an offer to transfer is not established in a casewhere the person that conducts the act of offering, by means such as advertising, differsfrom the person who makes the transfer, and therefore even if the Defendant, which is not
the seller, becomes involved in some way with regard to the portion sold by CJ Indonesia,that act does not constitute an offer to transfer.
However, in light of the purport that an offer to transfer is stipulated as an act ofworking that is separate from a transfer, even if an act of offering to transfer is notconducted by the seller, which is the transferor, it should be considered that the act ofoffering can be construed to be an offer to transfer as long as there are circumstances suchas that the act has been conducted by a person that has a certain relationship with theseller. Meanwhile, according to the results of the examination made in a. above, it can besaid that CJ Indonesia and the Defendant not only belong to the same corporate group,
but also had a close relationship with each other, in areas such as concluding a
Commission Contract and sharing profits with regard to the portion sold by CJ Indonesia.
Therefore, the fact that the party to the sales contract for the portion sold by CJ Indonesia
was CJ Indonesia and not the Defendant does not preclude the finding that the Defendant's
involvement mentioned in a. above constitutes an offer to transfer the MSG in question.
(b) The Defendant also alleges that, as a "transfer" under the Patent Act means a transfer
121

in Japan, and an "offer to transfer," which is an act to prepare for a transfer, also means
an offer to make a transfer in Japan, the Defendant's act with regard to the portion soldby CJ Indonesia does not constitute an offer to transfer.
Indeed, although the transfer of the MSG in question to the buyers with regard to the
portion sold by CJ Indonesia is found to have been conducted outside Japan as mentionedin (B) a. above, the portion sold by CJ Indonesia was all sold to buyers in Japan, and even
if the transfer of the MSG in question itself was conducted at the time of the shipping, theMSG in question was scheduled to be subsequently imported into Japan by the buyers'
side. In light of the purport that an offer to transfer is stipulated as a separate act ofworking from a transfer, regarding products like the MSG in question relating to the
portion sold by CJ Indonesia, which are sold to buyers in Japan and imported into Japanas a result of sales activities conducted in Japan, it is not reasonable if the conclusion as
to whether or not the sales activities constitute an offer to transfer made in Japan would
differ solely based on whether the transfer to the buyers is conducted outside Japan orinside Japan. As mentioned in a. above, it is reasonable to construe that the act of the
Defendant and CJ Indonesia to jointly conduct sales activities relating to the portion soldby CJ Indonesia, in Japan, constitutes an "offer to transfer" by the Defendant, theDefendant's allegation relating to this point cannot be adopted.
D. Regarding joint tort by the Defendant and CJ Indonesia(A) As mentioned in B. and C. above, during the period of use of the Defendant's
Production Processes, the Defendant worked the inventions relating to Patent Right 1 orPatent Right 2 by way of importing, transferring, and offering to transfer the MSG inquestion with regard to the portion sold by the Defendant, and by way of offering totransfer the MSG in question with regard to the portion sold by CJ Indonesia.
(B) As mentioned in A. (A) above, the Defendant and CJ Indonesia belong to the CJ
Group which is centered around CJCJ, the wholly owning parent company of CJ
Indonesia, and in that group, the group companies shared roles in producing and selling
CJ-brand and other products in countries around the world.
With regard to sales of the MSG in question during the period of use of the
Defendant's Production Processes, two modes of contract coexisted: one for the portion
sold by the Defendant and the other for the portion sold by CJ Indonesia. Although theydiffered on whether or not the Defendant was included in the sales channel when products
were sold to Japanese customers, they both sold the same product, the MSG in question
produced by CJ Indonesia, to the Japanese market, and as examined in C. (C) above, theDefendant, which is not party to the sales contract, is found to have conducted sales
activities also with regard to the portion sold by CJ Indonesia, while receiving a share of122the profits therefrom, and to have engaged in delivery of samples and recovery ofdefective products with regard to that portion, in the same manner as those for the portion
sold by the Defendant. In addition, as mentioned in A. (D) above, it is found that, in the
Defendant's accounting, overall amounts of transportation expenses, warehouse expenses,
etc. concerning sales of the MSG in question were distributed between the portion soldby the Defendant and the portion sold by CJ Indonesia based on instructions by CJCJ, theparent company of CJ Indonesia.
In light of these facts, it is reasonable to find that the Defendant and CJ Indonesia are
not merely in a relationship of a supplier/customer of the MSG in question with regard to
the portion traded by the Defendant, but are in an integral relationship with each other inselling the MSG in question produced by CJ Indonesia targeting the Japanese market,
regardless of the selling modes, i.e. that for the portion sold by the Defendant and that forthe portion sold by CJ Indonesia, and that they jointly conduct the respective acts ofworking the inventions mentioned in (A) above with regard to the portion sold by the
Defendant and the portion sold by CJ Indonesia, and it is reasonable to find that joint tort
will be established for the Defendant, etc. in regard to these acts.
(3) Regarding the value of damages under Article 102, paragraph (2) of the Patent Act for
the entire portion sold by the Defendant, etc. (Attachment 11-1 "Allegation on the Valueof Damages 1")
As mentioned in No. 3, 15. [Plaintiff's allegations] (3) above, the Plaintiff has madeallegations on the value of damages sustained by the Plaintiff, based on the four
calculation methods described in the [Plaintiff's allegations] columns in Attachment 11-1
"Allegation on the Value of Damages 1" through Attachment 11-4 "Allegation on the
Value of Damages 4." First, the allegation on the value of damages under Article 102,
paragraph (2) of the Patent Act for the entire portion sold by the Defendant, etc., relating
to Attachment 11-1"Allegation on the Value of Damages 1," will be examined.
A. Meaning of "profit" under Article 102, paragraph (2) of the Patent Act
The amount of profit which the infringer has made from the infringement prescribedin Article 102, paragraph (2) of the Patent Act is the amount of marginal profit obtainedby deducting expenses that became additionally required in direct association with thesale of the infringing goods by the infringer as a result of selling the infringing goods
from the amount of sales of infringing goods by the infringer.
B. Method of calculation of the profit under Article 102, paragraph (2) of the Patent Act
made through joint tort by the Defendant, etc.
(A) As mentioned in (2) above, the inventions relating to Patent Right 1 or Patent Right
2 have been worked by way of importing, transferring, and offering to transfer the MSG123

in question with regard to the portion sold by the Defendant, and by way of offering totransfer the MSG in question with regard to the portion sold by CJ Indonesia, and joint
tort by the Defendant and CJ Indonesia is found to be established for these acts.
Accordingly, in presuming the value of damages under Article 102, paragraph (2) of the
Patent Act as well, it should be said that the total amount of the marginal profits of the
Defendant, etc. calculated by the calculation methods mentioned in A. above would bepresumed to be the value of damages sustained by the Plaintiff with regard to theabovementioned infringing acts relating to the portion sold by the Defendant, etc.
Also considering that the Defendant and CJ Indonesia were in an integral relationshipwith each other in selling the MSG in question produced by CJ Indonesia targeting theJapanese market, regardless of the selling modes, i.e. that for the portion sold by the
Defendant and that for the portion sold by CJ Indonesia, as mentioned in (2) above, it isreasonable to calculate the total amount of the marginal profits made by the Defendant,
etc. through the respective infringing acts relating to the portion sold by the Defendant,
etc. by deducting overall expenses that became additionally required by Defendant, etc.
in direct association with the sale of the relevant portion from the overall sales amount of
the portion sold by the Defendant, etc.
(B) Regarding this point, the Defendant alleges that the value of damages from the "offer
to transfer," which is an act of working the inventions in relation to the portion sold by
CJ Indonesia, should not be calculated based on the sales amount of the portion sold by
CJ Indonesia, but instead should remain within the scope specific to the "offer to transfer."
However, because joint tort is established for the Defendant and CJ Indonesia withregard to the portion sold by CJ Indonesia as mentioned in (2) D. above., not only the
profits made by the Defendant but also those made by CJ Indonesia would be consideredin calculating the value of damages, and, as examined in (2) C. (C) b. (b) above, theportion sold by CJ Indonesia was all sold to buyers in Japan, and was scheduled to be
subsequently imported into Japan after being transferred to the buyers. Therefore, it isreasonable to find that, even if the "transfer" itself was conducted outside Japan, theprofits made by the Defendant, etc. calculated based on the sales amount of the portion
sold by CJ Indonesia are the profits received by the Defendant, etc. from the "offer totransfer" made in Japan, in applying Article 102, paragraph (2) of the Patent Act, and the
Defendant's abovementioned allegation cannot be adopted.
C. Sales amount of the portion sold by the Defendant, etc.
As mentioned in (2) A. (E) above, the sales values of the Defendant's Products relating
to the portion sold by the Defendant, etc. during the period of use of the Defendant's
Production Processes (2011 through 2017) are as shown in 1. of Attachment 10 "List of124

Sales Values/Volumes."
D. Expenses borne by the Defendant, etc. to be deducted from the sales amount(A) Sales volumes
As mentioned in (2) A. (E) above, the sales volumes of the Defendant's Products
relating to the portion sold by the Defendant, etc. during the period of use of the
Defendant's Production Processes are as shown in 2. of Attachment 10 "List of Sales
Values/Volumes."(B) Regarding the production expenses for the MSG in question
a. Eligibility of expenses
As the MSG in question relating to the portion sold by the Defendant, etc. was all
produced by CJ Indonesia, it should be said that its production expenses are eligible as
expenses to be deducted in calculating the marginal profits of the Defendant, etc. as
expenses that became additionally required in direct association with the sale of the
portion sold by the Defendant, etc.
b. Production expenses for the MSG in question per ton
According to evidence (Exhibit Otsu 107) and the entire import of oral arguments, it
is reasonable to find that, as the production expenses for the MSG in question during theperiod of use of the Defendant's Production Processes, the annual total amounts offermentation material expenses, purification material expenses, utility expenses, andpackaging expenses per ton of the MSG in question were the amounts stated in [Plaintiff's
allegations] 2. (2) B. (A) of Attachment 11-1 "Allegation on the Value of Damages 1"(Value of Damages under Article 102, Paragraph (2) of the Patent Act for the Overall
Portion Sold by the Defendant, etc.) (on a U.S. dollar basis; values for 2015 through 2017
are the same as the value for 2014).
Meanwhile, the abovementioned evidence (Exhibit Otsu 107) disclosed by the
Defendant also states expenses titled "fixed expenses (labor, depreciation, and others)" as
expenses relating to production of the MSG in question, but as they cannot be considered
as expenses in direct association with the sale of the MSG in question in light of theirnature, they should not be considered in calculating the marginal profits.
c. Exchange rates
According to evidence (Exhibit Ko 106), it is found that the exchange rates of U.S.
dollars to yen during the period of use of the Defendant's Production Processes were asshown in [Plaintiff's allegations] 2. (2) B. (B) of Attachment 11-1 "Allegation on the Valueof Damages 1" (Value of Damages under Article 102, Paragraph (2) of the Patent Act for
the Overall Portion Sold by the Defendant, etc.).
d. Amounts of expenses to be deducted125

Then, when the sales volumes of the portion sold by the Defendant, etc. mentioned in(A) above are multiplied by the production expenses per ton mentioned in b. above andthe exchange rates mentioned in c. above, the production expenses for the MSG inquestion during the period of use of the Defendant's Production Processes in relation to
the portion sold by the Defendant, etc., which should be deducted when calculating themarginal profits, would be as shown in 3. (i)-3 of Attachment 15 "Calculation Table of
the Value of Damages under Article 102, Paragraph (2) of the Patent Act for the Portion
Sold by the Defendant, etc."(C) Regarding expenses for transportation of the MSG in question to Japan
a. Eligibility of expenses
As the MSG in question relating to the portion sold by the Defendant, etc. was all
produced at CJ Indonesia's factory in Indonesia and was sent to Japan, and, according to
the statements in the invoices mentioned in (2) A. (C) b. above, CIF or CFR trade terms
under which CJ Indonesia, the seller, was to bear the cost of transporting the products tothe port of discharge with regard to the portion sold by CJ Indonesia are considered tohave been adopted, it should be said that the expenses for transporting the MSG inquestion to Japan in relation to the portion sold by the Defendant, etc. are eligible as
expenses to be deducted in calculating the marginal profits of the Defendant, etc. as
expenses that became additionally required in direct association with the sale of the
portion sold by the Defendant, etc.
b. Per-ton expenses for transporting the MSG in question to Japan
According to evidence (Exhibit Otsu 113) and the entire import of oral arguments, it
is reasonable to find that the annual average amounts of the per-ton expenses fortransporting the MSG in question to Japan relating to the portion sold by the Defendant,
etc. during the period of use of the Defendant's Production Processes were the amounts
stated in [Plaintiff's allegations] 2. (2) C. (A) of Attachment 11-1 "Allegation on the Valueof Damages 1" (Value of Damages under Article 102, Paragraph (2) of the Patent Act for
the Overall Portion Sold by the Defendant, etc.).
c. Amounts of expenses to be deducted
When the sales volumes of the portion sold by the Defendant, etc. mentioned in (A)above are multiplied by the per-ton expenses for transportation mentioned in b. above andthe exchange rates mentioned in (B) c. above, the expenses for transporting the MSG inquestion to Japan during the period of use of the Defendant's Production Processes inrelation to the portion sold by the Defendant, etc., which should be deducted whencalculating the marginal profits, would be as shown in 3. (ii) of Attachment 15"Calculation Table of the Value of Damages under Article 102, Paragraph (2) of the Patent126

Act for the Portion Sold by the Defendant, etc."(D) Regarding domestic freight expenses and other selling, general, and administrative
expenses
As mentioned in (2) A. (D) above, the Defendant had recorded expenses under the
expense items "freight expenses," "warehouse expenses," "personnel expenses," "service
expenses," and "others" as selling, general, and administrative expenses relating to the
portion sold by the Defendant, etc. The eligibility of these expense items as expenses andthe amounts of expenses to be deducted will be examined.
a. Regarding "freight expenses" and "warehouse expenses"
According to evidence (Exhibits Otsu 99 and 101) and the entire import of oral
arguments, it is found that the amounts of expenses recorded as "freight expenses" and"warehouse expenses" for the MSG in question relating to the portion sold by the
Defendant, etc. during the period of use of the Defendant's Production Processes were theamounts respectively shown for the portion sold by the Defendant and the portion soldby CJ Indonesia in 3. (iii) of Attachment 15 "Calculation Table of the Value of Damages
under Article 102, Paragraph (2) of the Patent Act for the Portion Sold by the Defendant,etc." (the values for the portion sold by the Defendant are the same as those stated in[Plaintiff's allegations] 2. (2) D. of Attachment 11-1 "Allegation on the Value of Damages1" (Value of Damages under Article 102, Paragraph (2) of the Patent Act for the Overall
Portion Sold by the Defendant, etc.)). Meanwhile, the respective warehouse expenses for2017 found above are those after correcting the distribution of the respective expenses
stated in Reference Materials 3 attached to Exhibit Otsu 101 between the portion sold by
the Defendant and the portion sold by CJ Indonesia, without changing the total amounts
for the two portions, according to the allegation made in Defendant's Brief 19.
In addition, according to evidence (Exhibits Otsu 99 and 101) and the entire importof oral arguments, these expenses recorded as "freight expenses" and "warehouse
expenses" are found to be those wherein the total amount of the expenses actually requiredby the Defendant for transporting and storing the MSG in question relating to the portion
sold by the Defendant, etc. was distributed between the portion sold by the Defendant andthe portion sold by CJ Indonesia in accounting, as mentioned in (2) A. (D) b. above.
Therefore, it should be said that the amount obtained by adding the amount of expenses
distributed to the portion sold by the Defendant, which the Plaintiff recognized as
expenses, and the amount of expenses distributed to the portion sold by CJ Indonesia iseligible as expenses to be deducted in calculating the marginal profits of the Defendant,
etc. as expenses that became additionally required in direct association with the sale ofthe portion sold by the Defendant, etc.
127

b. Regarding other selling, general, and administrative expenses
As for the expense items "personnel expenses," "service expenses," and "others,"
which the Defendant recorded as selling, general, and administrative expenses relating to
the sale of the MSG in question, the relevant portion of the expenses, such as "personnel
expenses," that arose in the Defendant's organization overall was distributed to the
Defendant's "bio" department in accounting, and a part thereof was further distributed
between the portion sold by the Defendant and the portion sold by CJ Indonesia as
mentioned in (2) A. (D) c. above. As they cannot be considered as expenses in direct
association with the sale of the MSG in question in light of their nature, they are noteligible as expenses to be deducted in calculating the marginal profits of the Defendant,etc.
The Defendant alleges that, while "personnel expenses" include the personnel
expenses of operation departments and those of indirect sections, at least the personnel
expenses of operation departments depend on the workload of sales operations, so theyshould be deducted as expenses that became additionally required in direct association
with the sale of the MSG in question. However, given the method by which the amountof the "personnel expenses" recorded for the portion sold by the Defendant, etc. has been
decided, the personnel expenses of operation departments, which constitute a part thereof,
also cannot be considered as expenses in direct association with the sale of the portion
sold by the Defendant, etc. in light of their nature, and hence the Defendant's allegation
cannot be adopted.
(E) Regarding purchasing expenses
The Defendant alleges that, when considering the profits for the portion sold by the
Defendant, the expenses required for their purchase should be taken into consideration.
As mentioned in B. (B) above, the Defendant, etc. are in an integral relationship withregard to the portion sold by the Defendant, etc., and joint tort by the Defendant, etc. isestablished for the portion sold by the Defendant, etc. Thus, the total amount of profits
made by the Defendant and CJ Indonesia should also be calculated when considering the
profits for the portion sold by the Defendant. Consequently, is should be said that thepurchasing expenses that arose when the Defendant purchased the MSG in question fromCJ Indonesia with regard to the portion sold by the Defendant are not eligible as expenses
to be deducted from the sales amount of the portion sold by the Defendant whencalculating the total amount of profits made by the Defendant, etc. with regard to the
portion sold by the Defendant.
(F) Summary
According to the above, expenses to be deducted from the sales amount of the portion128

sold by the Defendant, etc. when calculating the total amount of profits made by the
Defendant, etc. are the production expenses for the MSG in question mentioned in (B)above, the expenses for transportation of the MSG in question to Japan mentioned in (C)above, and the domestic freight expenses and warehouse expenses mentioned in (D) a.
above (the total amounts of the respective expenses to be deducted are as shown in 4. of
Attachment 15 "Calculation Table of the Value of Damages under Article 102, Paragraph(2) of the Patent Act for the Portion Sold by the Defendant, etc.").
E. Total amount of marginal profits made by the Defendant, etc.
According to the above, the total amount of the marginal profits made by the
Defendant, etc. through the respective infringing acts relating to the portion sold by the
Defendant, etc. during the period of use of the Defendant's Production Processes is theamount obtained by deducting, from the sales amount of the portion sold by the Defendant,
etc. mentioned in C. above, the expenses to be deducted mentioned in D. (F) above, andas shown in 4. of Attachment 15 "Calculation Table of the Value of Damages under Article
102, Paragraph (2) of the Patent Act for the Portion Sold by the Defendant, etc.," the total
amount throughout the period comes to 3,857,313,065 yen.
F. Regarding grounds for overturning the presumption(A) Regarding the sale of competitive products (reducing the amount according to market
share)
a. According to evidence (Exhibits Otsu 101 and 117) and the entire import of oral
arguments, the volumes of monosodium glutamate imported from the respective countries
into Japan during the period of use of the Defendant's Production Processes are as shownin column (i) through column (x) of Attachment 16 "Situation of Import of Monosodium
Glutamate During the Period of Use of the Defendant's Production Processes." Of these,
the entire volumes of the imports from (i) Thailand and (ii) Brazil are found to be thoseimported and sold in Japan by the Plaintiff, and the imports from (iii) Indonesia are foundto include not only the portion sold by the Defendant, etc. ((iii) A.), but also thoseimported by the Plaintiff. Meanwhile, in calculating the volumes for 2017, the volumes
stated in Reference Materials 7 attached to Exhibit Otsu 101 were adopted instead ofthose in Reference Materials 6 attached to the same as reference materials similar to thereference materials for 2011 through 2016 (Exhibit Otsu 117).
b. The MSG in question is monosodium glutamate, and as mentioned in a. above, themonosodium glutamate mentioned in a. above, which was imported during the period ofuse of the Defendant's Production Processes, is all found to constitute products that are incompetitive relationship with the MSG in question relating to the portion sold by the
Defendant, etc. as equivalents thereto in the Japanese market.
129

In this manner, the fact that the abovementioned competitive products existed apartfrom the portion sold by the Defendant, etc. and the portion imported and sold by the
Plaintiff should be considered as circumstances for overturning the presumption under
Article 102, paragraph (2) of the Patent Act, with regard to a part of the total amount ofmarginal profits made by the Defendant, etc. mentioned in E. above.
c. The percentage to be overturned due to the abovementioned circumstances will be
examined below.
With regard to the volume ((iii) B) other than the portion sold by the Defendant, etc.
((iii) A) out of the volume imported from (iii) Indonesia mentioned in a. above, theDefendant alleges that half of such other portion is presumed to have been imported bybusiness operators other than the Plaintiff. However, there is no evidence supporting thevolume imported from Indonesia by business operators other than the Plaintiff.
Considering the Plaintiff's allegation that most of that volume ((iii) B) was importedby the Plaintiff, the portion imported and sold by the Plaintiff ((xi)) was calculated byassuming that volume to have been imported by the Plaintiff. Then, the percentage of the
portion imported and sold by the Plaintiff ((xi)) in the volume obtained by deducting the
portion sold by the Defendant, etc. ((iii) A) from the total import volume ((x)) was
calculated. As a result, the annual percentages during the period of use of the Defendant's
Production Processes came to ●(omitted)●% to ●(omitted)●% as stated in the
Attachment, and the average of the annual percentages came to 44.7%.
With regard to the reference materials of trade statics by the Ministry of Financesubmitted by the Defendant (Reference Materials 3 through 10 of Exhibit Otsu 117), thePlaintiff also alleges that these reference materials do not include the volume of importsthat are imported as glutamic acid and processed into monosodium glutamate in Japan,
and that most of those were imports by the Plaintiff. However, the Plaintiff has not
submitted any specific reference material to support this allegation, and evidence(Reference Materials 7 attached to Exhibit Otsu 101) suggests that the import volume ofsuch glutamic acid was small compared to the import volume of monosodium glutamate.
Then, even by considering the Plaintiff's allegation, it is reasonable to find that, even
if the portion sold by the Defendant, etc. was not sold during the period of use of the
Defendant's Production Processes, at least about 50% of the demand that had been
applicable to the MSG in question relating to the portion sold by the Defendant, etc. would
have been applicable to the monosodium glutamate imported and sold by business
operators other than the Plaintiff, and accordingly, it is reasonable to find that thepresumption is to be overturned by 50%.
(B) Regarding the degree of contribution to infringement of the Patent Rights
130

The Defendant alleges that the inventions claimed in the Patents only contribute to alimited part of the processes for producing glutamic acid comprising complicated anddiverse steps and the degree of their respective contribution to production of the MSG inquestion is low, and that therefore, in calculating damages under Article 102, paragraph(2) of the Patent Act, the degree of contribution of the Patents should be considered orthat the overturning of the presumption according to the degree of contribution should beallowed.
However, as Invention 1-1 (Corrected Invention 1-1), Invention 1-2 (Corrected
Invention 1-2), and Invention 1-4 (Corrected Invention 1-3), as well as Invention 2-5(Corrected Invention 2-5), which are found to have been worked during the period of useof the Defendant's Production Processes, are all inventions relating to process for
producing glutamic acid (amino acid) by using strains having specific characteristics, if
the working of the abovementioned patented inventions is found as a result of the sale,etc. of the MSG in question produced by using those strains, it should be said that thepatented inventions have all been worked for the entirety of the MSG in question sold.
Even if production of glutamic acid by the fermentation method comprises thefermentation phase and the purification phase, and further, multiple reactions occur in thestrains in the fermentation phase, and the abovementioned patented inventions only relate
to a specific phase or reaction in that process, the degree of contribution of theabovementioned patented inventions to production of the MSG in question cannot befound to be low based on such reason. In addition, no other circumstances are found, inthe evidence of the present case, due to which the presumption under Article 102,
paragraph (2) of the Patent Act should be overturned with regard to the claims based on
the Patent Rights based on the degree of contribution by the abovementioned patented
inventions. Therefore, the Defendant's abovementioned allegation cannot be adopted.
G. Summary on the value of damages under Article 102, paragraph (2) of the Patent Act
for the overall portion sold by the Defendant, etc.
(A) If the grounds for overturning the presumption mentioned in F. above are taken into
consideration with regard to the total amount of the marginal profits made by the
Defendant, etc. mentioned in E. above, the value of damages sustained by the Plaintiff
under Article 102, paragraph (2) of the Patent Act comes to a total of 1,928,656,532 yen.
As this amount exceeds the value of damages of 900 million yen sought by the
Plaintiff as the partial claim for the value of damages calculated under Article 102,
paragraph (2) or (3) of the Patent Act, there is no need to determine the appropriatenessof overlapped application of Article 102, paragraph (3) of the Patent Act for theoverturned part mentioned in F. above or the allegations stated in the [Plaintiff's
131allegations] columns in Attachment 11-2 "Allegation on the Value of Damages 2" throughAttachment 11-4 "Allegation on the Value of Damages 4."(B) When the period for which exercise of rights based on the Patent Rights mentionedin (1) above is taken into consideration with regard to the value of damages mentioned in(A) above, and the correspondence between the damages that occurred in the respective
periods and the Patent Rights is examined, the following can be derived (the respective
calculation results are as shown in 5. of Attachment 15 "Calculation Table of the Value ofDamages under Article 102, Paragraph (2) of the Patent Act for the Portion Sold by the
Defendant, etc.").
a. January 2011 through August 22, 2013
A period during which Patent Right 1 was worked; a total of 466,184,463 yen
b. August 23, 2013 (the date of registration of establishment of Patent Right 2) throughJune 2016
A period during which Patent Rights 1 and 2 were worked; a total of 897,628,767 yenc. July 2016 through December 2017
A period during which Patent Right 2 was worked; a total of 564,843,302 yen(C) With regard to the relationship between the claims based on Patent Right 1 and Patent
Right 2, the Plaintiff has selectively made claims based on each patent right within thescope of 900 million yen, which is the amount of the partial claim. According to (B) above,
the values of damages caused by working Patent Right 1 and Patent Right 2 arerespectively found to exceed 450 million yen, which is half the amount of 900 million
yen. Therefore, it is reasonable to distribute the value of damages mentioned in (A) aboveby the ratio of 1:1, which is the ratio of the amounts of partial claims, and to affirm 450
million yen for the claim based on Patent Right 1 and 450 million yen for the claim basedon Patent Right 2.
(4) Regarding lawyer fees and patent attorney fees
Considering the complexity of the present case, the amounts of partial claims in theclaims for compensation for damages in the present case, and the amounts affirmed
mentioned in (3) above, among other matters, it is reasonable to find that lawyer fees andpatent attorney fees that have causal links with the joint tort of infringement of the Patent
Rights by the Defendant, etc. are 90 million yen (45 million yen for infringement of Patent
Right 1 and 45 million yen for infringement of Patent Right 2).
(5) Summary on the claim for compensation for damages
According to the above, without having to make determinations on other points, thePlaintiff's claims for compensation for damages against the Defendant based on tort ofinfringement of the Patent Rights (a partial claim for the total value of damages of 990
132

million yen, and a claim for delay damages accrued thereon at the rate of 5% per annum
as prescribed in the Civil Code for the period from August 10, 2016, the day following
the date of service of the complaint against this value) are all found to be well-grounded.
9. Regarding Issue 9 (whether or not the claim seeking injunction and the claim seeking
disposal are appropriate)(1) Regarding the claims based on Patent Right 1
As mentioned in No. 2, 2. (3) above, the term of Patent Right 1 expired on September
22, 2019. Therefore, the claim seeking injunction and the claim seeking disposal basedon Patent Right 1 are both groundless.
(2) Regarding the claims based on Patent Right 2
A. With regard to the period of use of the Defendant's Production Processes, as examinedin 1. (4) above, it is found that the Defendant's Production Process 1 was used for theperiods of use of Strains (ii) and (x) stated in Attachment 9 "Comparison Table ofAllegations concerning Processes for Producing the MSG in Question," and other periodsof use are as shown in the "Plaintiff's alternative claims" column of that Attachment. Afterthe use of Strain (xiii) ended in December 2017, the Defendant's Production Processes
that are included in the technical scope of Invention 2-5 (Corrected Invention 2-5) are notfound to have been used for producing the MSG in question.
However, given that the strains used for producing the MSG in question were
frequently changed during the period of use of the Defendant's Production Processes asmentioned above, it is considered to have been easy for CJ Indonesia to change the strain
again and produce the MSG in question by the Defendant's Production Processes.
Consequently, even at this point of time, there is found to be a need for the Plaintiff tosuspend transfer, import, or offer to transfer (including display for the purpose of transfer)of the MSG in question produced by the Defendant's Production Processes and seek theirdisposal under Article 100, paragraph (1) and paragraph (2) of the Patent Act based on
Patent Right 2.
B. Products subject to injunction, etc.
As mentioned in 3. above, the strains used in Defendant's Production Processes 1
through 3 are all found to fall within the technical scope of Invention 2-5 asCorynebacterium glutamicum into which a yggB gene, wherein alanine at position 100
in the amino acid sequence of SEQ ID NO: 6 in Description 2 has been substituted withthreonine, has been introduced.
In addition, as mentioned in 4. above, the strains used in Defendant's Production
Process 4 (Strains (xii) and (xiii)) are both found to fall within the technical scope ofInvention 2-5 as Corynebacterium glutamicum into which a yggB gene, wherein alanine133

at position 98 of the amino acid sequence encoded by a yggB gene derived from the wildtype Corynebacterium callunae DSM20147 strain has been substituted with threonineand valine at position 241 of the sequence has been substituted with isoleucine, has been
introduced.
Accordingly, it should be said that the products for which the injunction and disposal
mentioned in A. above should be sought based on Patent Right 2 would be specified asshown in 1. and 2. of Attachment 17 "List of Products Subject to Injunction" (meanwhile,
the amino acid sequence indicated in the sequence listing attached to that Attachment is
the amino acid sequence of SEQ ID NO: 6 in Description 2).
10. Conclusion
According to above, the Plaintiff's claim seeking injunction and disposal based on the
Patent Rights filed against the Defendant are well-grounded to the extent of seeking
injunction and disposal with regard to the act of transferring, importing, and offering totransfer the monosodium glutamate mentioned in 1. and 2. of Attachment 17 "List of
Products Subject to Injunction" based on Patent Right 2, and the Plaintiff's claims forcompensation for damages caused by infringement of the Patent Rights are all wellgrounded to the extent of the Plaintiff filing partial claims in the present case, whereas
the Plaintiff's other claims are all groundless; therefore, the judgment is rendered asindicated in the main text. As it is not reasonable to attach a declaration of provisional
execution to paragraph (2) of the main text, the declaration shall not be attached to thatparagraph.
Tokyo District Court, 29th Civil Division
Judge: YANO Norio
Presiding judge YAMADA Maki and judge NISHIYAMA Yoshiki were unable to signand seal due to a transfer of position.
Judge: YANO Norio
List of Attachments(omitted)

134

Attachment 1 "List of Parties"
Plaintiff: Ajinomoto Co., Inc.
(omitted)
Defendant: CJ Japan Corp.
(omitted)

135

Attachment 2 "List of the Defendant's Products"
Monosodium glutamate with the product name "MI-POONG"
which satisfies the following requirements:
1. Defendant's Product 1
One produced by using Corynebacterium glutamicum wherein a TTGTCA sequence
has been introduced in the -35 region and a TATAAT sequence has been introduced in the-10 region of the promoter sequence of the glutamate dehydrogenase (GDH) gene (theGDH gene), a TATAAT sequence has been introduced in the -10 region of the promoter
sequence of the citrate synthetase (CS) gene (the gltA gene), and alanine at position 100of the amino acid sequence encoded by a yggB gene has been substituted with threonine
2. Defendant's Product 2
One produced by using Corynebacterium glutamicum wherein alanine at position 100of the amino acid sequence encoded by a yggB gene has been substituted with threonine
3. Defendant's Product 3
One produced by using Corynebacterium glutamicum wherein a TATAAT sequence
has been introduced in the -10 region of the promoter sequence of the citrate synthetase(CS) gene (the gltA gene), and alanine at position 100 of the amino acid sequence encoded
by a yggB gene has been substituted with threonine
4. Defendant's Product 4
One produced by using Corynebacterium glutamicum into which a yggB gene of
Corynebacterium callunae, wherein alanine at position 98 of the amino acid sequence
encoded by a yggB gene has been substituted with threonine, has been introduced
End

136

Attachment 3 "List of the Defendant's Production Processes"
1. A process of producing monosodium glutamate which is the product mentioned in 1.
of Attachment 2 "List of the Defendant's Products," comprising the following steps:
Corynebacterium glutamicum, which is a coryneform bacterium having thecharacteristics mentioned in 1. of Attachment 2 "List of the Defendant's Products" in thegene on the chromosome, is placed in a fermentation vessel, or, in a medium containing
ingredients, such as the carbon source, and are propagated; L-glutamic acid is producedby the fermentation method whereby the carbon source is converted into L-glutamic acidthrough metabolic reactions within the bacterial cells; after the end of this fermentation
process, the supernatant liquid containing the fermentation bacterium (bacterial cells) andL-glutamic acid are separated and collected; and the collected glutamic acid is neutralizedby caustic soda
2. A process of producing monosodium glutamate which is the product mentioned in 2.
of Attachment 2 "List of the Defendant's Products" by using the same process as in 1.
above, except for using Corynebacterium glutamicum which is a coryneform bacterium
having the characteristics mentioned in 2. of Attachment 2 "List of the Defendant's
Products" in the gene on the chromosome
3. A process of producing monosodium glutamate which is the product mentioned in 3.
of Attachment 2 "List of the Defendant's Products" by using the same process as in 1.
above, except for using Corynebacterium glutamicum which is a coryneform bacterium
having the characteristics mentioned in 3. of Attachment 2 "List of the Defendant's
Products" in the gene on the chromosome
4. A process of producing monosodium glutamate which is the product mentioned in 4.
of Attachment 2 "List of the Defendant's Products" by using the same process as in 1.
above, except for using Corynebacterium glutamicum which is a coryneform bacterium
having the characteristics mentioned in 4. of Attachment 2 "List of the Defendant's
Products" in the gene on the chromosome
End

137

Attachment 4-1 "Claims (Patent 1)"[Claim 1] (Invention 1-1)
A process for producing L-glutamic acid or L-arginine by the fermentation, which
comprises the steps of culturing a bacterium in a medium to form and also accumulate Lglutamic acid or L-arginine in the medium, and collecting L-glutamic acid or L-argininefrom the medium, wherein the bacterium is a coryneform bacterium in which at least one
DNA sequence selected from the group consisting of TTGTCA, TTGACA, TTGCTA,
and TTGCCA is introduced into -35 region of the promoter sequence of a gene selectedfrom the group consisting of glutamate dehydrogenase (GDH), citrate synthase (CS),isocitrate dehydrogenase (ICDH), pyruvate dehydrogenase (PDH), and argininosuccinate
synthase on a chromosome of the coryneform bacterium and/or TATAAT sequence or
TATAAC sequence is introduced into -10 region of that promoter sequence.
[Claim 2] (Invention 1-2)
A process according to Claim 1, wherein a promoter of glutamate dehydrogenase (GDH)producing gene has at least one DNA sequence selected from the group consisting of
TTGTCA, TTGACA, and TTGCCA in -35 region and/or TATAAT sequence in -10 region.
[Claim 3] (Invention 1-3)
A process according to Claim 2, wherein a promoter of GDH has TTGACA sequence or
TTGCCA sequence in -35 region and/or TATAAT in -10 region.
[Claim 4] (Invention 1-4)
A process according to Claim 1, wherein a promoter of CS has TTGACA sequence in 35 region and/or TATAAT sequence or TATAAC sequence in -10 region.
End

138

Attachment 4-2 "Corrected Claims (Patent 1)"[Claim 1] (Corrected Invention 1-1)
A process for producing L-glutamic acid by the fermentation, which comprises the stepsof culturing a bacterium in a medium to form and also accumulate L-glutamic acid in themedium, and collecting L-glutamic acid from the medium, wherein the bacterium is acoryneform bacterium in which TTGTCA sequence is introduced into -35 region and
TATAAT sequence is introduced into -10 region of the promoter sequence of glutamate
dehydrogenase (GDH) gene on a chromosome of the coryneform bacterium and/or
TATAAT sequence is introduced into -10 region of the promoter sequence of citratesynthase (CS) gene on a chromosome of that bacterium.
[Claim 2] (Corrected Invention 1-2)
A process according to Claim 1, wherein the promoter of GDH gene has TTGTCA
sequence in -35 region and TATAAT sequence in -10 region.
[Claim 3] (Deleted)[Claim 4] (Corrected Invention 1-3)
A process according to Claim 1, wherein the promoter of CS gene has TATAAT sequencein -10 region or has TTGACA sequence in -35 region and TATAAT sequence in -10 region,and the promoter of GDH gene has TTGTCA sequence in -35 region and TATAAT
sequence in -10 region.
End

139

Attachment 4-3 "Division into Constituent Features (Invention 1)"
Invention 1-1 (Claim 1)
1-B
A process for producing L-glutamic acid or L-arginine by the fermentation,
which comprises the steps of culturing a bacterium in a medium to form andalso accumulate L-glutamic acid or L-arginine in the medium, and collecting
L-glutamic acid or L-arginine from the medium,
1-A-3
wherein the bacterium is a coryneform bacterium in which
1-A-1
at least one DNA sequence selected from the group consisting of TTGTCA,TTGACA, TTGCTA, and TTGCCA is introduced into -35 region of the

1-A-2

promoter sequence of a gene selected from the group consisting of glutamate
dehydrogenase (GDH), citrate synthase (CS), isocitrate dehydrogenase(ICDH), pyruvate dehydrogenase (PDH), and argininosuccinate synthase ona chromosome of the coryneform bacterium and/or
TATAAT sequence or TATAAC sequence is introduced into -10 region ofthat promoter sequence.

Invention 1-2 (Claim 2)
1-D
A process according to Claim 1,
1-C-1
wherein a promoter of glutamate dehydrogenase (GDH)-producing gene has

1-C-2

at least one DNA sequence selected from the group consisting of TTGTCA,TTGACA, and TTGCCA in 35 region and/or
TATAAT sequence in -10 region.

Invention 1-3 (Claim 3)
1-F
A process according to Claim 2,
1-E-1
wherein a promoter of GDH has TTGACA sequence or TTGCCA sequencein -35 region and/or
1-E-2
TATAAT in -10 region.
Invention 1-4 (Claim 4)
1-H
1-G-1
1-G-2

A process according to Claim 1,
wherein a promoter of CS has TTGACA sequence in -35 region and/or
TATAAT sequence or TATAAC sequence in -10 region.

End
140

Attachment 4-4 "Division into Constituent Features (Corrected Invention 1)"
Corrected Invention 1-1 (Claim 1)
1-B'
A process for producing L-glutamic acid by the fermentation, which
comprises the steps of culturing a bacterium in a medium to form and also
accumulate L-glutamic acid in the medium, and collecting L-glutamic acidfrom the medium,
1-A-3
wherein the bacterium is a coryneform bacterium in which
1-A'-1TTGTCA sequence is introduced into -35 region and TATAAT sequence isintroduced into -10 region of the promoter sequence of glutamate

1-A'-2dehydrogenase (GDH) gene on a chromosome of the coryneform bacterium
and/orTATAAT sequence is introduced into -10 region of the promoter sequenceof citrate synthase (CS) gene on a chromosome of that bacterium.

Corrected Invention 1-2 (Claim 2)
1-D
A process according to Claim 1,
1-C'-1
wherein the promoter of GDH gene has TTGTCA sequence in -35 region
and
1-C'-2TATAAT sequence in -10 region.
Corrected Invention 1-3 (Claim 4)
1-H
A process according to Claim 1,
1-G'
wherein the promoter of CS gene has TATAAT sequence in -10 region or
has TTGACA sequence in -35 region and TATAAT sequence in -10 region,
and
1-G''
the promoter of GDH gene has TTGTCA sequence in -35 region and
TATAAT sequence in -10 region.

141

Attachment 5-1 "Claims (Patent 2)"[Claim 1] (Invention 2-1)
A coryneform bacterium having an L-glutamic acid-producing ability,
wherein a mutant-type yggB gene is introduced into the coryneform bacterium, andthereby the L-glutamic acid-producing ability of the coryneform bacterium is enhanced
as compared to a non-modified strain,
wherein any of the following mutations (i), (i'), (i''), or (ii) is introduced into the mutanttype yggB gene:(i) deletion of a region of amino acid numbers 419 to 533 of an amino acid sequence of
SEQ ID NO: 6, 68, 84, or 85, or a region of amino acid numbers 419 to 529 of SEQ ID
NO: 62;(i') insertion of an insertion sequence or a transposon into a region of amino acid numbers
419 to 533 of an amino acid sequence of SEQ ID NO: 6, 68, 84, or 85, or a region ofamino acid numbers 419 to 529 of SEQ ID NO: 62;(i'') a mutation in which proline in a region of amino acid numbers 419 to 533 of an aminoacid sequence of SEQ ID NO: 6, 68, 84, or 85, or a region of amino acid numbers 419 to
529 of SEQ ID NO: 62 is substituted with another amino acid;or(ii) substitution, deletion, or insertion of one or several amino acids in a region selectedfrom the group consisting of amino acid numbers 1 to 23, 86 to 108, and 110 to 132 of anamino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85.
[Claim 4] (Invention 2-2)
The coryneform bacterium according to Claim 1, wherein the mutation (ii) is a mutationin which alanine at position 100 and/or alanine at position 111 is substituted with anotheramino acid, in the amino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85.
[Claim 6] (Invention 2-3)
The coryneform bacterium according to any of Claims 1 to 5, wherein the mutant-type
yggB gene is a mutant-type yggB gene selected from the group consisting of the following(a) to (n):(a) DNA encoding an amino acid sequence of SEQ ID NO: 8;(b) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 8, wherein the DNA is introduced into the coryneform bacterium, and thereby the Lglutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;
142

(c) DNA encoding an amino acid sequence of SEQ ID NO: 20;(d) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 20, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(e) DNA encoding an amino acid sequence of SEQ ID NO: 22;(f) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 22, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(g) DNA encoding an amino acid sequence of SEQ ID NO: 24;(h) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 24, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(i) DNA encoding an amino acid sequence of SEQ ID NO: 64;(j) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 64, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(k) DNA encoding an amino acid sequence of SEQ ID NO: 70;(l) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 70, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(m) DNA encoding an amino acid sequence of SEQ ID NO: 74; and(n) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 74, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin.
143

[Claim 10] (Invention 2-4)
The coryneform bacterium according to any of Claims 1 to 9, wherein the coryneformbacterium belongs to the genus Corynebacterium or the genus Brevibacterium.
[Claim 11] (Invention 2-5)
A process for producing L-glutamic acid, comprising culturing a coryneform bacterium
according to any of Claims 1 to 10 in a medium so as to cause production andaccumulation of L-glutamic acid in the medium or the bacterium, and collecting the Lglutamic acid from the medium or the bacterium.
End

144

Attachment 5-2 "Corrected Claims (Patent 2)"[Claim 1] (Corrected Invention 2-1)
A coryneform bacterium having an L-glutamic acid-producing ability,
wherein a mutant-type yggB gene is introduced into the coryneform bacterium, andthereby the L-glutamic acid-producing ability of the coryneform bacterium is enhanced
as compared to a non-modified strain,
wherein any of the following mutations (i), (i'), (i''), or (ii) is introduced into the mutanttype yggB gene:(i) deletion of a region of amino acid numbers 419 to 533 of an amino acid sequence of
SEQ ID NO: 6, 68, 84, or 85, or a region of amino acid numbers 419 to 529 of SEQ ID
NO: 62;(i') insertion of an insertion sequence or a transposon into a region of amino acid numbers
419 to 533 of an amino acid sequence of SEQ ID NO: 6, 68, 84, or 85, or a region ofamino acid numbers 419 to 529 of SEQ ID NO: 62;(i'') a mutation in which proline in a region of amino acid numbers 419 to 533 of an aminoacid sequence of SEQ ID NO: 6, 68, 84, or 85, or a region of amino acid numbers 419 to
529 of SEQ ID NO: 62 is substituted with another amino acid;or(ii) substitution, deletion, or insertion of one to five amino acids in a region selected fromthe group consisting of amino acid numbers 1 to 23, 86 to 108, and 110 to 132 of an aminoacid sequence of SEQ ID NO: 6, 62, 68, 84, or 85.
[Claim 4] (Corrected Invention 2-2)
A coryneform bacterium having an L-glutamic acid-producing ability,
wherein a mutant-type yggB gene is introduced into the coryneform bacterium, andthereby the L-glutamic acid-producing ability of the coryneform bacterium is enhanced
as compared to a non-modified strain,
wherein the mutation in the mutant-type yggB gene is a mutation in which alanine atposition 100 is substituted with threonine, and/or alanine at position 111 is substituted
with threonine or valine, in the amino acid sequence SEQ ID NO: 6, 62, 68, 84, or 85.
[Claim 6] (Corrected Invention 2-3)
The coryneform bacterium according to any of Claims 1, 2, 4, and 5, wherein the mutanttype yggB gene is a mutant-type yggB gene selected from the group consisting of thefollowing (a) to (n):(a) DNA encoding an amino acid sequence of SEQ ID NO: 8;(b) DNA encoding a protein having an amino acid sequence in which one to five amino145

acids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 8, wherein the DNA is introduced into the coryneform bacterium, and thereby the Lglutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(c) DNA encoding an amino acid sequence of SEQ ID NO: 20;(d) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 20, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(e) DNA encoding an amino acid sequence of SEQ ID NO: 22;(f) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 22, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(g) DNA encoding an amino acid sequence of SEQ ID NO: 24;(h) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 24, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(i) DNA encoding an amino acid sequence of SEQ ID NO: 64;(j) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 64, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(k) DNA encoding an amino acid sequence of SEQ ID NO: 70;(l) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 70, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(m) DNA encoding an amino acid sequence of SEQ ID NO: 74; and(n) DNA encoding a protein having an amino acid sequence in which one to five amino146acids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 74, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin.
[Claim 10] (Corrected Invention 2-4)
The coryneform bacterium according to any of Claims 1, 2, and 4 to 9, wherein the
coryneform bacterium belongs to the genus Corynebacterium or the genus
Brevibacterium.
[Claim 11] (Corrected Invention 2-5)
A process for producing L-glutamic acid, comprising culturing a coryneform bacterium
according to any of Claims 1, 2, and 4 to 10 in a medium so as to cause production andaccumulation of L-glutamic acid in the medium, and collecting the L-glutamic acid fromthe medium.

147

Attachment 5-3 "Re-corrected Claims (Patent 2)"[Claim 4] (Re-corrected Invention 2-2) (the same contents as Corrected Invention 2-2)
A coryneform bacterium having an L-glutamic acid-producing ability,
wherein a mutant-type yggB gene is introduced into the coryneform bacterium, andthereby the L-glutamic acid-producing ability of the coryneform bacterium is enhanced
as compared to a non-modified strain,
wherein the mutation in the mutant-type yggB gene is a mutation in which alanine atposition 100 is substituted with threonine, and/or alanine at position 111 is substituted
with threonine or valine, in the amino acid sequence SEQ ID NO: 6, 62, 68, 84, or 85.
[Claim 13] (Re-corrected Invention 2-3)
The coryneform bacterium according to Claim 4, wherein the mutant-type yggB gene isa mutant-type yggB gene selected from the group consisting of the following (e) to (j):(e) DNA encoding an amino acid sequence of SEQ ID NO: 22;(f) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 22, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(g) DNA encoding an amino acid sequence of SEQ ID NO: 24;(h) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 24, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin;(i) DNA encoding an amino acid sequence of SEQ ID NO: 64; and(j) DNA encoding a protein having an amino acid sequence in which one to five aminoacids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ ID
NO: 64, wherein the DNA is introduced into the coryneform bacterium, and thereby the
L-glutamic acid-producing ability of the coryneform bacterium is enhanced when the
coryneform bacterium is cultured in a medium containing an excess of biotin.
[Claim 14] (Re-corrected Invention 2-6)
A process for producing L-glutamic acid, comprising culturing a coryneform bacterium
according to Claim 13 in a medium so as to cause production and accumulation of Lglutamic acid in the medium, and collecting the L-glutamic acid from the medium.

148

End

149

Attachment 5-4 "Division into Constituent Features (Invention 2)"
Invention 2-1 (Claim 1)
2-A
A coryneform bacterium having an L-glutamic acid-producing ability,
2-B
wherein a mutant-type yggB gene is introduced into the coryneformbacterium, and thereby the L-glutamic acid-producing ability of thecoryneform bacterium is enhanced as compared to a non-modified strain,
2-C
wherein any of the following mutations (i), (i'), (i''), or (ii) is introduced into
the mutant-type yggB gene:(i) deletion of a region of amino acid numbers 419 to 533 of an amino acidsequence of SEQ ID NO: 6, 68, 84, or 85, or a region of amino acidnumbers 419 to 529 of SEQ ID NO: 62;(i') insertion of an insertion sequence or a transposon into a region of aminoacid numbers 419 to 533 of an amino acid sequence of SEQ ID NO: 6, 68,
84, or 85, or a region of amino acid numbers 419 to 529 of SEQ ID NO:
62;(i'') a mutation in which proline in a region of amino acid numbers 419 to
533 of an amino acid sequence of SEQ ID NO: 6, 68, 84, or 85, or a regionof amino acid numbers 419 to 529 of SEQ ID NO: 62 is substituted withanother amino acid;or(ii) substitution, deletion, or insertion of one or several amino acids in aregion selected from the group consisting of amino acid numbers 1 to 23,
86 to 108, and 110 to 132 of an amino acid sequence of SEQ ID NO: 6, 62,
68, 84, or 85.
Invention 2-2 (Claim 4)
2-E
The coryneform bacterium according to Claim 1,
2-D
wherein the mutation (ii) is a mutation in which alanine at position 100
and/or alanine at position 111 is substituted with another amino acid, in theamino acid sequence of SEQ ID NO: 6, 62, 68, 84, or 85.
Invention 2-3 (Claim 6)
2-F-2
The coryneform bacterium according to any of Claims 1 to 5,
2-F-1
wherein the mutant-type yggB gene is a mutant-type yggB gene selectedfrom the group consisting of the following (a) to (n):150

2-F-3(a) DNA encoding an amino acid sequence of SEQ ID NO: 8;(b) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 8, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(c) DNA encoding an amino acid sequence of SEQ ID NO: 20;(d) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 20, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(e) DNA encoding an amino acid sequence of SEQ ID NO: 22;(f) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 22, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(g) DNA encoding an amino acid sequence of SEQ ID NO: 24;(h) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 24, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(i) DNA encoding an amino acid sequence of SEQ ID NO: 64;(j) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 64, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(k) DNA encoding an amino acid sequence of SEQ ID NO: 70;151

(l) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 70, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(m) DNA encoding an amino acid sequence of SEQ ID NO: 74; and(n) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 74, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin.
Invention 2-4 (Claim 10)
2-G
The coryneform bacterium according to any of Claims 1 to 9, wherein the
coryneform bacterium belongs to the genus Corynebacterium or the genus
Brevibacterium.
Invention 2-5 (Claim 11)
2-J
2-H
2-I

A process for producing L-glutamic acid,comprising culturing a coryneform bacterium according to any of Claims 1
to 10 in a medium
so as to cause production and accumulation of L-glutamic acid in themedium or the bacterium, and collecting the L-glutamic acid from themedium or the bacterium.

End

152

Attachment 5-5 "Division into Constituent Features (Corrected Invention 2)"
Corrected Invention 2-1 (Claim 1)
2-A
A coryneform bacterium having an L-glutamic acid-producing ability,
2-B
wherein a mutant-type yggB gene is introduced into the coryneformbacterium, and thereby the L-glutamic acid-producing ability of thecoryneform bacterium is enhanced as compared to a non-modified strain,
2-C'
wherein any of the following mutations (i), (i'), (i''), or (ii) is introduced into
the mutant-type yggB gene:(i) deletion of a region of amino acid numbers 419 to 533 of an amino acidsequence of SEQ ID NO: 6, 68, 84, or 85, or a region of amino acidnumbers 419 to 529 of SEQ ID NO: 62;(i') insertion of an insertion sequence or a transposon into a region of aminoacid numbers 419 to 533 of an amino acid sequence of SEQ ID NO: 6, 68,
84, or 85, or a region of amino acid numbers 419 to 529 of SEQ ID NO:
62;(i'') a mutation in which proline in a region of amino acid numbers 419 to
533 of an amino acid sequence of SEQ ID NO: 6, 68, 84, or 85, or a regionof amino acid numbers 419 to 529 of SEQ ID NO: 62 is substituted withanother amino acid;or(ii) substitution, deletion, or insertion of one to five amino acids in a region
selected from the group consisting of amino acid numbers 1 to 23, 86 to
108, and 110 to 132 of an amino acid sequence of SEQ ID NO: 6, 62, 68,
84, or 85.
Corrected Invention 2-2 (Claim 4)
2-E'-1
A coryneform bacterium having an L-glutamic acid-producing ability,
2-E'-2wherein a mutant-type yggB gene is introduced into the coryneformbacterium, and thereby the L-glutamic acid-producing ability of thecoryneform bacterium is enhanced as compared to a non-modified strain,
2-D'

wherein the mutation in the mutant-type yggB gene is a mutation in whichalanine at position 100 is substituted with threonine, and/or alanine atposition 111 is substituted with threonine or valine, in the amino acidsequence SEQ ID NO: 6, 62, 68, 84, or 85.

153

Corrected Invention 2-3 (Claim 6)
2-F'-2The coryneform bacterium according to any of Claims 1, 2, 4, and 5,
2-F-1
wherein the mutant-type yggB gene is a mutant-type yggB gene selectedfrom the group consisting of the following (a) to (n):2-F-3(a) DNA encoding an amino acid sequence of SEQ ID NO: 8;(b) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 8, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(c) DNA encoding an amino acid sequence of SEQ ID NO: 20;(d) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 20, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(e) DNA encoding an amino acid sequence of SEQ ID NO: 22;(f) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 22, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(g) DNA encoding an amino acid sequence of SEQ ID NO: 24;(h) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 24, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(i) DNA encoding an amino acid sequence of SEQ ID NO: 64;(j) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 64, wherein the DNA is introduced into the154coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(k) DNA encoding an amino acid sequence of SEQ ID NO: 70;(l) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 70, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(m) DNA encoding an amino acid sequence of SEQ ID NO: 74; and(n) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 74, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin.
Corrected Invention 2-4 (Claim 10)
2-G'
The coryneform bacterium according to any of Claims 1, 2, and 4 to 9,
wherein the coryneform bacterium belongs to the genus Corynebacteriumor the genus Brevibacterium.
Corrected Invention 2-5 (Claim 11)
2-J
A process for producing L-glutamic acid,
2-H'
comprising culturing a coryneform bacterium according to any of Claims 1,
2, and 4 to 10 in a medium
2-I'
so as to cause production and accumulation of L-glutamic acid in themedium, and collecting the L-glutamic acid from the medium.
End

155

Attachment 5-6 "Division into Constituent Features (Re-corrected Invention 2)"
Re-corrected Invention 2-2 (Claim 4) (the same contents as Corrected Invention 2-2)
2-E'-1
A coryneform bacterium having an L-glutamic acid-producing ability,
2-E'-2wherein a mutant-type yggB gene is introduced into the coryneformbacterium, and thereby the L-glutamic acid-producing ability of thecoryneform bacterium is enhanced as compared to a non-modified strain
2-D'
wherein the mutation in the mutant-type yggB gene is a mutation in whichalanine at position 100 is substituted with threonine, and/or alanine atposition 111 is substituted with threonine or valine, in the amino acidsequence SEQ ID NO: 6, 62, 68, 84, or 85.
Re-corrected Invention 2-3 (Claim 13)
2-F''-2
The coryneform bacterium according to Claim 4,2-F'-1
wherein the mutant-type yggB gene is a mutant-type yggB gene selectedfrom the group consisting of the following (e) to (j):2-F'-3(e) DNA encoding an amino acid sequence of SEQ ID NO: 22;(f) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 22, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(g) DNA encoding an amino acid sequence of SEQ ID NO: 24;(h) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 24, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium
is cultured in a medium containing an excess of biotin;(i) DNA encoding an amino acid sequence of SEQ ID NO: 64; and(j) DNA encoding a protein having an amino acid sequence in which one
to five amino acids are substituted, deleted, inserted, or added in an aminoacid sequence of SEQ ID NO: 64, wherein the DNA is introduced into the
coryneform bacterium, and thereby the L-glutamic acid-producing abilityof the coryneform bacterium is enhanced when the coryneform bacterium156

is cultured in a medium containing an excess of biotin.
Re-corrected Invention 2-6 (Claim 14)
2-M
A process for producing L-glutamic acid,
2-K
comprising culturing a coryneform bacterium according to Claim 13 in a
medium2-L
so as to cause production and accumulation of L-glutamic acid in themedium, and collecting the L-glutamic acid from the medium.
End

157

Attachment 6-1 "Comparison between Defendant's Production Processes 1 and 3 andInvention 1"
1. Regarding Defendant's Production Process 1
As mentioned below, Defendant's Production Process 1 falls within the technicalscopes of all of Inventions 1-1 through 1-4.
(1) Comparison with Invention 1-1
A. Regarding Constituent Feature 1-A-1
The Defendant's Production Process 1 uses "Corynebacterium glutamicum," which isa coryneform bacterium, in which a TTGTCA sequence is introduced into the -35 regionof the promoter sequence of the GDH gene, which is a gene on a chromosome, andtherefore it fulfills Constituent Feature 1-A-1.
B. Regarding Constituent Feature 1-A-2
The Defendant's Production Process 1 uses "Corynebacterium glutamicum," which isa coryneform bacterium, in which a TATAAT sequence is introduced into the -10 regionof the promoter sequence of the GDH gene, which is a gene on a chromosome, and a
TATAAT sequence is introduced into the -10 region of the promoter sequence of the CS
gene, which is a gene on a chromosome.
Accordingly, Defendant's Production Process 1 fulfills Constituent Feature 1-A-2.
C. Regarding Constituent Feature 1-A-3
As mentioned in A. and B. above, the Defendant's Production Process 1 uses
Corynebacterium glutamicum, which is a coryneform bacterium, and therefore
Defendant's Production Process 1 fulfills Constituent Feature 1-A-3.
D. Regarding Constituent Feature 1-B
The Defendant's Production Process 1 uses the fermentation method comprising thesteps of culturing "Corynebacterium glutamicum," which is a coryneform bacterium, in aliquid medium containing carbon sources, etc. to form and accumulate glutamic acid, andcollecting glutamic acid from the medium by a separation operation.
Accordingly, Defendant's Production Process 1 fulfills Constituent Feature 1-B.
E. Summary
As mentioned in A. through D. above, Defendant's Production Process 1 fulfills
Constituent Features 1-A-1 through 1-A-3 and 1-B, and therefore it falls within thetechnical scope of Invention 1-1.
(2) Comparison with Invention 1-2
A. Regarding Constituent Features 1-C-1 and 1-C-2
158

As mentioned in (1) A. and B. above, Defendant's Production Process 1 uses
"Corynebacterium glutamicum," which is a coryneform bacterium, in which a TTGTCA
sequence is introduced into the -35 region and a TATAAT sequence is introduced into the-10 region of the promoter sequence of the GDH gene, which is a gene on a chromosomeof the coryneform bacterium.
Accordingly, Defendant's Production Process 1 fulfills Constituent Features 1-C-1
and 1-C-2.
B. Regarding Constituent Feature 1-D
As mentioned in 1. (1) above, Defendant's Production Process 1 uses the method
described in Claim 1 of Patent 1, and therefore it fulfills Constituent Feature 1-D.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 1 fulfills
Constituent Features 1-C-1, 1-C-2, and 1-D, and therefore it falls within the technicalscope of Invention 1-2.
(3) Comparison with Invention 1-3
A. Regarding Constituent Features 1-E-1 and 1-E-2
As mentioned in (1) B. above, in Defendant's Production Process 1, the promoter
sequence of the GDH gene, which is a gene on a chromosome of "Corynebacterium
glutamicum," which is a coryneform bacterium, has a TATAAT sequence in the -10 region,and therefore Defendant's Production Process 1 fulfills Constituent Feature 1-E-2.
B. Regarding Constituent Feature 1-F
As mentioned in (1) and (2) above, Defendant's Production Process 1 uses the method
described in Claim 2 of Patent 1, and therefore it fulfills Constituent Feature 1-F.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 1 fulfills
Constituent Features 1-E-2 and 1-F, and therefore it falls within the technical scope ofInvention 1-3.
(4) Comparison with Invention 1-4
A. Regarding Constituent Features 1-G-1 and 1-G-2
As mentioned in (1) B. above, in Defendant's Production Process 1, the promoter
sequence of the CS gene, which is a gene on a chromosome of a coryneform bacterium,
has a TATAAT sequence in the -10 region, and therefore Defendant's Production Process
1 fulfills Constituent Feature 1-G-2.
B. Regarding Constituent Feature 1-H
159

As mentioned in (1) above, Defendant's Production Process 1 uses the method
described in Claim 1 of Patent 1, and therefore it fulfills Constituent Feature 1-H.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 1 fulfills
Constituent Features 1-G-2 and 1-H, and therefore it falls within the technical scope ofInvention 1-4.
2 Regarding Defendant's Production Process 3
In relation to Invention 1, Defendant's Production Process 3 differs from Defendant's
Production Process 1 in respect to the promoter sequence of the GDH gene of
Corynebacterium glutamicum used in the process, and it is the same as Defendant's
Production Process 1 in other respects.
As mentioned below, Defendant's Production Process 3 falls within the technicalscope of Inventions 1-1 and 1-4.
(1) Comparison with Invention 1-1
A. Regarding Constituent Features 1-A-1 and 1-A-2
Defendant's Production Process 3 uses "Corynebacterium glutamicum," which is acoryneform bacterium, in which a TATAAT sequence is introduced into the -10 region of
the promoter sequence of the CS gene, which is a gene on a chromosome of thecoryneform bacterium.
Accordingly, Defendant's Production Process 3 fulfills Constituent Feature 1-A-2.
B. Regarding Constituent Features 1-A-3 and 1-B
As is the case with Defendant's Production Process 1, Defendant's Production Process
3 fulfills Constituent Features 1-A-3 and 1-B.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 3 fulfills
Constituent Features 1-A-2, 1-A-3, and 1-B, and therefore it falls within the technicalscope of Invention 1-1.
(2) Comparison with Invention 1-4
A. Regarding Constituent Features 1-G-1 and 1-G-2
As is the case with Defendant's Production Process 1, in Defendant's Production
Process 3, the promoter sequence of the CS gene, which is a gene on a chromosome of acoryneform bacterium, has a TATAAT sequence in the -10 region, and therefore it fulfills
Constituent Feature 1-G-2.
B. Regarding Constituent Feature 1-H
160

As is the case with Defendant's Production Process 1, Defendant's Production Process
3 fulfills Constituent Feature 1-H.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 3 fulfills
Constituent Features 1-G-2 and 1-H, and therefore it falls within the technical scope ofInvention 1-4.

161

Attachment 6-2 "Comparison between Defendant's Production Processes 1 and 3 andCorrected Invention 1"
1. Regarding Defendant's Production Process 1
As mentioned below, Defendant's Production Process 1 falls within the technicalscopes of all of Corrected Invention 1-1 through 1-3.
(1) Comparison with Corrected Invention 1-1
A. Regarding Constituent Feature 1-A'-1
Defendant's Production Process 1 uses "Corynebacterium glutamicum," which is acoryneform bacterium, in which a TTGTCA sequence is introduced into the -35 region
and a TATAAT sequence is introduced into the -10 region of the promoter sequence of the
GDH gene, which is a gene on a chromosome, and therefore it fulfills Constituent Feature
1-A'-1.
B. Regarding Constituent Feature 1-A'-2
The Defendant's Production Process 1 uses "Corynebacterium glutamicum," which isa coryneform bacterium, in which a TATAAT sequence is introduced into the -10 regionof the promoter sequence of the CS gene, which is a gene on a chromosome, and therefore
Defendant's Production Process 1 fulfills Constituent Feature 1-A'-2.
C. Regarding Constituent Feature 1-A-3
As mentioned in A. and B. above, the Defendant's Production Process 1 uses
"Corynebacterium glutamicum," which is a coryneform bacterium, and therefore
Defendant's Production Process 1 fulfills Constituent Feature 1-A-3.
D. Regarding Constituent Feature 1-B'
The Defendant's Production Process 1 uses the fermentation method comprising thesteps of culturing "Corynebacterium glutamicum," which is a coryneform bacterium, in aliquid medium containing carbon sources, etc. to form and accumulate glutamic acid, andcollecting glutamic acid from the medium by a separation operation, and therefore
Defendant's Production Process 1 fulfills Constituent Feature 1-B'.
E. Summary
As mentioned in A. through D. above, Defendant's Production Process 1 fulfills
Constituent Features 1-A'-1, 1-A'-2, 1-A-3, and 1-B', and therefore it falls within thetechnical scope of Corrected Invention 1-1.
(2) Comparison with Corrected Invention 1-2
A. Regarding Constituent Features 1-C'-1 and 1-C'-2Defendant's Production Process 1 uses Corynebacterium glutamicum in which the162sequence mentioned in (1) A. above is introduced into the promoter sequence of the GDH
gene, which is a gene on a chromosome, and therefore Defendant's Production Process 1
fulfills Constituent Features 1-C'-1 and 1-C'-2.
B. Regarding Constituent Feature 1-D
As mentioned in (1) above, Defendant's Production Process 1 uses the method
described in Claim 1 of Patent 1 after Correction 1, and therefore it fulfills Constituent
Feature 1-D.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 1 fulfills
Constituent Features 1-C'-1, 1-C'-2, and 1-D, and therefore it falls within the technicalscope of Corrected Invention 1-2.
(3) Comparison with Corrected Invention 1-3
A. Regarding Constituent Feature 1-G'
As mentioned in (1) B. above, in Defendant's Production Process 1, the promoter
sequence of the CS gene, which is a gene on a chromosome of a coryneform bacterium,
has a TATAAT sequence in the -10 region, and therefore it fulfills Constituent Feature 1G'.
B. Regarding Constituent Feature 1-G''
Defendant's Production Process 1 uses Corynebacterium glutamicum in which thesequence mentioned in (1) A. above is introduced into the promoter sequence of the GDH
gene, which is a gene on a chromosome, and therefore Defendant's Production Process 1
fulfills Constituent Feature 1-G''.
C. Regarding Constituent Feature 1-H
As mentioned in (1) above, Defendant's Production Process 1 uses the method
described in Claim 1 of Patent 1 after Correction 1, and therefore it fulfills Constituent
Feature 1-H.
D. Summary
As mentioned in A. through C. above, Defendant's Production Process 1 fulfills
Constituent Features 1-G', 1-G'', and 1-H, and therefore it falls within the technical scopeof Corrected Invention 1-3.
2. Regarding Defendant's Production Process 3
In relation to Corrected Invention 1, Defendant's Production Process 3 differs from
Defendant's Production Process 1 in respect to the promoter sequence of the GDH geneof Corynebacterium glutamicum used in the process, and it is the same as Defendant's
163

Production Process 1 in other respects.
As mentioned below, Defendant's Production Process 3 falls within the technicalscope of Corrected Invention 1-1.
(1) Comparison with Corrected Invention 1-1
A. Regarding Constituent Features 1-A'-1 and 1-A'-2
The Defendant's Production Process 3 uses "Corynebacterium glutamicum," which isa coryneform bacterium, in which a TATAAT sequence is introduced into the -10 regionof the promoter sequence of the CS gene, which is a gene on a chromosome, and therefore
Defendant's Production Process 3 fulfills Constituent Feature 1-A'-2.
B. Regarding Constituent Features 1-A-3 and 1-B'
As is the case with Defendant's Production Process 1, Defendant's Production Process
3 fulfills Constituent Features 1-A-3 and 1-B'.
C. Summary
As mentioned in A. and B. above, Defendant's Production Process 3 fulfills
Constituent Features 1-A'-2, 1-A-3, and 1-B', and therefore it falls within the technicalscope of Corrected Invention 1-1.
(2) Comparison with Corrected Invention 1-3
Defendant's Production Process 3 does not fulfill Constituent Feature 1-G'', andtherefore it does not fall within the technical scope of Corrected Invention 1-3.

164

Attachment 7-1 "Comparison between Defendant's Production Processes 1 through 3 andInvention 2"
1. Regarding Defendant's Production Process 1
As mentioned below, the bacterium used in Defendant's Production Process 1 falls
within the technical scopes of Inventions 2-1 (Claim 1), 2-2 (Claim 4), 2-3 (Claim 6), and
2-4 (Claim 10), and therefore Defendant's Production Process 1, which uses thatbacterium, falls within the technical scope of Invention 2-5 (Claim 11).
(1) Comparison between the bacterium used in Defendant's Production Process 1 andInvention 2-1
A. Regarding Constituent Feature 2-A
Defendant's Production Process 1 uses "Corynebacterium glutamicum," which is acoryneform bacterium having a glutamic acid-producing ability, and therefore thebacterium used in Defendant's Production Process 1 fulfills Constituent Feature 2-A.
B. Regarding Constituent Feature 2-B
Defendant's Production Process 1 uses Corynebacterium glutamicum into which amutant-type yggB gene, wherein the amino acid at position 100 is substituted withthreonine in the amino acid sequence of the yggB gene, is introduced, and thereby the Lglutamic acid-producing ability of the bacterium is enhanced as compared to a nonmodified strain, and therefore the bacterium used in Defendant's Production Process 1
fulfills Constituent Feature 2-B.
C. Regarding Constituent Feature 2-C
The mutant-type yggB gene introduced into Corynebacterium glutamicum, which isa coryneform bacterium used in Defendant's Production Process 1, is one into which amutation wherein alanine at position 100 in the amino acid sequence of SEQ ID NO: 6 in
Description 2 is substituted with threonine is introduced (the mutation mentioned inConstituent Feature 2-C (ii)).
Accordingly, the coryneform bacterium used in Defendant's Production Process 1
fulfills Constituent Feature 2-C.
D. Summary
As mentioned in A. through C. above, the coryneform bacterium used in Defendant's
Production Process 1 fulfills Constituent Features 2-A through 2-C, and therefore it
constitutes the coryneform bacterium described in Claim 1 of Patent 2.
(2) Comparison between the bacterium used in Defendant's Production Process 1 andInvention 2-2
165

A. Regarding Constituent Feature 2-D
As mentioned in (1) C. above, the mutant-type yggB gene introduced into
Corynebacterium glutamicum used in Defendant's Production Process 1 is one into which
a mutation wherein alanine at position 100 in the amino acid sequence of SEQ ID NO: 6
in Description 2 is substituted with threonine is introduced, and therefore it fulfills
Constituent Feature 2-D.
B. Regarding Constituent Feature 2-E
As mentioned in (1) above, the bacterium used in Defendant's Production Process 1
is Corynebacterium glutamicum, which constitutes the coryneform bacterium describedin Claim 1 of Patent 2, and therefore it fulfills Constituent Feature 2-E.
C. As mentioned in A. and B. above, the coryneform bacterium used in Defendant's
Production Process 1 fulfills Constituent Features 2-D and 2-E of Invention 2-2, andtherefore it constitutes the coryneform bacterium described in Claim 4 of Patent 2.
(3) Comparison between the bacterium used in Defendant's Production Process 1 andInvention 2-3
A. Regarding Constituent Features 2-F-1 and 2-F-3
As mentioned in (1) C. above, the mutant-type yggB gene introduced into
Corynebacterium glutamicum, which is a coryneform bacterium that is used in
Defendant's Production Process 1, is one into which a mutation wherein alanine atposition 100 in the amino acid sequence of SEQ ID NO: 6 in Description 2 is substituted
with threonine is introduced, and it encodes an amino acid sequence of SEQ ID NO: 22
in Description 2 (the amino acid sequence mentioned in 2-F-3 (e)).
Accordingly, this bacterium fulfills Constituent Features 2-F-1 and 2-F-3.
B. Regarding Constituent Feature 2-F-2
As mentioned in (1) and (2) above, the coryneform bacterium used in Defendant's
Production Process 1 constitutes the coryneform bacterium described in Claims 1 and 4
of Patent 2, and therefore it fulfills Constituent Feature 2-F-2.
C. Summary
As mentioned above, the coryneform bacterium used in Defendant's Production
Process 1 fulfills Constituent Features 2-F-1, 2-F-2, and 2-F-3 of Invention 2-3, andtherefore it constitutes the coryneform bacterium described in Claim 6 of Patent 2.
(4) Comparison between the bacterium used in Defendant's Production Process 1 andInvention 2-4
A. Regarding Constituent Feature 2-G
166

As mentioned in (1) through (3) above, the coryneform bacterium used in Defendant's
Production Process 1 is "Corynebacterium glutamicum," which constitutes the
coryneform bacterium described in Claims 1, 4, and 6 of Patent 2, and this bacterium is acoryneform bacterium which belongs to the genus Corynebacterium, and therefore it
fulfills Constituent Feature 2-G.
B. Summary
As mentioned above, the coryneform bacterium used in Defendant's Production
Process 1 fulfills Constituent Feature 2-G of Invention 2-4, and therefore it constitutes
the coryneform bacterium described in Claim 10 of Patent 2.
(5) Comparison between Defendant's Production Process 1 and Invention 2-5
A. Regarding Constituent Feature 2-H
As mentioned in (1) through (4) above, Defendant's Production Process 1 uses
"Corynebacterium glutamicum," which constitutes the coryneform bacterium describedin Claims 1, 4, 6, and 10 of Patent 2, and this bacterium is cultured in a medium containing
various ingredients including carbon sources.
Accordingly, Defendant's Production Process 1 fulfills Constituent Feature 2-H.
B. Regarding Constituent Feature 2-I
Defendant's Production Process 1 collects glutamic acid from fermented liquid thatserves as the medium in which Corynebacterium glutamicum, which is a coryneformbacterium, was cultured, and therefore it fulfills Constituent Feature 2-I.
C. Regarding Constituent Feature 2-J
Defendant's Production Process 1 is a process for producing glutamic acid, andtherefore it fulfills Constituent Feature 2-J.
D. As mentioned in A. through C. above, Defendant's Production Process 1 fulfills
Constituent Features 2-H through 2-J, and therefore it falls within the technical scope ofInvention 2-5.
2 Regarding Defendant's Production Processes 2 and 3
Defendant's Production Processes 2 and 3 use Corynebacterium glutamicum into
which the same mutant-type yggB gene as that in Defendant's Production Process 1 isintroduced, and therefore in relation to Invention 2, they fulfill the respective Constituent
Features in the same manner as Defendant's Production Process 1.
Accordingly, both Defendant's Production Processes 2 and 3 fall within the technicalscope of Invention 2-5.

167

Attachment 7-2 "Comparison between Defendant's Production Processes 1 through 3 andCorrected Invention 2"
1. Regarding Defendant's Production Process 1
As mentioned below, the bacterium used in Defendant's Production Process 1 falls
within the technical scopes of Corrected Inventions 2-1 (Claim 1), 2-2 (Claim 4), 2-3(Claim 6), and 2-4 (Claim 10), and therefore Defendant's Production Process 1, whichuses that bacterium, falls within the technical scope of Corrected Invention 2-5 (Claim
11).
(1) Comparison between the bacterium used in Defendant's Production Process 1 andInvention 2-1
A. Regarding Constituent Feature 2-A
Defendant's Production Process 1 uses "Corynebacterium glutamicum," which is acoryneform bacterium having a glutamic acid-producing ability, and therefore thebacterium used in Defendant's Production Process 1 fulfills Constituent Feature 2-A.
B. Regarding Constituent Feature 2-B
Defendant's Production Process 1 uses Corynebacterium glutamicum into which amutant-type yggB gene, wherein the amino acid at position 100 is substituted withthreonine in the amino acid sequence of the yggB gene, is introduced, and thereby the Lglutamic acid-producing ability of the bacterium is enhanced as compared to a nonmodified strain, and therefore the bacterium used in Defendant's Production Process 1
fulfills Constituent Feature 2-B.
C. Regarding Constituent Feature 2-C'
The mutant-type yggB gene introduced into Corynebacterium glutamicum, which isa coryneform bacterium that is used in Defendant's Production Process 1, is one into
which a mutation wherein alanine at position 100 in the amino acid sequence of SEQ ID
NO: 6 in Description 2 is substituted with threonine is introduced (the mutation mentionedin Constituent Feature 2-C' (ii)).
Accordingly, the coryneform bacterium used in Defendant's Production Process 1
fulfills Constituent Feature 2-C'.
D. Summary
As mentioned in A. through C. above, the coryneform bacterium used in Defendant's
Production Process 1 fulfills Constituent Features 2-A, 2-B, and 2-C', and therefore it
constitutes the coryneform bacterium described in Claim 1 of Patent 2 after Correction 2.
(2) Comparison between the bacterium used in Defendant's Production Process 1 and168

Corrected Invention 2-2
A. Regarding Constituent Features 2-E'-1 and 2-E'-2
As mentioned in (1) A. and B. above, Defendant's Production Process 1 uses
Corynebacterium glutamicum into which a mutant-type yggB gene is introduced, andtherefore the bacterium used in Defendant's Production Process 1 fulfills Constituent
Features 2-E'-1 and 2-E'-2.
B. Regarding Constituent Feature 2-D'
As mentioned in (1) C. above, the mutant-type yggB gene introduced into
Corynebacterium glutamicum, which is a coryneform bacterium that is used in
Defendant's Production Process 1, is one into which a mutation wherein alanine atposition 100 in the amino acid sequence of SEQ ID NO: 6 in Description 2 is substituted
with threonine is introduced, and therefore the coryneform bacterium used in Defendant's
Production Process 1 fulfills Constituent Feature 2-D'.
C. As mentioned in A. and B. above, the coryneform bacterium used in Defendant's
Production Process 1 fulfills Constituent Features 2-E'-1, 2-E'-2, and 2-D' of Corrected
Invention 2-2, and therefore it constitutes the coryneform bacterium described in Claim
4 of Patent 2 after Correction 2.
(3) Comparison between the bacterium used in Defendant's Production Process 1 andCorrected Invention 2-3
A. Regarding Constituent Features 2-F-1 and 2-F-3
As mentioned in (1) C. above, the mutant-type yggB gene introduced into
Corynebacterium glutamicum, which is a coryneform bacterium that is used in
Defendant's Production Process 1, is one into which a mutation wherein alanine atposition 100 in the amino acid sequence of SEQ ID NO: 6 in Description 2 is substituted
with threonine is introduced, and it encodes an amino acid sequence of SEQ ID NO: 22
in Description 2 (the amino acid sequence mentioned in 2-F-3 (e)).
Accordingly, this bacterium fulfills Constituent Features 2-F-1 and 2-F-3.
B. Regarding Constituent Feature 2-F'-2
As mentioned in (1) and (2) above, the coryneform bacterium used in Defendant's
Production Process 1 constitutes the coryneform bacterium described in Claims 1 and 4
of Patent 2 after Correction 2, and therefore it fulfills Constituent Feature 2-F'-2.
C. Summary
As mentioned above, the coryneform bacterium used in Defendant's Production
Process 1 fulfills Constituent Features 2-F-1, 2-F'-2, and 2-F-3 of Corrected Invention 23, and therefore it constitutes the coryneform bacterium described in Claim 6 after
169

Correction 2.
(4) Comparison between the bacterium used in Defendant's Production Process 1 andCorrected Invention 2-4
A. Regarding Constituent Feature 2-G'
As mentioned in (1) through (3) above, the coryneform bacterium used in Defendant's
Production Process 1 is "Corynebacterium glutamicum," which constitutes the
coryneform bacterium described in Claims 1, 4, and 6 of Patent 2 after Correction 2, andthis bacterium is a coryneform bacterium which belongs to the genus Corynebacterium,
and therefore it fulfills Constituent Feature 2-G'.
B. Summary
As mentioned above, the coryneform bacterium used in Defendant's Production
Process 1 fulfills Constituent Feature 2-G' of Invention 2-4, and therefore it constitutes
the coryneform bacterium described in Claim 10 of Patent 2.
(5) Comparison between Defendant's Production Process 1 and Corrected Invention 2-5
A. Regarding Constituent Feature 2-H'
As mentioned in (1) through (4) above, Defendant's Production Process 1 uses
"Corynebacterium glutamicum," which constitutes the coryneform bacterium describedin Claims 1, 4, 6, and 10 of Patent 2 after Correction 2, and this bacterium is cultured ina medium containing various ingredients including carbon sources.
Accordingly, Defendant's Production Process 1 fulfills Constituent Feature 2-H'.
B. Regarding Constituent Feature 2-I'
Defendant's Production Process 1 collects glutamic acid from fermented liquid thatserves as the medium in which Corynebacterium glutamicum, which is a coryneformbacterium, was cultured, and therefore it fulfills Constituent Feature 2-I'.
C. Regarding Constituent Feature 2-J
Defendant's Production Process 1 is a process for producing glutamic acid, andtherefore it fulfills Constituent Feature 2-J.
D. As mentioned in A. through C. above, Defendant's Production Process 1 fulfills
Constituent Features 2-H', 2-I', and 2-J, and therefore it falls within the technical scopeof Corrected Invention 2-5.
2. Regarding Defendant's Production Processes 2 and 3
Defendant's Production Processes 2 and 3 use Corynebacterium glutamicum into
which the same mutant-type yggB gene as that in Defendant's Production Process 1 is170

introduced, and therefore in relation to Corrected Invention 2, they fulfill the respective
Constituent Features in the same manner as Defendant's Production Process 1.
Accordingly, both Defendant's Production Processes 2 and 3 fall within the technicalscope of Corrected Invention 2-5.

171

Attachment 7-3 "Comparison between Defendant's Production Processes 1 through 3
and Re-corrected Invention 2"
1. Regarding Defendant's Production Process 1
As mentioned below, the bacterium used in Defendant's Production Process 1 falls
within the technical scopes of Re-corrected Inventions 2-2 (Claim 4) and 2-3 (Claim 13),and therefore Defendant's Production Process 1, which uses that bacterium, falls within
the technical scope of Re-corrected Invention 2-6 (Claim 14).
(1) Comparison between the bacterium used in Defendant's Production Process 1 and Recorrected Invention 2-2
While the contents of Re-corrected Invention 2-2 are the same as the contents ofCorrected Invention 2-2, as mentioned in 1. (2) of Attachment 7-2 "Comparison between
Defendant's Production Processes 1 through 3 and Corrected Invention 2," thecoryneform bacterium used in Defendant's Production Process 1 fulfills Constituent
Features 2-E'-1, 2-E'-2, and 2-D', and therefore, it constitutes the coryneform bacterium
described in Claim 4 of Patent 2 after Correction 2.
(2) Comparison between the bacterium used in Defendant's Production Process 1 and Recorrected Invention 2-3
A. Regarding Constituent Features 2-F'-1 and 2-F'-3
The mutant-type yggB gene introduced into Corynebacterium glutamicum, which isa coryneform bacterium that is used in Defendant's Production Process 1, is one into
which a mutation wherein alanine at position 100 in the amino acid sequence of SEQ ID
NO: 6 in Description 2 is substituted with threonine is introduced, and it encodes an aminoacid sequence of SEQ ID NO: 22 in Description 2 (the amino acid sequence mentioned
in 2-F'-3 (e)).
Accordingly, this bacterium fulfills Constituent Features 2-F'-1 and 2-F'-3.
B. Regarding Constituent Feature 2-F''-2
As mentioned in (1) above, the coryneform bacterium used in Defendant's Production
Process 1 constitutes the coryneform bacterium described in Claim 4 of Patent 2 after the
re-correction, and therefore it fulfills Constituent Feature 2-F''-2.
C. Summary
As mentioned in A. and B. above, the coryneform bacterium used in Defendant's
Production Process 1 fulfills Constituent Features 2-F'-1, 2-F''-2, and 2-F'-3 of Recorrected Invention 2-3, and therefore it constitutes the coryneform bacterium describedin Claim 13 of Patent 2 after the re-correction.
172

(3) Comparison between Defendant's Production Process 1 and Re-corrected Invention
2-6
A. Regarding Constituent Feature 2-Ｋ
As mentioned in (2) above, Defendant's Production Process 1 uses Corynebacterium
glutamicum, which constitutes the coryneform bacterium described in Claim 13 of Patent
2 after the re-correction, and this bacterium is cultured in a medium containing various
ingredients including carbon sources.
Accordingly, Defendant's Production Process 1 fulfills Constituent Feature 2-K.
B. Regarding Constituent Feature 2-L
Defendant's Production Process 1 collects glutamic acid from fermented liquid thatserves as the medium in which Corynebacterium glutamicum, which is a coryneformbacterium, was cultured, and therefore it fulfills Constituent Feature 2-L.
C. Regarding Constituent Feature 2-M
Defendant's Production Process 1 is a process for producing glutamic acid, andtherefore it fulfills Constituent Feature 2-M.
D. As mentioned in A. through C. above, Defendant's Production Process 1 fulfills
Constituent Features 2-K, 2-L, and 2-M, and therefore it falls within the technical scopeof Re-corrected Invention 2-6.
2. Regarding Defendant's Production Processes 2 and 3
Defendant's Production Processes 2 and 3 use Corynebacterium glutamicum into
which the same mutant-type yggB gene as that in Defendant's Production Process 1 isintroduced, and therefore in relation to Re-corrected Invention 2, they fulfill therespective Constituent Features in the same manner as Defendant's Production Process 1.
Accordingly, both Defendant's Production Processes 2 and 3 fall within the technicalscope of Re-corrected Invention 2-6.

173

Attachment 8-1 "Comparison between Defendant's Production Process 4 and Invention
2"
1. Comparison between the strains used in Defendant's Production Process 4 andInventions 2-1, 2-2, and 2-3(1) Comparison between the strains used in Defendant's Production Process 4 andInvention 2-1
A. Regarding Constituent Feature 2-A
Defendant's Production Process 4 uses "Corynebacterium glutamicum," which is acoryneform bacterium having a glutamic acid-producing ability, and therefore the strainsused in Defendant's Production Process 4 fulfill Constituent Feature 2-A.
B. Regarding Constituent Feature 2-B
A mutant-type yggB gene, wherein alanine at position 98 is substituted with threonine(A98T mutation) and valine at position 241 is substituted with isoleucine (V241I
mutation) in YggB of wild-type Corynebacterium callunae, is introduced into the strainsused in Defendant's Production Process 4. In addition, the glutamic acid production
amount has increased as compared to a non-modified strain, and therefore, the strains
fulfill Constituent Feature 2-B.
C. Regarding Constituent Feature 2-C
The strains used in Defendant's Production Process 4 are those of Corynebacterium
glutamicum, which is a coryneform bacterium, into which the mutant-type yggB gene
mentioned in B. above is introduced. This mutant-type yggB gene does not coincide with
the DNA encoding an amino acid sequence in which alanine at position 100 is substituted
with threonine in the amino acid sequence of SEQ ID NO: 6 in Description 2 (themutation mentioned in (ii)), and it is not one into which any of the other mutationsprescribed in Constituent Feature 2-C is introduced.
Accordingly, the strains used in Defendant's Production Process 4 do not fulfill
Constituent Feature 2-C.
(2) Comparison between the strains used in Defendant's Production Process 4 andInvention 2-2
A. Regarding Constituent Feature 2-D
Since the strains used in Defendant's Production Process 4 are as mentioned in (1) B.
above, their mutation does not coincide with one in which alanine at position 100 is
substituted with threonine in the amino acid sequence of SEQ ID NO: 6 in Description 2,
and the strains are not those into which any of the other mutations prescribed in
174Constituent Feature 2-D is introduced.
B. Regarding Constituent Feature 2-E
As mentioned in (1) above, the strains used in Defendant's Production Process 4 do
not fulfill the part of Constituent Feature 2-E relating to Constituent Feature 2-C ofInvention 2-1.
(3) Comparison between the strains used in Defendant's Production Process 4 andInvention 2-3
As mentioned in (1) B. above, the mutant-type yggB gene of the strains used in
Defendant's Production Process 4 does not coincide with the DNA encoding an aminoacid sequence in which alanine at position 100 is substituted with threonine in the aminoacid sequence of SEQ ID NO: 6 in Description 2 (the amino acid sequence of SEQ ID
NO: 22 in 2-F-3 (e)), and it is not one into which any of the other mutations prescribed inConstituent Feature 2-F-3 is introduced.
In addition, according to (1) and (2) above, it also does not fulfill Constituent Feature
2-F-2.
Accordingly, the strains used in Defendant's Production Process 4 do not fulfill
Constituent Features 2-F-1, 2-F-2, and 2-F-3.
2. Comparison between Defendant's Production Process 4 and Invention 2-5(1) Regarding Constituent Feature 2-H
In Defendant's Production Process 4, the coryneform bacterium used is cultured in a
medium containing various ingredients including carbon sources.
Accordingly, if the strains used are equivalent to the coryneform bacterium describedin Claim 6 (Invention 2-3), they fulfill Constituent Feature 2-H.
(2) Regarding Constituent Feature 2-I
Defendant's Production Process 4 collects glutamic acid from fermented liquid thatserves as the medium in which the coryneform bacterium was cultured, and therefore it
fulfills Constituent Feature 2-I.
(3) Regarding Constituent Feature 2-J
Defendant's Production Process 4 is a process for producing glutamic acid, andtherefore it fulfills Constituent Feature 2-J.

175

Attachment 8-2 "Comparison between Defendant's Production Process 4 and Corrected
Invention 2"
1. Comparison between the strains used in Defendant's Production Process 4 andCorrected Inventions 2-2 and 2-3(1) Comparison between the strains used in Defendant's Production Process 4 andCorrected Invention 2-2
A. Regarding Constituent Feature 2-E'-1
Defendant's Production Process 4 uses "Corynebacterium glutamicum," which is acoryneform bacterium having a glutamic acid-producing ability, and therefore the strainsused in Defendant's Production Process 4 fulfill Constituent Feature 2-E'-1.
B. Regarding Constituent Feature 2-E'-2
A mutant-type yggB gene, wherein alanine at position 98 is substituted with threonine(A98T mutation) and valine at position 241 is substituted with isoleucine (V241I
mutation) in YggB of wild-type Corynebacterium callunae, is introduced into the strainsused in Defendant's Production Process 4. In addition, the glutamic acid production
amount has increased as compared to a non-modified strain, and therefore, the strains
fulfill Constituent Feature 2-E'-2.
C. Regarding Constituent Feature 2-D'
Since the strains used in Defendant's Production Process 4 are as mentioned in B.
above, their mutation does not coincide with one in which alanine at position 100 is
substituted with threonine in the amino acid sequence of SEQ ID NO: 6 in Description 2,
and the strains are not those into which any of the other mutations prescribed inConstituent Feature 2-D' is introduced, and therefore the strains do not fulfill Constituent
Feature 2-D'.
(2) Comparison between the strains used in Defendant's Production Process 4 andCorrected Invention 2-3
As mentioned in (1) B. above, the mutant-type yggB gene of the strains used in
Defendant's Production Process 4 does not coincide with the DNA encoding an aminoacid sequence in which alanine at position 100 is substituted with threonine in the aminoacid sequence of SEQ ID NO: 6 in Description 2 (the amino acid sequence of SEQ ID
NO: 22 in 2-F-3 (e)), and it is not one into which any of the other mutations prescribed inConstituent Feature 2-F-3 is introduced.
In addition, according to (1) above, it also does not fulfill Constituent Feature 2-F'-2.
Accordingly, the strains used in Defendant's Production Process 4 do not fulfill
176Constituent Features 2-F-1, 2-F'-2, and 2-F-3.
2. Comparison between Defendant's Production Process 4 and Corrected Invention 2-5(1) Regarding Constituent Feature 2-H'
In Defendant's Production Process 4, the coryneform bacterium used is cultured in a
medium containing various ingredients including carbon sources.
Accordingly, if the strains used are equivalent to the coryneform bacterium describedin Claim 6 after the correction (Corrected Invention 2-3), they fulfill Constituent Feature
2-H'.
(2) Regarding Constituent Feature 2-I'
Defendant's Production Process 4 collects glutamic acid from fermented liquid thatserves as the medium in which the coryneform bacterium was cultured, and therefore it
fulfills Constituent Feature 2-I'.
(3) Regarding Constituent Feature 2-J
Defendant's Production Process 4 is a process for producing glutamic acid, andtherefore it fulfills Constituent Feature 2-J.

177

Attachment 8-3 "Comparison between Defendant's Production Process 4 and Recorrected Invention 2"
1. Comparison between the strains used in Defendant's Production Process 4 and Recorrected Inventions 2-2 and 2-3(1) Comparison between the strains used in Defendant's Production Process 4 and Recorrected Invention 2-2Re-corrected Invention 2-2 is identical to Corrected Invention 2-2. Accordingly, as
mentioned in 1. (1) of Attachment 8-2 "Comparison between Defendant's Production
Process 4 and Corrected Invention 2," the strains used in Defendant's Production Process
4 fulfill Constituent Features 2-E'-1 and 2-E'-2, and do not fulfill Constituent Feature 2D'.
(2) Comparison between the strains used in Defendant's Production Process 4 and Recorrected Invention 2-3
The strains used in Defendant's Production Process 4 are those of Corynebacterium
glutamicum, which is a coryneform bacterium, into which a mutant-type yggB gene,
wherein alanine at position 98 is substituted with threonine (A98T mutation) and valine
at position 241 is substituted with isoleucine (V241I mutation) in YggB of wild-type
Corynebacterium callunae, is introduced. This mutant-type yggB gene does not coincidewith the DNA encoding an amino acid sequence in which alanine at position 100 is
substituted with threonine in the amino acid sequence of SEQ ID NO: 6 in Description 2(the amino acid sequence of SEQ ID NO: 22 in 2-F'-3 (e)), and it is not one into which
any of the other mutations prescribed in Constituent Feature 2-F'-3 is introduced.
In addition, according to (1) above, it also does not fulfill Constituent Feature 2-F''-2.
Accordingly, the strains used in Defendant's Production Process 4 do not fulfill
Constituent Features 2-F'-1, 2-F''-2, and 2-F'-3.
2. Comparison between Defendant's Production Process 4 and Re-corrected Invention 26(1) Regarding Constituent Feature 2-Ｋ
In Defendant's Production Process 4, the coryneform bacterium used is cultured in a
medium containing various ingredients including carbon sources.
Accordingly, if the strains used are equivalent to the coryneform bacterium describedin Claim 13 of Patent 2 after the re-correction (Re-corrected Invention 2-3), they fulfill
Constituent Feature 2-K.
178

(2) Regarding Constituent Feature 2-L
Defendant's Production Process 4 collects glutamic acid from fermented liquid thatserves as the medium in which the coryneform bacterium was cultured, and therefore it
fulfills Constituent Feature 2-L.
(3) Regarding Constituent Feature 2-M
Defendant's Production Process 4 is a process for producing glutamic acid, andtherefore it fulfills Constituent Feature 2-M.

179

Attachment 9 "Comparison Table of Allegations concerning Processes for Producing the
MSG in Question"
Defendant's allegations
Period●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●

No.

Strain

CS

No.

-10

[i]

[iv]

TATAAT

[vi]

[vii]

[ix]

[x]

[xi]

TTGTCATATAAT●●●(omitted)(omitted)(omitted)(omitted)

●

●

●

●

●

●

●

●

(omitted)(omitted)(omitted)(omitted)

●

●

●

●

●

●

(omitted)(omitted)

●

●

●

●

●

●

(omitted)(omitted)

●

●

●

●

●

●

(omitted)(omitted)●●(omitted)(omitted)(omitted)

TATAATTATAATTATAAT●[viii]

-10

●

●
[v]

-35

Plaintiff's

ygg

alternative

principal

BclaimsclaimsA100

Defendant's

T

Production*

Process 1

Unknown due to lack of data

[ii]

[iii]

GDH

Plaintiff's

A100
T

A100
T

A100
T

A100
T

A100
T

Defendant's
Production
Process 2
Defendant's
Production
Process 2
Defendant's
Production
Process 3
Defendant's

Defendant's

Production

Production

Process 3

Process 1

Defendant's
Production
Process 3
Defendant's

●

●

●

●

(omitted)(omitted)(omitted)(omitted)

●

●

●

●

●

●

●

●

(omitted)(omitted)(omitted)(omitted)

●

●

●

●

●

●

●

●

(omitted)(omitted)(omitted)(omitted)

●

●

●

●

●

●

●

●

A100

Defendant's(omitted)(omitted)(omitted)(omitted)T

Production

180

A100
T

A100
T

A100
T

Production
Process 2
Defendant's
Production
Process 2
Defendant's
Production
Process 2

●(omitted)●●(omitted)●●(omitted)
●

[xii]

[xiii]

[xiv]

●

●

●

●

Process 2

●

●

●

●

Defendant's(omitted)(omitted)(omitted)(omitted)

●

●

●

●

Process 4

●

●

●

●

Defendant's(omitted)(omitted)(omitted)(omitted)

●

●

●

●

●

●

●

●

(omitted)(omitted)(omitted)(omitted)

●

●

●

●

**

**

Production

Production
Process 4Nomutation* Alanine at position 100 is substituted with threonine.
** ●(omitted)● in yggB of Corynebacterium glutamicum, and alanine at position 98
is substituted with threonine and valine at position 241 is substituted with isoleucine in
yggB derived from Corynebacterium callunae.

181

Attachment 10 "List of Sales Values/Volumes"
1. Sales values (for 2011 through 2017)
2011

2012

2013

2014

2015

2016

2017

Total

Portion sold by●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)●
the DefendantPortion sold by●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)●CJ Indonesia
Total sales value●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)●(Unit: 1,000 yen)
2. Sales volumes (for 2011 through 2017)
2011

2012

2013

2014

2015

2016

2017

Total

Portion sold by●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)●
the DefendantPortion sold by●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)●CJ Indonesia
Total sales value●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)● ●(omitted)●(Unit: MT)

182

Attachment 11-1 "Allegations on the Value of Damages 1" (Value of Damages under
Article 102, Paragraph (2) of the Patent Act for the Overall Portion Sold by the Defendant,etc.)[Plaintiff's allegations]
1. Outline of the allegations
As mentioned in No. 3, 15. [Plaintiff's allegations] (1) of the text, joint tort is foundto be established between the Defendant and CJ Indonesia with regard to the overall
portion sold by the Defendant, etc. Therefore, the Defendant is liable for tort due to theinfringement of the respective Patent Rights with regard to the overall portion sold by the
Defendant, etc.
Therefore, the total profits made by the Defendant, etc. in relation to the overall
portion sold by the Defendant, etc. during the subject period (as mentioned in 2. below,
in relation to Patent Right 2, the period on and after August 23, 2013 within that period)
will be the value of damages to be compensated by the Defendant pursuant to Article 102,
paragraph (2) of the Patent Act.
2. Profits made by the Defendant, etc. during the subject period(1) Sales amountThe total sales amount relating to the portion sold by the Defendant, etc. during the
subject period is ●(omitted)● yen (No. 3, 15. [Plaintiff's allegations] (2) of the text).
(2) Regarding the expenses borne by the Defendant, etc.
A. Sales volumes of the Defendant, etc.
The sales volumes of the Defendant, etc. during the subject period are as shown in
No. 3, 15. [Plaintiff's allegations] (2) of the text.
B. Production cost for the MSG in question(A) Production costs per ton
The production costs per ton (unit: U.S. dollars) of the MSG in question for 2009
through 2014 are as shown in the table below.
They indicate the total amounts of the fermentation material expenses, purificationmaterial expenses, utility expenses, and packaging expenses out of the expenses stated inmaterial disclosed by the Defendant (Exhibit Otsu 107). Meanwhile, the expenses statedin that material as fixed expenses (labor, depreciation, and others) should not be included
as expenses in calculating the profit under Article 102, paragraph (2) of the Patent Act.
As the costs for other years are not stated in that material, the costs for 2007 and 2008
are deemed to be the same amount as the cost for 2009, and the costs for 2015 through2017 are deemed to be the same amount as the cost for 2014 in the calculation.
183

2009

2010

2011

2012

2013

2014

●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●(B) Yen-dollar exchange rates
The exchange rates of U.S. dollars to yen during the subject period are as shown in
the table below.
2007

2008

2009

2010

2011

2012

117.8

103.4

93.6

87.8

79.8

79.8

2013

2014

2015

2016

2017

97.6

105.9

121.0

108.8

112.2

(C) Production costs in yen
When the sales volumes mentioned in A. above are multiplied by the production costs
per ton mentioned in (A) above and the exchange rates mentioned in (B) above, theproduction costs relating to the portion sold by the Defendant, etc. during the subject
period come to a total of ●(omitted)● yen.
C. Expenses for international transportation
The expenses for international transportation (meaning the expenses fortransportation from the factory in Indonesia to Japan; the same applies hereinafter) per
ton of the MSG in question during the subject period are calculated as follows.
(A) The expenses for international transportation per ton in U.S. dollars are as shown in
the table below (unit: U.S. dollars).
2011

2012

2013

2014

2015

2016

2017

●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●(B) When the amounts of expenses in U.S. dollars mentioned in (A) above are converted
into yen by using the exchange rates mentioned in B. (B) above, the average amount ofexpenses per ton will be ●(omitted)● yen, and it is reasonable to use that amount forcalculating the expenses for international transportation during the subject period.
Accordingly, the expenses for international transportation for the sales volumes of the
Defendant, etc. during the subject period mentioned in A. above come to a total of ●(omitted)● yen.
D. Domestic freight expenses and other selling, general, and administrative expenses
From among the selling, general, and administrative expenses of the Defendant statedin material disclosed by the Defendant (Exhibit Otsu 99), those that should be found to
be expenses in calculating the profit under Article 102, paragraph (2) of the Patent Act
are only the freight expenses and warehouse expenses shown in the table below. As theamounts of these expenses for 2007 through 2010 are unknown, they are deemed to be
the same amounts as those for 2011 in the calculation.
184

Thus, the amount that should be found to be expenses as the selling, general, andadministrative expenses relating to the portion sold by the Defendant, etc. during the
subject period comes to a total of ●(omitted)● yen.
2011

2012

2013

2014

2015

2016

2017

Freight●(omitted)●(omitted)●(omitted)●(omitted)●(omitted)●(omitted)●(omitted)

expenses

●

●

●

●

●

●

●

Warehouse●(omitted)●(omitted)●(omitted)●(omitted)●(omitted)●(omitted)●(omitted)

expenses

●

●

●

●

●

●

●

(unit: 1,000 yen)
E. Total amount of expenses
The total amount of expenses that should be taken into consideration for the portion
sold by the Defendant, etc. during the subject period will be the sum of the amounts
mentioned in B. through D. above, which comes to ●(omitted)● yen.
(3) Profits made by the Defendant, etc.
When the total amount of expenses mentioned in (2) E. above is deducted from thesales amount mentioned in (1) above, the amount of profits made by the Defendant, etc.
in relation to the portion sold by the Defendant, etc. during the subject period comes to4,994,885,970 yen.
3. Value of damages during the subject period for Patent Right 2 (August 23, 2013 throughDecember 31, 2017)(1) Sales amountOf the sales amount relating to the portion sold by the Defendant, etc. for 2013, whichis ●(omitted)● yen, the portion for the period from August 23, 2013 through the last
day of that year is presumed to be ●(omitted)● yen, as calculated pro rata based on thenumber of days.
As the sales amounts for 2014 through 2017 are as shown in 1. of Attachment 10 "Listof Sales Values/Volumes," the sales amount relating to the portion sold by the Defendant,
etc. during the subject period for Patent Right 2 comes to ●(omitted)● yen.
(2) Regarding the expenses borne by the Defendant, etc.
When the expenses for 2013 are calculated for the period on and after August 23 ofthat year pro rata based on the number of days, and the expenses for 2014 through 2017
are calculated by the method stated in 2. (2) above, the amount that should be found to be
expenses with regard to the portion sold by the Defendant, etc. during the subject period
for Patent Right 2 will approximately be as follows:
A. Production cost ●(omitted)● yen
B. Expenses for international transportation ●(omitted)● yen
185

C. Domestic freight expenses, warehouse expenses, other selling, general, andadministrative expenses ●(omitted)● yen(3) Profits made by the Defendant, etc.
When the expenses mentioned in (2) above are deducted from the sales amountmentioned in (1) above, the amount of profits made by the Defendant, etc. in relation to
the portion sold by the Defendant, etc. during the subject period for Patent Right 2 comes
to 2,917,455,000 yen.
4. Relationship between the claims for compensation for damages caused by the
infringement of the Patent Rights
Of the subject period, during the period from January 1, 2007 through August 22,
2013, only Patent Right 1 was infringed, and during the subject period for Patent Right 2
from August 23, 2013 onward, both of the Patent Rights were infringed.
In presuming the value of damages under Article 102, paragraph (2) of the Patent Act,with regard to the period in which only Patent Right 1 was infringed, the entire amountof the profits made by the Defendant, etc. during that period are found to be the damages
caused by the infringement of Patent Right 1. With regard to the period in which both of
the Patent Rights were infringed, the entire amount of profits made by the Defendant, etc.
during that period are presumed to be the damages for the infringement of the respective
Patent Rights, but in light of the purport of Article 102 of the Patent Act, the obligationof the Defendant to make payment remains within the scope of the entire profits made by
the Defendant, etc. during that period.
With regard to the infringement of Patent Right 1, the Plaintiff is presumed to havesustained damages in an amount equal to the amount of profits made by the Defendant,
etc. during the entire subject period, that is, the period during which the relevant patentright was infringed, which is 4,994,885,970 yen (mentioned in 2. above). With regard to
the infringement of Patent Right 2, the Plaintiff is presumed to have sustained damages
in an amount equal to the profits made by the Defendant, etc. during the subject period
for Patent Right 2, that is, the period during which the relevant patent right was infringed,
which is 2,917,455,000 yen (mentioned in 3. above), but because this amount overlaps
with the damages caused by the infringement of Patent Right 1 during that period, thePlaintiff selectively makes claims based on the Patent Rights within the scope of
2,917,455,000 yen, with regard to the infringement of the Patent Rights during that period.
As damages throughout the subject period, the Plaintiff selectively makes claims
based on the Patent Rights within the scope of 4,994,885,970 yen as mentioned in 2.
above. In this lawsuit, the Plaintiff claims 900 million yen from that amount as partial
claims for the damages.
186

5. Regarding grounds for the rebuttal of the presumption(1) Regarding the rebuttal of the presumption based on the sale of competitive products(reduction of the amount according to the market share)
The Defendant alleges that the amount of damages should be reduced according to
the market share of monosodium glutamate. However, in order to rebut the presumption
under Article 102, paragraph (2) of the Patent Act, the Defendant needs to specifically
prove the grounds for which the Plaintiff would not have been able to sell all of the portion
sold by the Defendant, etc., and therefore the rebuttal of the presumption based on themarket share is unreasonable.
The Defendant alleges that, out of the volume of monosodium glutamate imported
from Indonesia into Japan, excluding the portion sold by the Defendant, etc., half wasimported by the Plaintiff. In reality, however, most of that volume was imported by the
Plaintiff, and the Defendant's calculation of the Plaintiff's share in the import volume isunreasonable. It is also unreasonable that, in the analysis of Exhibit Otsu 117, the volumeof imports that were imported as glutamic acid and processed into monosodium glutamate
in Japan (most of which were imports by the Plaintiff) are not taken into consideration.
(2) Regarding the rebuttal of the presumption based on the degree of contribution of the
Patent Rights to damages
The Defendant makes allegations concerning the degree of contribution made by the
Patents by strictly distinguishing the production processes, etc. of glutamic acid. However,as the Patents are processes for producing glutamic acid by using specific bacteria, andthe Defendant, etc. sell glutamic acid produced by using those specific bacteria, it should
be said that the degree of contribution of the Patents is 100%, and there is no groundregarding the rebuttal of the presumption based on the degree of contribution.
6. Regarding overlapped application of Article 102, paragraph (3) of the Patent Act for
the rebutted part
Supposing that the rebuttal of the presumption of damages were to be allowed basedon the existence of competitive products, the value of damages in an amount equivalent
to royalties under Article 102, paragraph (3) of the Patent Act, as mentioned in
Attachment 11-2, should be recognized for the rebutted part.
[Defendant's allegations]
1. Outline of the allegations
The Defendant disputes the Plaintiff's allegations concerning damages.
As mentioned in No. 3, 15. [Defendant's allegations] (1) of the text, the Defendant is
not liable for compensation for damages with regard to the portion sold by CJ Indonesia.
Therefore, when calculating the value of damages under Article 102, paragraph (2) of the187Patent Act, the portion sold by CJ Indonesia should not be taken into consideration.
In addition, as mentioned in No. 3, 15. [Defendant's allegations] (2) of the text, thePlaintiff's allegations concerning the sales values/volumes for 2007 through 2010 are
unreasonable.
2. Regarding the expenses to be deducted
The Defendant disputes the Plaintiff's allegations concerning the amount of expenses
to be deducted.
(1) Regarding selling, general, and administrative expenses
As selling, general, and administrative expenses (Exhibit Otsu 101), not only thefreight expenses and warehouse expenses, but also the personnel expenses, service
expenses, and other expenses should be taken into consideration. The total amount ofselling, general, and administrative expenses for 2011 through 2017 is ●(omitted)●
yen for the portion sold by the Defendant and ●(omitted)● yen for the portion sold by
CJ Indonesia. Whereas Exhibit Otsu 101 only states the selling, general, andadministrative expenses for 2011 onward, if the profits for 2007 through 2010 were to betaken into consideration, the selling, general, and administrative expenses for that period
should also be estimated in a manner similar to that for the sales amounts, by a formulasimilar to that mentioned in No. 3, 15. [Defendant's allegations] (2) B. of the text, which
will come to a total of ●(omitted)● yen for the portion sold by the Defendant and a
total of ●(omitted)● yen for the portion sold by CJ Indonesia.
In accounting, the Defendant recorded the overall amounts of the freight expenses andwarehouse expenses concerning the MSG in question by distributing them between the
expenses for the portion sold by the Defendant and the expenses for the portion sold by
CJ Indonesia (which had not actually occurred).
As for expenses that are common to the respective businesses, such as personnel
expenses, the Defendant has proportionally distributed its overall amount of expenses
among the respective departments according to the internal standards, and from among
the expenses distributed to the department involved in the sale of the MSG in question,
the expenses concerning the MSG in question are calculated according to the proportionof the sales of the MSG in question in the department's total sales. Moreover, on an
accounting basis, expenses concerning the MSG in question are further distributed
between those relating to the portion sold by the Defendant and the portion sold by CJ
Indonesia. Also, while, from among these expenses, as personnel expenses include thepersonnel expenses of operation departments and those of indirect sections, at least thepersonnel expenses of operation departments shown in the table below depend on theworkload of sales operations, so they should be deducted as expenses that became
188additionally required in direct association with the sale of the MSG in question.
2011

2012

2013

2014

2015

2016

2017

●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●●(omitted)●(unit: 1,000 yen)
(2) Regarding purchasing expenses
When considering the profits for the portion sold by the Defendant, the expenses
required for their purchase should be taken into consideration.
The total purchasing expenses for 2011 through 2017 is ●(omitted)● yen (Exhibits
Otsu 99, 101). Whereas Exhibit Otsu 101 only states the selling, general, andadministrative expenses for 2011 onward, if the profits for 2007 through 2010 were to betaken into consideration, the purchasing expenses for that period should also be estimatedin a manner similar to that for the sales amounts, by a formula similar to that mentioned
in No. 3, 15. [Defendant's allegations] (2) B. of the text, which will come to a total of ●(omitted)● yen.
3. Relationship between the claims for compensation for damages caused by the
infringement of the Patent Rights
With regard to the value of damages presumed pursuant to Article 102, paragraph (2)of the Patent Act, the Plaintiff alleges that the value of damages will be the same between
a case where one patent right has been infringed and a case where two patent rights havebeen infringed, as the value of damages will be equal to the total amount of the infringer's
profits in both cases. However, if the number of patents infringed is smaller, the value ofdamages should also be smaller, so the Plaintiff's allegation is unreasonable.
When multiple patent rights have been infringed, it should be regarded that the profit
made by the infringer by the act of infringement as referred to in Article 102, paragraph(2) of the Patent Act with regard to the infringement of one patent right is not the entire
amount of profits of the infringer. Even if such interpretation is not adopted, at least the
rebuttal of the presumption should be allowed according to the number of patent rights
infringed and the degree of contribution of the patent rights, as mentioned in 4. (2) below.
4. Regarding grounds for the rebuttal of the presumption
The presumption of the value of damages under Article 102, paragraph (2) of the
Patent Act should be rebutted based on the following circumstances.
(1) Sale of competitive products (reduction of the amount according to the market share)
Regarding both the profits for the portion sold by the Defendant and the profits forthe portion sold by CJ Indonesia, the value of damages should be reduced according to
the market share
The MSG in question is a chemical substance called monosodium glutamate. As the189products do not change depending on their production process, customers are notinterested in the production process. Even if the Defendant or CJ Indonesia could not sell
the MSG in question, the Plaintiff would not have been able to sell all of the portion that
could not be sold by the Defendant or CJ Indonesia, but would only have been able to sell
the portion according to the Plaintiff's own market share.
When analysis is made based on the trade statics by the Ministry of Finance(Reference Materials 7 attached to Exhibit Otsu 101, Exhibit Otsu 117), out of themonosodium glutamate imported from Indonesia into Japan, excluding the portion soldby the Defendant, etc., half was produced and sold by the Plaintiff. When also taking
imports from other countries into consideration, out of the import volume of monosodium
glutamate, excluding the portion sold by the Defendant, etc., approximately ●(omitted)●% was produced and sold by the Plaintiff.
(2) Degree of contribution of the Patent Rights to damages
The Patents only contribute to a limited part of the processes for producing glutamic
acid that comprise complicated and diverse steps.
The production of glutamic acid is largely divided into the fermentation phase and thepurification phase, and the fermentation phase comprises at least six steps.
Patent 1 relates to a biosynthesis, which is one of the six steps of the fermentation
phase, and further, it relates to one of the four reactions of the biosynthesis. Therefore, its
contribution to the process for producing glutamic acid is approximately 2% (1/2 × 1/6 ×
1/4 = 1/48) at the most.
Patent 2 relates to technological development on efflux of glutamic acid to outside of
the bacterial cell, which is one of the six steps of the fermentation phase. Therefore, its
contribution to the process for producing glutamic acid is approximately 8.3% (1/2 × 1/6= 1/12) at the most.
Accordingly, when calculating the damages under Article 102, paragraph (2) of the
Patent Act, the abovementioned percentages should be taken into consideration as thecontribution rates of the Patents, or the rebuttal of the presumption to the extent of theabovementioned percentages should be allowed.
5. Regarding overlapped application of Article 102, paragraph (3) of the Patent Act for
the rebutted part
It should be regarded that, when presumption under Article 102, paragraph (2) of the
Patent Act is to be rebutted based on the existence of competitive products, the provisionsof paragraph (3) of that Article should not be allowed to be applied in an overlappedmanner to the portion to be rebutted.

190

End

191

Attachment 11-2 "Allegations on the Value of Damages 2" (Value of Damages under
Article 102, paragraph (3) of the Patent Act for the Overall Portion Sold by the Defendant,etc.)[Plaintiff's allegations]
1. Outline of the allegations
As mentioned in No. 3, 15. [Plaintiff's allegations] (1) of the text, joint tort is foundto be established between the Defendant and CJ Indonesia with regard to the overall
portion sold by the Defendant, etc. Therefore, the Defendant is liable for tort due to theinfringement of the respective Patent Rights with regard to the overall portion sold by the
Defendant, etc.
Therefore, the amount equivalent to royalties for the overall portion sold by the
Defendant, etc. during the subject period from January 1, 2007 through December 31,
2017 (as mentioned in 2. below, in relation to Patent Right 2, the period on and after
August 23, 2013 within that period) will be the value of damages to be compensated by
the Defendant pursuant to Article 102, paragraph (3) of the Patent Act.
2. Royalty rates for the Patent Rights
The reasonable royalty rates for the Patent Rights are 30 yen/kg of the sales volume,
respectively.
When converted into a rate against the sales amount for the portion sold by the
Defendant, 30 yen/kg will be approximately ●(omitted)●%.
According to the Royalty Rate Data Handbook, the average royalty rate forbiotechnology is 6.2%. However, as the royalty rate to be determined in a patent
infringement lawsuit should be higher than an ordinary royalty rate and given that theinventions claimed in the Patents have high values and significantly contribute to profits,high royalty rates should be determined. In particular, circumstances such as ●(omitted)● should also be taken into consideration when calculating the royalty rates in this case.
3. Amount equivalent to royalties for Patent Right 1
The total sales volume of the portion sold by the Defendant, etc. during the subject
period is ●(omitted)● tons, as mentioned in No. 3, 15. [Plaintiff's allegations] (2) of
the text, and the amount obtained by multiplying this volume by 30 yen/kg, which comesto ●(omitted)● yen, will be the amount equivalent to royalties for Patent Right 1.
4. Amount equivalent to royalties for Patent Right 2(1) Sales volumes during the subject period for Patent Right 2
Of the sales volume relating to the portion sold by the Defendant, etc. for 2013, whichis ●(omitted)● tons, the portion for the period from August 23, 2013 through the last192

day of that year is presumed to be ●(omitted)● tons, as calculated pro rata based on
the number of days.
As the sales volumes for 2014 through 2017 are as shown in 2. of Attachment 10 "Listof Sales Values/Volumes," the total sales volume relating to the portion sold by the
Defendant, etc. during the subject period for Patent Right 2 comes to ●(omitted)● tons.
(2) Amount equivalent to royalties for the subject period for Patent Right 2
The amount equivalent to royalties for Patent Right 2 will be the amount obtained bymultiplying the sales volume of ●(omitted)● tons mentioned in (1) above by 30 yen/kg,
which comes to ●(omitted)● yen.
5. Relationship between the claims for compensation for damages caused by the
infringement of the Patent Rights
During the subject period for Patent Right 2, both Patent Right 1 and Patent Right 2
were infringed, and the amount equivalent to royalties relating to the infringement ofPatent Right 1 is the same as the amount mentioned in 4. (2) above, but with regard to theinfringement of the Patent Rights during that period, the Plaintiff selectively makes claims
based on the Patent Rights within the scope of a total of ●(omitted)● yen.
As damages throughout the subject period, the Plaintiff selectively makes claims
based on the Patent Rights within the scope of a total of ●(omitted)● yen as mentioned
in 3. above. In this lawsuit, the Plaintiff claims 900 million yen from that amount as partial
claims for the damages.
[Defendant's allegations]
1. Outline of the allegations
As mentioned in No. 3, 15. [Defendant's allegations] (1) of the text, the Defendant is
not liable for compensation for damages with regard to the portion sold by CJ Indonesia.
Therefore, when calculating the value of damages under Article 102, paragraph (3) of the
Patent Act, the portion sold by CJ Indonesia should not be taken into consideration.
In addition, as mentioned in No. 3, 15. [Defendant's allegations] (2) of the text, thePlaintiff's allegations concerning the sales volumes for 2007 through 2010, which should
serve as the basis for calculation of the amount equivalent to royalties, are unreasonable.
2. Royalty rates for the Patent Rights
The Plaintiff alleges that the royalty rate should be as high as approximately 18.6%of the sales amount for each of the Patent Rights. However, as this rate considerably
deviates from the usual rate, the Defendant disputes the Plaintiff's allegations concerning
the royalty rates.
[i] The general royalty rate in the relevant technical field and [ii] the inventions
claimed in the Patents both relate to processes for producing general-purpose products,
193and alternative technologies exist. In addition, [iii] the inventions claimed in the Patents
are all used in only a part of the fermentation process in the overall process for producing
glutamic acid, and their contribution to profits and their effects are small. In light of thesefactors, the royalty rates for the Patent Rights should be low, and a rate not more than 1%
will be reasonable.
End

194

Attachment 11-3 "Allegations on the Value of Damages 3" (Value of Damages under
Article 102, paragraph (3) of the Patent Act for the Portion Sold by the Defendant andunder Paragraph (2) of that Article for the Portion Sold by CJ Indonesia)[Plaintiff's allegations]
1. Outline of the allegations
As mentioned in No. 3, 15. [Plaintiff's allegations] (1) of the text, joint tort is foundto be established between the Defendant and CJ Indonesia at least with regard to the
portion sold by CJ Indonesia.
Therefore, from among the portion sold by the Defendant, etc. during the subject
period (in relation to Patent Right 2, the period on and after August 23, 2013 within that
period), with regard to the portion sold by the Defendant, the amount equivalent toroyalties based on Article 102, paragraph (3) of the Patent Act (the royalty rate is as shownin [Plaintiff's allegations] 2. of Attachment 11-2) will be the value of damages to becompensated by the Defendant, and with regard to the portion sold by CJ Indonesia,
damages in an amount equivalent to the profits made by the Defendant, etc. under Article
102, paragraph (2) of the Patent Act will be the value of damages to be compensated by
the Defendant.
2. Amount equivalent to royalties for the portion sold by the Defendant(1) Amount equivalent to royalties for Patent Right 1
The sales volume of the portion sold by the Defendant during the subject period is a
total of ●(omitted)● tons, according to No. 3, 15. [Plaintiff's allegations] (2) A. and B.
(B) of the text, and the amount obtained by multiplying this volume by 30 yen/kg, whichcomes to ●(omitted)● yen, will be the amount equivalent to royalties for Patent Right
1.
(2) Amount equivalent to royalties for Patent Right 2
Of the sales volume relating to the portion sold by the Defendant for 2013, which is●(omitted)● tons, the portion for the period from August 23, 2013 through the last dayof that year is presumed to be ●(omitted)● tons, as calculated pro rata based on thenumber of days. As the sales volumes for 2014 through 2017 are as shown in 2. ofAttachment 10 "List of Sales Values/Volumes," the total sales volume relating to the
portion sold by the Defendant during the subject period for Patent Right 2 comes to ●(omitted)● tons.
The amount equivalent to royalties for Patent Right 2 will be the amount obtained bymultiplying this sales volume of ●(omitted)● tons by 30 yen/kg, which comes to ●(omitted)● yen.
195

3. Profits made by the Defendant, etc. during the subject period for the portion sold by CJ
Indonesia(1) Sales amount
According to No. 3, 15. [Plaintiff's allegations] (2) A. and B. (C) of the text, the total
sales amount relating to the portion sold by CJ Indonesia during the subject period is ●(omitted)● yen.
(2) Regarding the expenses borne by the Defendant, etc.
The amounts of expenses to be deducted are calculated by multiplying the amountsof the production cost for the MSG in question, expenses for international transportation,
expenses for domestic transportation, and other selling, general, and administrative
expenses mentioned in [Plaintiff's allegations] 2. (2) of Attachment 11-1 "Allegations on
the Value of Damages 1" by the percentage of the total sales volume of the portion soldby CJ Indonesia in the total sales volume of the portion sold by the Defendant, etc. during
the subject period.
The production cost for the MSG in question, expenses for domestic transportation,and other selling, general, and administrative expenses come to a total of ●(omitted)●yen, and the expenses for international transportation come to a total of ●(omitted)●yen.
(3) Profits made by the Defendant, etc.
When the expenses to be deducted mentioned in (2) above are deducted from the sales
amount mentioned in (1) above, the profits made by the Defendant, etc. in relation to the
portion sold by CJ Indonesia during the subject period come to a total of 3,110,343,000
yen.
4. Profits made by the Defendant, etc. during the subject period for Patent Right 2 (August23, 2013 through December 31, 2017) for the portion sold by CJ Indonesia(1) Sales amountOf the sales amount relating to the portion sold by CJ Indonesia for 2013, which is●(omitted)● yen, the portion for the period from August 23, 2013 through the last dayof that year is presumed to be ●(omitted)● yen, as calculated pro rata based on thenumber of days. As the sales volumes for 2014 through 2017 are as shown in 1. of
Attachment 10 "List of Sales Values/Volumes," the sales amount relating to the portion
sold by CJ Indonesia during the subject period for Patent Right 2 comes to ●(omitted)● yen.
(2) Regarding the expenses borne by the Defendant, etc.
When the amounts of expenses to be deducted in relation to the portion sold by CJ
Indonesia are calculated in a manner similar to [Plaintiff's allegations] 3. (2) of196

Attachment 11-1 "Allegations on the Value of Damages 1," the respective amounts will
be as follows:
Production cost for the MSG in question ●(omitted)● yen
Expenses for international transportation ●(omitted)● yen
Expenses for domestic transportation and other selling, general, and administrative
expenses ●(omitted)● yen(3) Profits made by the Defendant, etc.
When the expenses to be deducted mentioned in (2) above are deducted from the sales
amount mentioned in (1) above, the profits made by the Defendant, etc. in relation to the
portion sold by CJ Indonesia during the subject period for Patent Right 2 come to a totalof 1,745,876,000 yen.
5. Relationship between the claims for compensation for damages caused by the
infringement of the Patent Rights
The value of damages presumed with regard to the infringement of Patent Right 1 willbe the sum of the amount equivalent to royalties for the portion sold by the Defendantmentioned in 2. (1) above and the amount of profits made by the Defendant, etc. in
relation to the portion sold by CJ Indonesia mentioned in 3. (3) above, which comes to●(omitted)● yen.
The value of damages presumed with regard to the infringement of Patent Right 2 willbe the sum of the amount equivalent to royalties for the portion sold by the Defendantmentioned in 2. (2) above and the amount of profits made by the Defendant, etc. in
relation to the portion sold by CJ Indonesia mentioned in 4. (3) above, which comes to●(omitted)● yen.
With regard to damages for the subject period for Patent Right 2, during which bothof the Patent Rights were infringed, the Plaintiff selectively makes claims based on the
Patent Rights within the scope of the abovementioned total value of damages of ●(omitted)● yen.
As damages throughout the subject period, the Plaintiff selectively makes claims
based on the Patent Rights within the scope of a total of ●(omitted)● yen as mentioned
above. In this lawsuit, the Plaintiff claims 900 million yen from that amount as partial
claims for the damages.
6. Regarding grounds for the rebuttal of the presumption with regard to the portion soldby CJ Indonesia
There are no grounds based on which the presumption of damages under Article 102,
paragraph (2) of the Patent Act should be rebutted with regard to the portion sold by CJ
Indonesia, as mentioned in [Plaintiff's allegations] 5. of Attachment 11-1 "Allegations on
197

the Value of Damages 1." In addition, supposing that the rebuttal of the presumption ofdamages were to be allowed based on the existence of competitive products, the value ofdamages in an amount equivalent to royalties under Article 102, paragraph (3) of the
Patent Act should be recognized for the rebutted part, as mentioned in [Plaintiff's
allegations] 6. of that Attachment.
[Defendant's allegations]
As mentioned in No. 3, 15. [Defendant's allegations] (1) of the text, the Defendant is
not liable for compensation for damages with regard to the portion sold by CJ Indonesia.
Therefore, when calculating the value of damages under Article 102, paragraph (2) of the
Patent Act, the portion sold by CJ Indonesia should not be taken into consideration.
In addition, the Defendant makes allegations concerning the value of damages under
Article 102, paragraph (3) of the Patent Act with regard to the portion sold by the
Defendant as described in [Defendant's allegations] of Attachment 11-2 "Allegations on
the Value of Damages 2", and makes allegations concerning the value of damages underparagraph (2) of that Article with regard to the portion sold by CJ Indonesia as describedin [Defendant's allegations] of Attachment 11-1 "Allegations on the Value of Damages
1."
End

198

Attachment 11-4 "Allegations on the Value of Damages 4" (Value of Damages under
Article 102, Paragraph (3) of the Patent Act for the Portion Sold by the Defendant andunder Paragraph (2) of That Article for the Defendant's Commissions for the Portion Sold
by CJ Indonesia)[Plaintiff's allegations]
1. Outline of the allegations
Irrespective of whether joint tort may be established between the Defendant and CJ
Indonesia for the portion sold by CJ Indonesia, from among the portion sold by the
Defendant, etc. during the subject period (in relation to Patent Right 2, the period on andafter August 23, 2013 within that period), with regard to the portion sold by the Defendant,
compensation for damages in an amount equivalent to royalties based on Article 102,
paragraph (3) of the Patent Act may be claimed, and with regard to the portion sold by CJ
Indonesia, compensation for damages in an amount equivalent to commissions, which areprofits made by the Defendant in relation to that portion, may be claimed as the value ofdamages under Article 102, paragraph (2) of the Patent Act.
2. Amount equivalent to royalties for the portion sold by the Defendant
The same as in [Plaintiff's allegations] 2 of Attachment 11-3.
3. Profits made by the Defendant in an amount equivalent to commissions relating to the
portion sold by CJ Indonesia(1) Profits made by the Defendant (the amount of commissions) during the subject period
According to material disclosed by the Defendant (Reference Materials 3 attached to
Exhibit Otsu 101), the Defendant has received payment of commissions (fees) from CJ
Indonesia for the portion sold by CJ Indonesia, and they constitute profits (Article 102,
paragraph (2) of the Patent Act) made by the Defendant from the act of infringement,
which is an act of offering to transfer the products, conducted by the Defendant withregard to the portion sold by CJ Indonesia.
The total amount of commissions for 2011 through 2017 is ●(omitted)● yen, and
it is expected that the Defendant also received payment of commissions of at least thesame amount as that for 2011, which is ●(omitted)● yen, in each year from 2007
through 2010. Therefore, the total amount of commissions paid during the subject periodis ●(omitted)● yen, and this amount is presumed to be damages sustained by the
Plaintiff, pursuant to Article 102, paragraph (2) of the Patent Act.
(2) Profits made by the Defendant (the amount of commissions) during the subject period
for Patent Right 2 (August 23, 2013 through December 31, 2017)
Of the commissions for 2013, which is ●(omitted)● yen, the portion for the period
199

from August 23, 2013 through the last day of that year is presumed to be ●(omitted)●yen, as calculated pro rata based on the number of days.
When the commissions for 2014 through 2017 are added up, the total amount ofcommissions paid to the Defendant during the subject period for Patent Right 2 comes to●(omitted)● yen.
4. Relationship between the claims for compensation for damages caused by the
infringement of the Patent Rights
The value of damages presumed with regard to the infringement of Patent Right 1 willbe the sum of the amount equivalent to royalties for the portion sold by the Defendant([Plaintiff's allegations] 2. (1) of Attachment 11-3 "Allegations on the Value of Damages
3") and the amount of commissions mentioned in 3. (1) above, relating to the subject
period, which comes to ●(omitted)● yen.
The value of damages presumed with regard to the infringement of Patent Right 2 willbe the sum of the amount equivalent to royalties for the portion sold by the Defendant([Plaintiff's allegations] 2. (2) of Attachment 11-3 "Allegations on the Value of Damages
3") and the amount of commissions mentioned in 3. (2) above, relating to the subject
period for Patent Right 2, which comes to ●(omitted)● yen.
With regard to damages for the subject period for Patent Right 2, during which bothof the Patent Rights were infringed, the Plaintiff selectively makes claims based on the
Patent Rights within the scope of the abovementioned total value of damages of ●(omitted)● yen.
As damages throughout the subject period, the Plaintiff selectively makes claims
based on the Patent Rights within the scope of a total of ●(omitted)● yen as mentioned
above. In this lawsuit, the Plaintiff claims 900 million yen from that amount as partial
claims for the damages.
5. Regarding grounds for the rebuttal of the presumption with regard to the portion soldby CJ Indonesia
There are no grounds based on which the presumption of damages under Article 102,
paragraph (2) of the Patent Act should be rebutted with regard to the portion sold by CJ
Indonesia, as mentioned in [Plaintiff's allegations] 5. of Attachment 11-1 "Allegations on
the Value of Damages 1." In addition, supposing that the rebuttal of the presumption ofdamages were to be allowed based on the existence of competitive products, the value ofdamages in an amount equivalent to royalties under Article 102, paragraph (3) of the
Patent Act should be recognized for the rebutted part, as mentioned in [Plaintiff's
allegations] 6. of that Attachment.
[Defendant's allegations]200

1. Regarding the value of damages under Article 102, paragraph (3) of the Patent Act withregard to the portion sold by the Defendant
The Defendant makes allegations as described in [Defendant's allegations] of
Attachment 11-2 "Allegations on the Value of Damages 2."
2. Regarding allegations based on Article 102, paragraph (2) of the Patent Act treating
commissions as profits made by the Defendant in relation to the portion sold by CJ
Indonesia
Commissions are profits which the Defendant can independently gain based on acommission contract concluded with CJ Indonesia. On the other hand, the Plaintiff does
not conduct business relating to commissions. Therefore, the Plaintiff and the Defendant
are not in a competitive relationship in business relating to commissions, and the
Defendant's obtainment of commissions does not deprive the Plaintiff of the opportunity
to obtain commissions. Accordingly, commissions should not be taken into consideration
as the profits made by the Defendant under Article 102, paragraph (2) of the Patent Act.
Supposing that commissions were to be taken into consideration as profits made by
the Defendant, the amount of commissions received in each year from 2007 through 2010is not the same as the amount received in 2011. The commissions for 2007 through 2010
should also be estimated in a manner similar to that for the sales amounts, by a formulasimilar to that mentioned in No. 3, 15. [Defendant's allegations] (2) B. of the text, which
will come to a total of ●(omitted)● yen. In addition, the related expenses (the selling,
general, and administrative expenses for the portion sold by CJ Indonesia in [Defendant's
allegations] 2. (1) of Attachment 11-1 "Allegations on the Value of Damages 1") should
also be taken into consideration in relation to commissions.
Moreover, in a manner similar to [Defendant's allegations] 4. (1) of Attachment 11-1
"Allegations on the Value of Damages 1," the amount of profits made by the Defendant
through commissions should also be reduced (rebutted) based on the Plaintiff's market
share, and overlapped application of Article 102, paragraph (3) of the Patent Act should
not be allowed for the rebutted part, as in [Defendant's allegations] 5. of that Attachment.
End

201

Attachment 12 "Statements in Description 1"
The detailed explanation of the invention in Description 1 concerning Corrected
Invention 1 contains the following statements:
1. Technical Field[0001]The present invention relates to a method of constructing a mutant strain capable ofproducing amino acids in a high yield, and a method of producing L-amino acids by thefermentation with the mutant.
2. Background Art[0002]
Methods of constructing mutant strains usable for the production of amino acids by
the fermentation can be roughly classified into two methods. One of them comprises
introducing random mutations into DNA with a chemical mutagen, and the othercomprises the genetic recombination. In the latter method, a strain having an improved
capacity of producing an intended substance can be developed by enhancing a gene on ametabolic pathway relating to the biosynthesis of an intended substance, or by weakening
a gene of an enzyme relating to the destruction. In this connection, for enhancing an
intended gene, a plasmid capable of autonomously replicating independently from thechromosome in a cell has been mainly used.
However, the method of enhancing the intended gene with a plasmid has problems.
In particular, the degree of enrichment of the intended gene is variable depending on thenumber of copies of the plasmid itself. Therefore, for some kinds of intended genes, thecopies are often too many in number and, as a result, the expression becomes excessive,the growth is seriously inhibited or the capacity of producing the intended substance islowered. In such a case, although the degree of the enhancement of the intended gene canbe lowered by using a plasmid of a small number of the copies, the variety of the plasmid
is limited in many cases, and the intended control of the expression level of the intended
gene is impossible.
[0003]
Another problem is that since the replication of the plasmid is often unstable, theplasmid is eliminated.
3. Problem to be Solved by the Invention[0006]The object of the present invention is to provide a method of constructing a mutant
202

capable of suitably enhancing or controlling the expression of an intended gene without
using a plasmid and also capable of producing amino acids in a high yield, by generecombination or mutation.
Another object of the present invention is to provide a promoter for GDH capable ofimparting a capability of producing glutamic acid in a high yield to a Corynebacterium
strain without seriously increasing the amount of by-produced aspartic acid and alanine.
Still another object of the present invention is to provide a GDH gene having asequence of the above-described promoter for GDH.
A further object of the present invention is to provide a Corynebacterium strain having
the above-described gene and capable of producing L-glutamic acid.
A further object of the present invention is to provide a method of producing aminoacids by fermentation wherein amino acid-producing microorganism thus constructed isused.
A further object of the present invention is to provide a fermentation method of
producing glutamic acid at a low cost by increasing the yield of glutamic acid by using a
glutamic acid-producing coryneform bacterium.
4. Means for Solving the Problem[0007]The present invention has been completed on the basis of a finding that the abovedescribed problems can be efficiently solved by variously modifying the promoter ofamino acid-biosynthesizing genes on a chromosome to control the amount of theexpression of the intended genes. Particularly, the invention has been completed on thebasis of a finding that the above-described problem can be efficiently solved byintroducing a specific mutation into -35 region or -10 region which is a specific region of
the promoter.
Namely, the present invention provides a method of producing coryneform bacteria
having an improved amino acid- or nucleic acid-productivity, which comprises the steps
of introducing a mutation in a promoter sequence of amino acid- or nucleic acidbiosynthesizing genes on a chromosome of a coryneform bacterium to make it close to aconsensus sequence or introducing a change in a promoter sequence of amino acid- or
nucleic acid-biosynthesizing genes on a chromosome of a coryneform bacterium by generecombination to make it close to a consensus sequence, to obtain mutants of thecoryneform amino acid- or nucleic acid-producing microorganism, culturing the mutants,
and selecting a mutant capable of producing the intended amino acid or nucleic acid in alarge amount.
[0008]
203

The present invention also provides a promoter for glutamate dehydrogenase (GDH)producing gene, which has the sequence of (i) at least one DNA sequence selected fromthe group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in -35 region, (ii)
TATAAT sequence or the same TATAAT sequence but in which the base of ATAAT is
replaced with another base in -10 region, or (iii) a combination of (i) and (ii), wherein thesequence does not inhibit the promoter function.
The present invention also provides a glutamate dehydrogenase-producing gene
having the above-described promoter.
The present invention also provides a coryneform L-glutamate-producing
microorganism having the above-described gene.
[0009]The present invention also provides a process for producing an amino acid by thefermentation, which comprises the steps of culturing a coryneform bacterium constructedby the above-described method and having an improved amino acid-producing capacity
in a medium to form and also to accumulate the intended amino acid in the medium, andcollecting the amino acid from the medium.
The present invention also provides a process for producing L-glutamic acid by thefermentation, which comprises the steps of culturing a coryneform L-glutamic acidproducing microorganism resistant to 4-fluoroglutamic acid in a liquid medium to form
and also to accumulate L-glutamic acid in the medium, and collecting L-glutamic acidfrom the medium.
5. Best Mode for Carrying Out the Invention[0010]The term "coryneform glutamic acid producing microorganism" as used herein
includes also bacteria which were classified to be the genus Brevibacterium before but
now integrated into the genus Corynebacterium [Int. J. Syst. Bacteriol., 41, 255 (1981)]
and also bacteria of the genus Brevibacterium which are very close to those of the genus
Corynebacterium. Therefore, the mutants used in the present invention can be derived
from the coryneform glutamic acid-producing bacteria of the genus Brevibacterium or
Corynebacterium shown below. Bacteria of the genus Corynebacterium and those of thegenus Brevibacterium will be collectively referred to as "coryneform bacteria" so far as
they do not concern the glutamic acid productivity.
[0011]... Corynebacterium glutamicum
ATCC13032... Brevibacterium lactofermentum
ATCC13869...
[0012]
204

The amino acids to be produced are not particularly limited so far as the genes
concerning the biosynthesis and promoters thereof have been elucidated. Examples ofeffective enzymes concerning the biosynthesis include GDH, citrate synthase (CS),isocitrate dehydrogenase (ICDH), pyruvate dehydrogenase (PDH) and aconitase (ACO)
for glutamic acid fermentation. ...
[0015]In the present invention, a mutant of a coryneform amino acid-producing bacterium
is obtained by, introducing a mutation in a promoter sequence of desired amino acidbiosynthesizing genes on a chromosome of a coryneform amino acid-producing
bacterium, such as the above-described promoter sequence for GDH, to make it close toa consensus sequence with a chemical or by introducing the mutation by the genetic
recombination to obtain a mutant of the coryneform amino acid-producing
microorganism.
The term "consensus sequence" is a sequence which appears most frequently invarious promoter sequences. Such consensus sequences include, for example, those of E.
coli and Bacillus subtilis. The consensus sequence of E. coli is described in Diane K.
Hawley and William R. McClure Nuc. Acid. Res. 11:2237-2255 (1983), and that of B.
subtilis is described in Charles et al. Mol. Gen. Genet 186:339-346 (1982).
The mutation may be caused in either only one promoter sequence such as that for
GDH or two or more promoter sequences such as those for GDH, citrate synthase (citratesynthesizing enzyme) (CS) and isocitrate dehydronenase (isocitrate-synthesizing
enzyme) (ICDH).
In the present invention, the mutant thus obtained is cultured to obtain the mutant
capable of producing a large amount of an intended amino acid.
[0016]
It was already elucidated that in the fermentation of glutamic acid, GDH derived froma coryneform glutamate-producing microorganism has its own promoter sequence in
upstream region thereof [Sahm et al., Molecular Microbiology (1992), 6, 317-326].
For example, the promoter for GDH of the present invention, GDH gene having thepromoter sequence for GDH and L-glutamate-producing Corynebacterium strain having
this gene can be obtained by, for example, the following methods:
Namely, the strain is subjected to a mutagenesis treatment such as the irradiation with
UV, X-rays or radiation, or treatment with a mutagen to obtain a strain resistant to 4fluoroglutamic acid on an agar plate culture medium containing 4-fluoroglutamic acid.
Namely, the mutagenized cells are spread on an agar plate culture medium containing 4fluoroglutamic acid in such a concentration that it inhibits the growth of the parent, and205

the mutant thus grown is separated.
Further, the promoter sequence of GDH genes can be replaced with variously
modified sequences by site directed mutagenesis, and the relationship between therespective sequences and GDH activity is examined so as to select the ones having a highL-glutamate-productivity.
[0017]
It is particularly preferred in the present invention that the DNA sequence in -35
region of the promoter for GDH-producing gene is at least one DNA sequence selectedfrom the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA and/or the
DNA sequence in -10 region of the promoter is TATAAT, or the bases of ATAAT in
TATAAT sequence in -10 region is replaced with another base, while they do not inhibit
the promoter function. The reason why the strain in which the bases of ATAAT in TATAAT
sequence in -10 region is replaced with another base and the promoter function is notinhibited can be selected is as follows: Because a remarkable increase in the specificactivity of GDH was observed by merely replacing the first "C" of CATAAT with "T" inwild type -10 sequence (refer to p6-4 in Table 1), it was considered that such a
replacement with another base is possible.
The promoter sequence of GDH gene is described in, for example, the abovedescribed Sahm et al., Molecular Microbiology (1992), 6, 317-326. It is described therein
as Seq ID No. 1. The sequence of GDH gene itself is also described in Sahm et al.,
Molecular Microbiology (1992), 6, 317-326 to be Seq ID No. 1.
Similarly, the mutation can be introduced in the promoter for citrate-synthesizing
enzyme (CS) or isocitrate dehydrogenase (ICDH).
Thus, the promoters for GDH are those having at least one DNA sequence selectedfrom the group consisting of CGGTCA, TTGTCA, TTGACA and TTGCCA in -35 region
and/or TATAAT sequence or the TATAAT sequence but in which the base of ATAAT is
replaced with another base in -10 region, wherein they do not inhibit the promoter
function. Genes for producing glutamate dehydrogenase, which have the above-describedpromoter, are also provided.
[0018]The promoters for CS are those having TTGACA sequence in -35 region and/or
TATAAT sequence in -10 region, which do not inhibit the promoter function. CS genes
having the above-described promoter are also provided. ...
The present invention also provides coryneform L-glutamate-producing bacterium
having the above-described genes.
[0020]
206

The coryneform bacteria usually produce L-glutamic acid under reduced biotin
condition. Therefore, the amount of biotin in the medium is restricted or a substanceinhibiting the effect of biotin such as a surfactant or penicillin is added.
The fermentation is preferably conducted by shaking the culture or agitating theculture with aeration while the pH of the culture liquid is kept in the range of 5 to 9 for 2
to 7 days. The pH is preferably controlled with urea, calcium carbonate, gaseous ammonia,
ammonia water or the like. The culture temperature is preferably 24 to 37°C.
L-glutamic acid thus produced and accumulated in the culture liquid is collected byan ordinary method such as ion-exchange resin method or crystallization method.
Specifically, L-glutamic acid is separated by the adsorption on an anion-exchange resin
or by the neutralization crystallization.
According to the present invention, the intended amino acid can be obtained in a highyield by introducing a mutation into a promoter region of amino acid-biosynthesizing
genes of a coryneform amino acid-producing bacterium to control the expression of theintended genes. In addition, since any elimination of the intended gene does not occur inthe bacteria according to the present invention, contrary to the cases using plasmid, theintended amino acid can be stably obtained in a high yield. Thus, the industrial merit of
the invention is great.
[0021]The present invention provides various promoters, particularly, promoters for GDH,
capable of imparting a power of producing amino acids, particularly glutamic acid, in ahigh yield to Corynebacterium strains without increasing the amount of by-produced
aspartic acid and alanine.
In the present invention, a coryneform L-glutamate-producing bacterium ismutagenized, a strain in which the mutation is introduced in a promoter region of GDHgene and which is resistant to 4-fluoroglutamic acid is collected, and the strain is cultured
to obtain glutamic acid in a high yield. Thus, the present invention is industrially very
advantageous.
6. Working Examples(1) Example 1 Production of Mutant GDH Promoter[0022]... A mutant GDH promoter was prepared by site-directed mutagenesis method as follows:(1) Preparation of GDH Genes Having Various Mutant Promoters:
The wild type sequence in -35 region and -10 region of a promoter of GDH gene of acoryneform bacteria is shown in sequence 1. The promoter sequence of wild type has
already been reported [Molecular Microbiology (1992), 6, 317-326].
207

The method of preparing a plasmid carrying GDH gene having a mutant promoter is
as follows:
As shown in FIG. 1, a chromosomal gene of a wild type strain of a coryneformbacterium ATCC13869 prepared with "Bacterial Genome DNA purification kit"
(Advanced Genetic Technologies Corp.) was used as the template for PCR. The gene
amplification was conducted by PCR using upstream and downstream sequences of GDH
gene. Both ends were blunt-ended. The product thus obtained was inserted in SmaI siteof plasmid pHSG399 (a product of Takara Shuzo Co., Ltd.). Then a replication origin
taken from plasmid pSAK4 having the replication origin capable of replicating in acoryneform bacterium was introduced into Sal I site of the plasmid to obtain plasmid
pGDH. By this method, GDH genes having each above-described promoter sequence canbe obtained by using a primer having each of the sequence of Seq ID No. 1 to Seq ID No.
6 shown in the Sequence Listing as the upstream primer for GDH gene, respectively. It
was confirmed by sequencing the PCR amplified fragment that any mutation, other than
the introduced mutation in the promoter sequence, did not occur in the amplified fragment.
pSAK4 is constructed as follows: previously obtained plasmid pHK4 (Japanese
Unexamined Patent Application Publication No. 1993-7491) having an autonomous
replication origin derived from plasmid pHM1519 [Agric. Biol. Chem., 48, 2901-2903(1984)] which is capable of autonomously replicating in Corynebacterium
microorganism, is digested with restriction enzymes BamHI and KpnI to obtain a DNA
fragment having the replication origin. Then the fragment thus obtained is blunt-ended
with DNA-Blunting Kit (Blunting kit of Takara Shuzo Co., Ltd.). After the ligation withSal I linker (a product of Takara Shuzo Co., Ltd.), the product thus obtained was inserted
into Sal I site of pHSG299 to obtain plasmid pSAK4.
[0023](2) Comparison of the Degrees of Expression of GDH Having Each Promoter Sequence:
Each plasmid prepared as described above was introduced into wild type strain of
coryneform bacterium ATCC13869 by electroporation method (refer to Japanese
Unexamined Patent Application Publication No. 1990-207791). For comparing thedegrees of expression of GDH for these strains, the specific activity of GDH wasdetermined by the above-described method of Sahm et al. The results are shown in Table
1.
[0024]
Table 1
StrainPromoter sequence
-35

-10
208

Specific activityof GDH

Relativevalue

ATCC 13869
/pGDH
/p6-2
/p6-4
/p6-3
/p6-7
/p6-8

TGGTCATGGTCACGGTCATGGTCATTGACA
TTGCCA
TTGTCACATAATCATAATCATAATTATAATTATAATTATAATTATAAT

7.7
82.7
33.1
225.9
327.2
407.0
401.3

0.1
1.0
0.4
2.7
4.0
4.9
4.9

[0025]
ATCC 13869/p6-2 through ATCC 13869/p6-8 corresponded to the sequences of Seq
ID No. 2 through Seq ID No. 6, respectively. These sequences were the same as thesequence of Seq ID No. 1 (wild type) except that the underlined parts were changed asfollows:
Seq ID No. 1

5'-TTAATTCTTTGTGGTCATATCTGCGACACTGCCATAATTTGAACGT- 3'

Seq ID No. 2

CGGTCACATAAT

Seq ID No. 3

TGGTCATATAAT

Seq ID No. 4

TTGACATATAAT

Seq ID No. 5

TTGCCATATAAT

Seq ID No. 6

TTGTCATATAAT

These were those of synthetic linear doubled stranded DNA.
(2) Example 2 Preparation of Mutant Strains[0026]... (1) Preparation of Mutant Strains Resistant to 4-Fluoroglutamic Acid:
AJ13029 is a mutant strain producing glutamic acid and disclosed in WO96/06180.
Although it does not produce glutamic acid at a culture temperature of 31.5°C., itproduces glutamic acid even in the absence of a biotin-inhibitor when the culture
temperature is shifted to 37°C. In this Example, Brevibacterium lactofermentum AJ13029
strain was used as the parent strain for preparing the mutant strains. As a matter of course,
any of glutamic acid-producing strains other than AJ13029 can be used as a parent strain
for preparing mutant strains resistant to 4-fluoroglutamic acid.
AJ13029 was cultured on a CM2B agar medium (...) at 31.5°C. for 24 hours to obtain
the bacterial cells. The cells were treated with 250 μg/ml aqueous solution of N-methylN'-nitro-N-nitrosoguanidine at 30°C. for 30 minutes. Then a suspension of the cells
having a survival rate of 1% was spread on agar plates culture medium (...) containing 4fluoroglutamic acid (4FG). Colonies were formed after incubating the plate at 31.5°C. for
20 to 30 hours. In this experiment, a slant medium containing 1 mg/ml of 4FG was
prepared at first, and then a layer of the same medium without 4FG was formed thereon
209horizontally. Thus, 4FG concentration gradient was obtained on the surface of the agar
medium. When the plate was inoculated with the mutant cells obtained as described, aboundary line was formed at a border of the growing limit of the strain. Bacterial strains
which formed colonies in an area containing 4FG of a concentration higher than that of
the boundary line were taken. Thus, about 50 strains resistant to 4FG were obtained fromabout 10,000 mutagenized cells.
[0029](2) Confirmation of Capability of L-Glutamic Acid-Production of 4FG-Resistant Mutant
Strains:
The capability of glutamic acid-production of about 50 mutant strains obtained inabove (1) and parent AJ13029 strain were confirmed as described below.
AJ13029 and mutant strains were each ... After the completion of the culture, theculture was examined with a Biotic Analyzer (a product of Asahi Chemical Industry Co.,
Ltd.) to determine whether L-glutamic acid was produced or not. It was thus found thatwhen the 50 strains were cultured, two strains having a yield of glutamic acid higher thanthat obtained from the parent strains and a high GDH activity were separated (strain A
and strain B). GDH activity of each of them was determined to find that the specific GDH
activity of both of them was increased (Table 5). The GDH activity was determined by
the method of E. R. Bormann et al. [Molecular Microbiol., 6, 317-326 (1996)]. By
sequencing the GDH genes, it was identified that the mutation points were found only inthe promoter region of GDH (Table 6).
[0031]
Table 5 Glutamic acid formation and GDH activity of mutant strains
Strain
AJ13029
FGR1
FGR2

Glu (g/dl)

GDH specific activity

2.6
2.9
3.0

7.7
23.1
25.9

Relative value
1.0
3.0
3.4

[0032]
Table 6 DNA sequences in GDH promoter region of mutant strains
StrainGDH promoter sequence-35

AJ13029FGR1
FGR2

TGGTCATGGTCA
TTGTCA-10
TTCTGTGCGACACTGC
TTCTGTGCGACACTGC
T-CTGTGCGACACTGCCATAATTATAATTATAAT(3) Example 3 Introduction of Mutation into CS Gene Promoter Region of Coryneform
Glutamate-Producing Bacterium:210

[0033]... In this Example, a strain having an enhanced promoter for the genes which code
glutamate dehydrogenase (GDH) and citrate-synthesizing enzyme (CS) was produced.
(1) Cloning of gltA Gene:
The sequence of gltA gene of a coryneform bacterium, which codes citratesynthesizing enzyme, has already been elucidated [Microbial. 140, 1817-1828 (1994)].
On the basis of this sequence, primers shown in Seq ID No. 7 and Seq ID No. 8 weresynthesized. On the other hand, chromosomal DNA from Brevibacterium lactofermentum
ATCC13869 was prepared using Bacterial Genome DNA Purification Kit (Advanced
Genetic Technologies Corp.). Sterilized water was added to a mixture of 0.5 μg of thechromosomal DNA, 10 pmol of each of the oligonucleotides, 8 μl of dNTP mixture (2.5mM each), 5 μl of 10×La Taq Buffer (Takara Shuzo Co., Ltd.) and 2 U of LA Taq (Takara
Shuzo Co., Ltd.) to obtain 50 μl of PCR reaction cocktail. The reaction cocktail was
subjected to PCR. The PCR conditions were 30 cycles of denaturation at 94°C. for 30
seconds, annealing at 55°C. for 15 seconds and extension at 72°C. for 3 minutes using
Thermal Cycler TP240 (Takara Shuzo Co., Ltd.) to amplify about 3 Kbp of DNA
fragments containing gltA gene and promoter thereof. The amplified fragments thus
obtained were purified with SUPREC02 (Takara Shuzo Co., Ltd.) and then blunt-ended.
The blunting was conducted with Blunting Kit of Takara Shuzo Co., Ltd. The blunt-ended
fragment was mixed with pHSG399 (Takara Shuzo Co., Ltd.) completely digested with
SmaI to conduct the ligation. The ligation reaction was conducted with DNA Ligation Kit
ver 2 (Takara Shuzo Co., Ltd.). After the completion of the ligation, the transformation
was conducted with competent cells of E. coli JM109 (Takara Shuzo Co., Ltd.). The cells
were spread on L medium plates (comprising 10 g/l of bactotryptone, 5 g/l of bactoycast
extract, 5 g/l of NaCl and 15 g/l of agar; pH 7.2) containing 10 μg/ml of IPTG (isopropylβ-D-thiogalactopyranoside), 40 μg/ml of X-Gal (5-bromo-4-chloro-3-indoyl-β-Dgalactoside) and 40 μg/ml of chloramphenicol. After culturing them overnight, whitecolonies were taken to obtain the transformed strains after single colony isolation.
From the transformed strains, plasmids were prepared by the alkali method (Seibutsu
Kogaku Jikken-sho edited by Nippon Seibutsu Kogaku-kai and published by Baifukan, p.
105, 1992). Restriction enzyme maps were prepared, and the plasmid which has the same
restriction map as the map shown in FIG. 2 was named "pHSG399CS".
[0034](2) Introduction of Mutations into gltA Promoter:
Mutan-Super Express Km (Takara Shuzo Co., Ltd.) was used for the introduction of
mutation into gltA promoter region.
211

[0035]... The plasmids in which gltA promoter region was replaced with the sequence shown in
Table 7 were named pKF19CS1, pKF19CS2 and pKF19CS4, respectively.
[0036]
Table 7
pKF19CS
pKF19CS1
pKF19CS2
pKF19CS4-35 region-10 region

ATGGCT
ATGGCT
ATGGCTTTGACATATAGCTATAACTATAATTATAAT[0041](5) Introduction of Mutant gltA Gene into Temperature-Sensitive Plasmid:
For integrating mutant gltA promoter sequences into a chromosome, a method isknown wherein a plasmid of which replication in a coryneform bacterium is temperaturesensitive is used (Japanese Unexamined Patent Application Publication No. 19937491). ... From the transformants, plasmids were prepared. Temperature-sensitive shuttle
vectors containing gltA gene were named pSFKTC1, pSFKTC2 and pSFKTC4,
respectively.
[0042](6) Introduction of Mutant gltA Promoter into Chromosome:pSFKTC1, pSFKTC2 and pSFKTC4 were each introduced into Brevibacterium
lactofermentum FGR2 strain by electrical pulse method. ... It was thus proved that in thisstrain, mutant gltA gene derived from the temperature-sensitive plasmid was integrated
near gltA gene in the host chromosome by homologous recombination. Strains derived
from pSFKTC1, 2 and 4 were named BLCS11, BLCS12 and BLCS14, respectively.
[0043](7) Preparation of Substituted gltA Promoters:
First, kanamycin-sensitive strains were obtained from the strains BLCS11, BLCS12
and BLCS14 having mutant gltA gene integrated therein by homologous recombination. ...
The chromosome was extracted from the kanamycin-sensitive strain, and PCR was
conducted with primers having the sequence shown in Seq ID No. 7 and Seq ID No.8 toprepare gltA gene fragments. The amplified fragments thus obtained were purified with
SUPREC02 (Takara Shuzo Co., Ltd.) and then subjected to the sequencing reaction usinga primer of Seq ID No. 13 to determine the sequence in the promoter region thereof. As
a result, the strain having the same promoter sequence as that of pKF19CS1 in Table 7
was named GB01, the strain having the same promoter sequence as that of pKF19CS2
212

was named GB02 and the strain having the same promoter sequence as that of pKF19CS4
was named GB03. It was indicated that in these strains, the gltA gene of wild typeoriginally located on the chromosome was excised together with the vector plasmid while
the mutant gltA gene introduced by the plasmid remained on the chromosome when theplasmid and duplicated gltA gene were excised from the chromosome.
[0044](8) Determination of Activity of Citrate Synthase of Mutant gltA Promoter Strains:
The activities of the citrate synthase were determined by treating FGR2, GB01, GB02,
GB03 and FGR2/pSFKC strains obtained in step (7) ... The results are shown in Table 10.
It was confirmed that the citrate synthase activity of the substituted gltA promoter strain
was higher than that of the parent strains thereof.
[0045]
Table 10StraindABS/min/mg

Relative activity

7.9
9.5
15.0
31.6
61.6

1.0
1.2
1.9
4.0
7.8

FGR2
GB01
GB02
GB03
FGR2/pSFKC[0046](9) Results of Culture of Substituted gltA Promoter Strains:
Each of the strains obtained in above-described step (7) was ... The culture wasterminated ... and the quantity of L-glutamic acid formed and accumulated in the culture
liquid was determined.
As a result, the larger improvement in the yield of L-glutamic acid was confirmed
when each of the strains GB02 and GB03 rather than GB01 and FGR2/pSFKC was used
as shown in Table 12. From these facts, it was found that good results were obtained byintroducing the mutation into the gltA promoter to increase the CS activity to 2 to 4 times
for the improvement in the yield of glutamic acid produced by those strains.
[0048]
Table 12StrainL-glutamic acid (g/l)

FGR2
GB01
GB02
GB03

8.9
9.1
9.4
9.4
213

FGR2/pSFKC

9.1(4) Example 7 Introduction of Mutation into GDH Gene Promoter Region ofCoryneform Glutamate-Producing Bacterium[0087]... (1) Construction of Mutant gdh Plasmids:
Plasmids having GDH promoter sequence of FGR1 strain and FGR2 strain describedin Example 2 were constructed by site directed mutagenesis. For obtaining GDH
promoter sequence of FGR1 strain, PCR was conducted by using ... as the primers withchromosomal DNA of ATCC13869 as the template. Further, PCR was conducted byusing ... as the primers with a mixture of these PCR products as the template. The PCR
product thus obtained was inserted into SmaI site of pSFKT2 (Japanese Patent
Application No. 1999-69896) to construct pSFKTG11. To obtain GDH promoter
sequence of FGR2 strain, PCR was conducted by using ... as the primers andchromosomal DNA of ATCC13869 as the template; and on the other hand, PCR was
conducted by using ... as the primers and chromosomal DNA of ATCC13869 as the
template. Further, PCR was conducted by using ... as the primers and a mixture of these
PCR products as the template. The PCR product thus obtained was inserted into SmaI siteof pSFKT2 (Japanese Patent Application No. 1999-69896) to construct pSFKTG07. The
DNA sequences of the fragments inserted into SmaI sites of pSFKTG11 and pSFKTG07
were determined to confirm that no mutation was introduced into other regions than the
promoter region of GDH.
[0088](2) Construction of gdh Promoter Modified Strains:
Then, pSFKTG11 and pSFKTG07 were introduced into AJ13029 strain by electricalpulse method, and transformants which grew on CM2B plates containing 25 μg/ml of
kanamycin at 25°C. were selected. The transformants were cultured at 34°C. to selectstrains which were resistant to kanamycin at 34°C. The fact that a strain is resistant tokanamycin at 34°C. indicates that pSFKTG11 or pSFKTG07 was thus integrated on thechromosome of AJ13029 strain. Kanamycin-sensitive strains were obtained from thestrains in which the plasmid was integrated on the chromosome. The GDH promoter
sequences of these strains were determined. The strains having the same gdh promoter
sequence as those of pSFKTG11 and pSFKTG07 were named GA01 and GA02,
respectively.
(3) Confirmation of L-Glutamic Acid-Productivity of gdh Promoter Modified Strains:
The glutamic acid productivities of strains GA01 and GA02 and the parent strain
AJ13029 were confirmed in the same manner as that of Example 2 (2) given above. As a214

result, a remarkable improvement in the accumulation of glutamic acid was recognizedin GA01 and GA02 as shown in Table 23.
[0089]
Table 23
StrainGlu (g/dl)
Specific activity of GDH
Relative value
AJ13029
GA01
GA02

2.6
3.0
2.9

7.7
22.3
27.0

1.0
2.9
3.5

[0090](4) Construction of Self-Cloning Type gdh Plasmid:
First, self-cloning vector pAJ220 was constructed. ... This plasmid could
autonomously replicate in a coryneform bacterium, and it could afford trimethoprim
resistance to the host.
PCR reaction was conducted by using ... as the primers and chromosomal DNA ofwild-type coryneform bacterium strain ATCC13869 as the template. The gdh genefragment thus obtained was inserted in BalI site of pAJ220 to construct pAJ220G. ...
PAJ220G and pGDH were introduced into ATCC13869 strain by electrical pulse method.
GDH activities of the strains thus constructed were determined by the method stated inabove-described step (1). As a result, GDH activity of the strain into which pAJ220G had
been introduced was about 1.5 times as high as that of the strain into which dGDH had
been introduced as shown in Table 24.
[0091]
Table 24
StrainSpecific activity of GDH
ATTC13869
ATTC13869/pGDH
ATTC13869/pAJ220G

7.7
82.7
120.1

Relative value
1.0
10.7
15.6

[0092](5) Investigations on Influence of gdh Activity on the Yield and By-Produced Asp:
pGDH and pAJ220G were introduced into AJ13029 by electrical pulse method. Eachof these strains and those obtained in above-described step (2) was ... The culture wasterminated ... and the quantity of L-glutamic acid produced and accumulated in the culture
liquid were determined (Table 26). The GDH activity for obtaining the highest yield wasabout 3-times as high. When GDH activity was further elevated, the degree of the
improvement in the yield was reduced. When the GDH activity was elevated to about 16times, the yield was rather reduced. Amino acids produced as by-products were analyzed
215

with Hitachi Amino Acid Analyzer L-8500 to find that as GDH activity was elevated, theamount of accumulated aspartic acid and alanine was increased. These results proved thefollowing facts: For increasing the yield of glutamic acid, it is necessary to suitably
increase GDH activity so as not to cause a remarkable increase in the amount of aspartic
acid and alanine. One of the effective methods therefor comprises the introduction ofvarious mutations into gdh promoter to control GDH activity to about 3-times as high asthat of the parent strain.
[0094]
Table 26
StrainGlu
Asp
Ala
Relative activity
Relative
AJ 13029
GA01
GA02AJ13029/pGDH
AJ13029/pAJ220G(g/dl)(mg/dl)(mg/dl)of GDH

value

8.3
9.0
8.9
8.6
7.5

49
145
153
201
290

60
152
155
190
590

7.7
22.3
27.0
82.7
120.12

1.0
2.9
3.5
10.7
15.6

7. Drawings
FIG. 1 shows a flow of construction of GDH gene having a mutant promoter.

216

FIG. 2 shows a flow of construction of CS gene having a mutant promoter.

217

FIG. 3 shows a flow of construction of shuttle vector carrying lacZ as a reporter gene.

End

218

Attachment 13 "Statements in Description 2"
The detailed explanation of the invention in Description 2 contains the following
statements:
1. Technical Field[0001]The present invention relates to the fermentation industry. In detail, the present
invention relates to a process for producing L-glutamic acid and a bacterium used in thisprocess. L-glutamic acid is widely used as a raw material in the production of seasonings
and the like.
2. Background Art[0002]
L-glutamic acid has been conventionally produced on an industrial scale by afermentation method using coryneform bacteria which have L-glutamic acid-producing
ability, such as bacteria belonging to the genus Brevibacterium or the genus
Corynebacterium. As for these coryneform bacteria, strains isolated from nature or
artificial mutants of such strains are used in order to enhance productivity.
[0003]
Generally, a wild-type strain of a coryneform bacterium does not produce L-glutamic
acid under the condition where biotin is present. Accordingly, the production of Lglutamic acid by a coryneform bacterium is typically performed under a glutamic acidproduction-inducing condition such as a biotin-limited condition, a surfactant-added
condition, and a penicillin-added condition (Non-Patent Document 1). Further, as strains
which can produce L-glutamic acid in the presence of sufficient biotin without applying
these methods, a surfactant-temperature-sensitive strain (Patent Document 1), a
penicillin-sensitive strain (Patent Document 2), a cerulenin-sensitive strain (Patent
Document 3), and a lysozyme-sensitive strain (Patent Document 4), etc. have beendeveloped.
[0004]
However, these L-glutamic acid-producing bacteria which have been developed bythese methods often showed a decrease in fatty acid-producing ability or a decrease incell wall synthesis ability. Thus, it was highly probable that these strains caused reduced
adaptation to environmental changes in exchange for L-glutamic acid production.
Therefore, considerable efforts have been required for the development of a strain capableof accumulating a significant amount of L-glutamic acid when using these methods.
219

[0005]On the other hand, a strain which produces L-glutamic acid in the presence ofsufficient biotin can be achieved by deleting a gene encoding α-ketoglutarate
dehydrogenase (Patent Document 5). However, the α-ketoglutarate dehydrogenase genedeficient strain blocks the TCA cycle in the midway and thus grows slowly. Therefore, it
was difficult to surely obtain a sufficient amount of bacterial cells, which was one of the
problems.
[0006]The yggB gene of a coryneform bacterium is a homologue of the yggB gene of
Escherichia coli (Non-Patent Documents 2 and 3) and has been analyzed as a kind of
mechanosensitive channel (Non-Patent Document 4). However, the effect of the yggB
gene on L-glutamic acid production was not known.
3. Problem to be Solved by the Invention[0007]The problem to be solved by the present invention is to provide novel art to enhance
the L-glutamic acid-producing ability of a coryneform bacterium when L-glutamic acidis produced by using the coryneform bacterium.
4. Means for Solving the Problem[0008]The present researchers have made extensive studies to solve the above problem. As
a result, the present researchers have clarified that a yggB gene is involved in the
production of L-glutamine by a coryneform bacterium. In addition, the present
researchers have found that the L-glutamic acid-producing ability can be greatly
enhanced by modifying the coryneform bacterium by using the yggB gene. Finally, thepresent researchers have completed the present invention.
5. Advantageous Effect of the Invention[0010]
L-glutamic acid can be efficiently produced by using a coryneform bacterium which
is modified by using a yggB gene of the present invention.
6. Mode for Carrying Out the Invention(1) Coryneform Bacterium of the Present Invention[0011]... The coryneform bacterium of the present invention has an L-glutamic acidproducing ability, and is modified by using a yggB gene, and thereby the L-glutamic acidproducing ability of the coryneform bacterium is enhanced as compared to a nonmodified strain.
220[0012]In the present invention, the "coryneform bacterium" includes a bacterium which has
been conventionally classified into the genus Brevibacterium, but is currently classified
into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255(1991)), and also includes
the Brevibacterium bacteria which are very closely related to Corynebacterium bacteria.
Examples of such coryneform bacterium include the following.
... Corynebacterium callunae
Corynebacterium glutamicum ...
... Corynebacterium melassecola ...
... Brevibacterium flavum ...
[0013]
Specific examples of the coryneform bacteria are as follows:... Corynebacterium callunae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060 ...
... Corynebacterium melassecola ATCC17965 ...
... Brevibacterium flavum ATCC13826, ATCC14067 ...
[0015]In the present invention, the phrase "L-glutamic acid-producing ability" means anability to cause accumulation of L-glutamic acid in a medium or a bacterial cell when the
coryneform bacterium of the present invention is cultured in the medium. The "Lglutamic acid-producing ability" may be a property of a wild-type strain of thecoryneform bacterium, because most of the wild-type strains of coryneform bacterium
can produce L-glutamic acid under the "L-glutamic acid-producing condition" asmentioned below. However, the L-glutamic acid-producing ability may be imparted orenhanced by breeding, or may be imparted by modification by using a yggB gene, as
mentioned below.
The phrase "an L-glutamic acid-producing ability of a coryneform bacterium isenhanced" means that the L-glutamic acid-producing ability of the coryneform bacterium
is enhanced as compared to a non-modified strain such as a wild-type strain. In this regard,
examples of the wild-type strain of the coryneform bacterium include Corynebacterium
glutamicum ATCC13032 strain, 13869 strain, 14067 strain, and Corynebacterium
melassecola ATCC 17965 strain. The "non-modified strain" may also include a strain
which expresses the wild-type yggB gene at the same level as the wild-type strains or astrain in which a mutation is not introduced into the coding region of a yggB gene.
[0029]
The coryneform bacterium of the present invention can be obtained by modifying the221above-described coryneform bacterium possessing L-glutamic acid-producing ability
using a yggB gene so that the L-glutamic acid-producing ability is further enhanced.
Alternatively, modification using a yggB gene may be performed first, followed byadditional modification to impart or enhance L-glutamic acid-producing ability, as
mentioned above.
Modification using a yggB gene includes enhancing the expression level of a yggB
gene and introducing a mutant-type yggB gene, as described below.
(2) Enhancing Expression Level of yggB Gene
[0030]... A yggB gene encodes a protein which is known as a kind of mechanosensitive
channel, and which is also referred to as mscS (FEMS Microbiol Lett. 2003 Jan. 28;
218(2): 305-9.).
Enhancing the expression level of the yggB gene leads to improvement of the Lglutamic acid-producing ability of a coryneform bacterium as compared to a nonmodified strain. That is, by culturing a coryneform bacterium which has been modified
so that the expression level of the yggB gene is increased, the coryneform bacteriumcauses accumulation of a greater amount of L-glutamic acid than a non-modified strain,or alternatively, the coryneform bacterium produces L-glutamic acid at a higher rate thana non-modified strain. It is preferable that a yggB gene expression-enhancing strain
increases the L-glutamic acid yield (a yield per consumed sugar) by 2% or more, more
preferably 4% or more, and particularly preferably 6% or more, as compared to the parent
strain or a non-modified strain. The yggB gene expression-enhancing strain may increase
the bacterial cell production-subtracted yield as compared to a non-modified strain. The"bacterial cell production-subtracted yield" means a value calculated by subtracting thecarbon yield which is used for production of bacterial cells from the yield per consumed
sugar.
[0032]The L-glutamic acid-producing ability of the coryneform bacterium which has been
modified so that the expression level of the yggB gene increases may be enhanced ascompared to a non-modified strain under at least one condition of an L-glutamic acidproducing condition and a condition with an excess of biotin.
In this regard, the "L-glutamic acid producing condition" means a condition in which
a substance which induces L-glutamic acid production is added to a medium whichcontains carbon sources, nitrogen sources, inorganic salts, and, if necessary, organic
micronutrients such as amino acids and vitamins, or alternatively a condition in which an
amount of a substance which inhibits L-glutamic acid production is limited in a medium.
222

Examples of a substance which induces L-glutamic acid production include penicillin G
and surfactants containing saturated fatty acid such as Tween 40. Examples of a substancewhich is limited due to inhibiting L-glutamic acid production include biotin ("Amino San
Hakko (Amino Acid Fermentation)," Gakkai Shuppan Senta (Academic Society
Publishing Center), 1986 (in Japanese)). Regarding the concentration of these substances
which are added to the medium under the L-glutamic acid-producing condition, theconcentration of biotin is less than 15 μg/L, preferably less than 10 μg/L, and more
preferably less than 5 μg/L, and it is not necessary that any biotin is contained in themedium at all. The concentration of penicillin which is added to the medium is 0.1 U/ml
or more, preferably 0.2 U/ml or more, and more preferably 0.4 U/ml or more. The
concentration of surfactants which are added to the medium is 0.5 g/L or more, preferably
1 g/L or more, and more preferably 2 g/L or more.
On the other hand, the "condition with an excess of biotin" means, for example, acondition containing biotin of 30 μg/L or more, preferably 40 μg/L, and more preferably
50 μg/L, in the medium.
[0033] (Including a correction by Correction 2)
Examples of the yggB gene of the coryneform bacterium include a gene encoding aprotein having an amino acid sequence of SEQ ID NO: 6, 62, 68, or 84. More specifically,
examples of the yggB gene of the coryneform bacterium include: a gene having asequence of nucleotide numbers 1437 to 3035 of SEQ ID NO: 5; a gene having a sequenceof nucleotide numbers 507 to 2093 of SEQ ID NO: 61; a gene having a sequence of
nucleotide numbers 403 to 2001 of SEQ ID NO: 67; and a gene having a sequence of
nucleotide numbers 501 to 2099 of SEQ ID NO: 83. The gene having a sequence of
nucleotide numbers 1437 to 3035 of SEQ ID NO: 5 is a yggB gene of Corynebacterium
glutamicum ATCC13032 strain [Note by the Judgment: Corrected to "ATCC13869
strain" by Correction 2]. The gene having a sequence of nucleotide numbers 507 to 2093
of SEQ ID NO: 61 is a yggB gene of Corynebacterium glutamicum (Brevibacterium
flavum) ATCC14967 strain. The gene having a sequence of nucleotide numbers 403 to2001 of SEQ ID NO: 67 is a yggB gene of Corynebacterium melassecola ATCC17965
strain. The gene having a sequence of nucleotide numbers 501 to 2099 of SEQ ID NO:
83 is encoded to nucleotide numbers 1336092 to 1337693 in the genome sequence
registered as GenBank Accession No. NC_003450 of Corynebacterium glutamicum
ATCC13032, and is registered as NCgl 1221 (NP_600492. Reports small-conductance ...
[gi:19552490]).
Further, since the nucleotide sequence of the yggB gene may differ depending on thespecies and the strain of the coryneform bacterium, the yggB gene may be a variant of223

the nucleotide sequence consisting of nucleotide numbers 1437 to 3035 of SEQ ID NO:
5. A variant of the yggB gene can be searched by referring to the nucleotide sequence
consisting of nucleotide numbers 1437 to 3035 of SEQ ID NO: 5; for example, by meansof BLAST and the like (http://blast.genome.jp/). In addition, the variant of the yggB geneincludes a gene which can be amplified by PCR using yggB gene homologues, forexample, synthetic oligonucleotides of SEQ ID NOS: 75 and 76, for example, by using
coryneform bacterium chromosome as templates. Further, as the gene of the present
invention, a yggB gene of coryneform bacterium is preferable. However, a gene derived
from other microorganisms may be used so far as the gene has a function in a coryneformbacterium. A mutant-type yggB gene mentioned below may be used as a gene of thepresent invention.
[0034]The yggB gene may be a gene having an amino acid sequence in which one or severalamino acids are substituted, deleted, inserted, or added in an amino acid sequence of SEQ
ID NO: 6, 62, 68, or 84 so far as the yggB gene enhances the L-glutamic acid-producing
ability of the coryneform bacterium. The number of "several" as referred to herein means,for example, 2 to 20, preferably 2 to 10, and more preferably 2 to 5.
The above substitution is preferably a conservative substitution. Examples of theconservative substitution include: substitution of ala with ser or thr; substitution of arg
with gln, his, or lys; substitution of asn with glu, gln, lys, his, or asp; substitution of asp
with asn, glu, or gln; substitution of cys with ser or ala; substitution of gln with asn, glu,lys, his, asp, or arg; substitution of glu with gly, asn, gln, lys, or asp; substitution of gly
with pro; substitution of his with asn, lys, gln, arg, or tyr; substitution of ile with leu, met,val, or phe; substitution of leu with ile, met, val, or phe; substitution of lys with asn, glu,
gln, his, or arg; substitution of met with ile, leu, val, or phe; substitution of phe with trp,tyr, met, ile, or leu; substitution of ser with thr or ala; substitution of thr with ser or ala;
substitution of trp with phe or tyr; substitution of tyr with his, phe, or trp; and substitutionof val with met, ile, or leu.
The yggB gene of the present invention includes a gene which encodes a protein
which is at least 80% homology, preferably at least 90% homology, more preferably atleast 95% homology, and particularly preferably at least 97% homology, to the entire
amino acid sequence of SEQ ID NO: 6, 62, 68, or 84, and which enhances the L-glutamic
acid-producing ability of the coryneform bacterium. In this regard, the homology can becalculated by BLAST developed by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90,
5873 (1993)) and FASTA developed by Pearson (Methods Enzymol., 183, 63 (1990)).
The homology search programs (such as BLASTN and BLASTP) based on these224

algorithms are available from NCBI and others (//www.ncbi.nlm.nih.gov).
The substitutions, deletions, insertions, additions, or inversions of amino acids as
mentioned above include those caused by naturally occurring mutants or variants, suchas those caused by individual differences or by species differences in microorganisms
carrying the yggB gene.
[0035]
Especially, the amino acids at the following positions in the amino acid sequence of
SEQ ID NO: 6 may be substituted or deleted. The amino acid sequence of the YggB
protein which is conserved among coryneform bacteria is shown in SEQ ID NO: 85, in
which Xaa indicates a location where an amino acid may be substituted or deleted.
Glutamine residue at position 48 (preferably substituted with arginine residue)
Asparagine residue at position 275 (preferably substituted with serine residue)
Glutamic acid residue at position 298 (preferably substituted with alanine residue)
Alanine residue at position 343 (preferably substituted with valine residue)
Phenylalanine residue at position 396 (preferably substituted with isoleucine residue)
Serine residue at position 438 (preferably substituted with glycine residue)
Valine residue at position 445 (preferably substituted with alanine residue)
Alanine residue at position 454 (preferably substituted with valine residue)
Proline residue at position 457 (preferably substituted with serine residue)
Serine residue at position 474 (preferably substituted with asparagine residue)
Valine residue at position 517 (preferably deleted)
Glutamic acid residue at position 518 (preferably deleted)
Alanine residue at position 519 (preferably deleted)
Proline residue at position 520 (preferably deleted)[0036]
The above yggB gene homologue can be obtained by modifying the gene having asequence of nucleotide numbers 1437 to 3035 of the nucleotide sequence of SEQ ID NO:
5, the gene having a sequence of nucleotide numbers 507 to 2093 of the nucleotide
sequence of SEQ ID NO: 61, the gene having a sequence of nucleotide numbers 403 to2001 of the nucleotide sequence of SEQ ID NO: 67, or the gene having a sequence of
nucleotide numbers 501 to 2099 of the nucleotide sequence of SEQ ID NO: 83 by, forexample, the site-directed mutagenesis method, so that the amino acid residue at the
specific site in the encoded protein is substituted, deleted, inserted, or added.
Further, such genes can also be obtained by a conventionally known mutagenesis
treatment. Examples of the mutagenesis treatment include: a method of treating a gene
having a sequence of nucleotide numbers 1437 to 3035 of the nucleotide sequence of SEQ
225

ID NO: 5, a gene having a sequence of nucleotide numbers 507 to 2093 of the nucleotide
sequence of SEQ ID NO: 61, a gene having a sequence of nucleotide numbers 403 to2001 of the nucleotide sequence of SEQ ID NO: 67, or a gene having a sequence of
nucleotide numbers 501 to 2099 of the nucleotide sequence of SEQ ID NO: 83 in vitro
with hydroxylamine, etc.; and treating a microorganism, for example, an Escherichia
bacterium, carrying the gene with ultraviolet rays or a mutagenesis agent typically usedin mutagenesis treatments, such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl
methanesulfonate (EMS). The above substitutions, deletions, insertions, additions,inversions, etc. of amino acids include those caused by naturally occurring mutants orvariants, such as those caused by individual differences or by species differences in
microorganisms carrying the yggB gene. When these genes are introduced into a
coryneform bacterium, whether these genes enhance the L-glutamic acid-producing
ability of coryneform bacterium can be confirmed by introducing the genes into a wildtype strain of the coryneform bacterium and determining if the L-glutamic acid-producing
ability of the obtained coryneform bacterium is enhanced under the above condition, forexample.
[0039]
Enhancing the expression of the above-described yggB gene can be attained byincreasing the copy number of the yggB gene, modifying an expression regulatory
sequence of the yggB gene, amplifying a transcriptional activator of the yggB gene, or
reducing the activity of a transcriptional repressor of the yggB gene.
(3) Introduction of Mutant-Type yggB Gene
[0050]... The modification using the yggB gene also includes introduction of a mutant-type
yggB gene into a coryneform bacterium. "Introduction of a mutant-type yggB gene" asused herein includes: introduction of a mutation into a chromosomal yggB gene of acoryneform bacterium; introduction of a plasmid containing a mutant-type yggB gene
into a coryneform bacterium; and substitution of a chromosomal yggB gene with amutant-type yggB gene.
In the present invention, the "mutant-type yggB gene" means a yggB gene comprisinga mutation in a coding region of the yggB gene, in which the mutation imparts a function
to the yggB gene of enhancement of the L-glutamic acid-producing ability of acoryneform bacterium under a condition with an excess of biotin. In this regard, a mutanttype yggB gene may be a gene which enhances the L-glutamic acid-producing ability ofa coryneform bacterium not only under a condition with an excess of biotin when thegene is introduced into the coryneform bacterium, but also even under the L-glutamic
226acid-producing condition as mentioned above.
The phrase "enhance the L-glutamic acid-producing ability of a coryneformbacterium under a condition with an excess of biotin" means that when the coryneformbacterium of the present invention is cultured in a medium containing biotin at aconcentration in which a non-modified strain of the coryneform bacterium cannot cause
accumulation of L-glutamic acid, for example, in a medium containing 30 μg/L or moreof biotin, the coryneform bacterium of the present invention causes accumulation of more
L-glutamic acid in the medium than that of a non-modified strain, or the coryneformbacterium of the present invention produces L-glutamic acid at a higher rate than that of
the non-modified strain.
[0051]
Hereinafter, a method for obtaining a mutant-type yggB gene of the present invention
and a method for introducing a mutation into the yggB gene are stated. However, amethod for obtaining a mutant-type yggB gene of the present invention and a method for
introducing a mutation into the yggB gene are not limited to the following methods.
(II-1) Method of Utilizing odhA Gene-Deleted Strain
The inventors of the present invention have found that an odhA gene (a sucA gene)deleted strain (hereinafter referred to as "odhA-disrupted strain") can be utilized for amethod for obtaining a mutant-type yggB gene, in which the odhA gene encodes an E1osubunit of α-ketoglutarate dehydrogenase. The construction of the odhA-disrupted strain
can be performed by a method using the sacB gene as mentioned above.
[0058](II-2) Method of Utilizing a Transposable Element
A coryneform bacterium having a mutant-type yggB gene may be also screened byusing a transposable element which functions in the coryneform bacterium. The
transposable element includes an insertion sequence (IS element) and a transposon. The
mutant-type yggB gene may be a gene which can be obtained by allowing an insertion
sequence (IS element) and/or a transposon to be accidentally inserted into the wild-type
yggB gene. Alternatively, the mutant-type yggB gene may be a gene which is constructed
artificially by using an artificial transposon. A strain into which a transposable element isinserted can be selected by reference to an index, such as a decrease in sensitivity to Lglutamic acid analogues. As an L-glutamic acid analogue, 4-fluoroglutamic acid can beutilized. Further, a strain into which a transposable element is inserted can be selected by
selecting antibiotics-resistant strains at random with the use of an artificial transposon
containing an antibiotics-resistant gene, and confirming the length of the yggB gene of
the antibiotics-resistant strains by PCR.
227

[0064](II-3) Method of Introducing a Mutation into the yggB Gene In Vitro at Random
A mutation can be introduced into the yggB gene in vitro at random, among which
the mutant-type yggB gene can be selected from a clone which can produce L-glutamic
acid without the addition of surfactants. The parent strain useful for screening ispreferably a strain which cannot cause accumulation of L-glutamic acid under thecondition containing an excess of biotin; for example, C. glutamicum wildtype
ATCC13869 strain, ATCC13032 strain, ATCC14067 strain, and C. melassecola wildtype ATCC17965 strain.
[0068](II-4) Method for Obtaining L-glutamic Acid Analogue-Resistant Strains
Mutant-type yggB genes can also be obtained by culturing a coryneform bacterium
having a wild-type yggB gene in a medium containing an L-glutamic acid analogue, andobtaining L-glutamic acid analogue-resistant strains which can grow in the same medium.
The parent strain used for screening is preferably the wild-type strain of the coryneformbacterium as mentioned above, and may be any strains having a wild-type yggB gene. In
addition, a coryneform bacterium having a plasmid containing a wild-type yggB gene canalso be used. ...
(4) Mutant-Type yggB Gene of the Present Invention[0069]
Hereinafter, specific examples of the mutant-type yggB gene are stated. However, themutant-type yggB gene of the present invention is not particularly limited so far as themutant-type yggB gene has a mutation to enhance the L-glutamic acid producing abilityof a coryneform bacterium under a condition in which an excess of biotin is present.
A. Mutation in C-Terminal Side
[0070]... This mutation is a mutation which is introduced into a portion of a nucleotide
sequence of a region encoding a sequence of amino acid numbers 419 to 533 of SEQ ID
NO: 6, 68, 84, or 85, or a sequence of amino acid numbers 419 to 529 of SEQ ID NO: 62.
For example, in the nucleotide sequence of SEQ ID NO: 5, this region corresponds to theregion consisting of nucleotide numbers 2692 to 3035. This mutation may be of any typeso far as the mutation is introduced into at least a portion of the nucleotide sequence ofthe region. Among mutations, insertion of an insertion sequence (hereinafter referred toas IS) or a transposon is preferable. The mutation may be accompanied by an amino acidsubstitution (missense mutation), or a frameshift mutation by insertion of the above IS
may be introduced, or a nonsense mutation may be introduced, any of which is preferable.
228

(a) Mutation by Insertion of Transposable Element (2A-1-Type Mutation)[0071]... Examples of a mutation in the C-terminal side include a mutation in which an IS isinserted next to G at position 2691 of SEQ ID NO: 5. The nucleotide sequence of themutant-type yggB gene in which this mutation is introduced is shown in SEQ ID NO: 7,
and the amino acid sequence of the mutant-type YggB protein encoded by this mutanttype yggB gene is shown in SEQ ID NO: 8. In SEQ ID NO: 8, the region positioned
downstream from valine at position 419 of SEQ ID NO: 6 is substituted with a sequencewhich is derived from a short IS. In this regard, the IS inserted into SEQ ID NO: 7 hashigh homology to IS1207 (GenBank accession No. X96962) and IS719 (GenBank
accession No. E12759).
The above mutant-type yggB gene is called a 2A-1-type mutation. The 2A-1-type
mutation includes mutation in which the above region of the C-terminal side in SEQ ID
NOS: 6, 62, 68, 84, and 85 is deleted or substituted.
Further, the scope of the present invention includes a mutation in which another IS isinserted into the above region, for example, a mutation in which an IS encoding
transposase as mentioned above is inserted into the above region. The position into whichtransposase is introduced may be any position in the above region, and a location which
each transposase can easily recognize and a location of a hot spot into which an IS can beeasily inserted are preferable.
(b) Mutation in which Proline Residue is Substituted with Another Amino Acid (66-Type
Mutation, 22-Type Mutation)[0072]... In addition, examples of a mutation in the C-terminal side include a mutation inwhich proline which is present in a region in a sequence of amino acid numbers 419 to
533 of SEQ ID NO: 6, 68, 84, or 85, or a region of amino acid numbers 419 to 529 of
SEQ ID NO: 62 is substituted with another amino acid. The proline which may besubstituted is present in the following positions of SEQ ID NO: 6:
Proline residue at position 424 (424th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 437 (437th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 453 (453rd in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 457 (457th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 462 (462nd in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 469 (469th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 484 (484th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 489 (489th in SEQ ID NOS: 62, 68, 84, and 85);229

Proline residue at position 497 (497th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 515 (515th in SEQ ID NOS: 62, 68, 84, and 85);
Proline residue at position 529 (529th in SEQ ID NOS: 68, 84, and 85; and 525th in
SEQ ID NO: 62); andProline residue at position 533 (533rd in SEQ ID NOS: 68, 84, and 85; and 529th in
SEQ ID NO: 62).
It is considered that proline residues in the C-terminal side of the yggB gene play animportant role in maintaining a three-dimensional structure of the YggB protein (ProteinEng. 2002 Jan; 15(1): 29-33, J Biol Chem. 1991 Dec 25; 266(36): 24287-94.).
Particularly, proline at position 424 or 437 is preferably substituted with anotheramino acid.
In this regard, another amino acid may be any amino acid so far as such other aminoacid is a natural amino acid other than proline. Proline is preferably substituted into a
residue selected from Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met,
Phe, Trp, Tyr, Gly, Ala, and His. Particularly, proline at position 424 is preferably
substituted with a hydrophobic amino acid Ala, Gly, Val, Leu, or Ile. Among thesehydrophobic amino acids, proline at position 424 is more preferably substituted with abranched amino acid Leu, Val, or Ile (66-Type Mutation). Examples of a mutation inwhich proline at position 424 is substituted with leucine includes a mutation in which "C"at position 1673 is substituted with "T" in SEQ ID NO: 67. This mutant-type yggB geneis shown in SEQ ID NO: 69, and the amino acid sequence of the mutant-type YggB
protein encoded by this mutant-type yggB gene is shown in SEQ ID NO: 70.
Further, proline at position 437 is preferably substituted with an amino acid having ahydroxyl group in its side chain (Thr, Ser, Tyr). Among these, proline at position 437 ismost preferably substituted with Ser (22-Type Mutation). Examples of a mutation inwhich proline at position 437 is substituted with serine include a mutation in which C atposition 2745 is substituted with T in SEQ ID NO: 5. In addition, this mutation may be
accompanied with a mutation in which C at position 3060 is substituted with T in SEQ
ID NO: 5. This mutant-type yggB gene is shown in SEQ ID NO: 73, and the amino acidsequence of the mutant-type YggB protein encoded by this mutant-type yggB gene isshown in SEQ ID NO: 74.
B. Mutation in Transmembrane Region[0073]... The YggB protein encoded by the yggB gene is presumed to have fivetransmembrane regions. In the amino acid sequence of the wild-type YggB protein shown
in SEQ ID NOS: 6, 62, 68, 84, and 85, the transmembrane regions correspond to amino
230

acid numbers 1 to 23 (the first transmembrane region), amino acid numbers 25 to 47 (thesecond transmembrane region), amino acid numbers 62 to 84 (the third transmembraneregion), amino acid numbers 86 to 108 (the fourth transmembrane region), and aminoacid numbers 110 to 132 (the fifth transmembrane region). In SEQ ID NO: 5, DNA
encoding these regions corresponds to nucleotide numbers 1437 to 1505, nucleotide
numbers 1509 to 1577, nucleotide numbers 1620 to 1688, nucleotide numbers 1692 to1760, and nucleotide numbers 1764 to 1832, respectively. The mutation of the present
invention is preferably introduced into the DNA encoding these transmembrane regions.
The mutation which is introduced into these regions is preferably a mutation including
substitution, deletion, addition, insertion, or inversion of one or several amino acids
without involving a frame-shift mutation and a nonsense mutation. Accordingly, in caseof substitution in an amino acid sequence, a missense mutation involving amino acidsubstitution is preferable. The term "several" in the above-mentioned "substitution,
deletion, addition, insertion, or inversion of one or several amino acids" means 2 to 20,
preferably 2 to 10, more preferably 2 to 5, and still more preferably 2 or 3. The mutationof insertion and deletion of one or several amino acids includes introduction of point
mutations of one to several nucleotides and introduction of nucleotide sequence without
involving frame-shift mutation, and is, for example, insertion or deletion of 3, 6, 9, 12,
15, 18, or 21 nucleotides, preferably deletion or insertion of 3, 6, or 9 nucleotides, andmore preferably deletion or insertion of 3 nucleotides.
(a) Mutation in the First Transmembrane Region (A1-Type Mutation)[0074]For example, the following mutations are listed.
... This mutation is a mutation in which one to several amino acids are inserted
between leucine residue at position 14 and tryptophan residue at position 15 in an aminoacid sequence shown in any of SEQ ID NOS: 6, 62, 68, 84, and 85.
Specific examples of the above mutation include a mutation in which three aminoacids (for example, Cys-Ser-Leu) are inserted between leucine residue at position 14 andtryptophan residue at position 15. Examples of this mutation include a mutation in which
TTCATTGTG is inserted next to G at position 1480 in SEQ ID NO: 5. The nucleotide
sequence of the mutant-type yggB gene into which this mutation is introduced is shown
in SEQ ID NO: 19, and the amino acid sequence of the mutant-type YggB protein
encoded by this mutant-type yggB gene is shown in SEQ ID NO: 20.
(b) Mutation in the Fourth Transmembrane Region (19-Type Mutation)[0075]... This mutation is a mutation in which alanine residue at position 100 is substituted
231

with another amino acid in an amino acid sequence shown in any of SEQ ID NOS: 6, 62,
68, 84 and 85. "Another amino acid" may be any amino acid residue other than alanine,and means any of arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine,
glycine, histidine, isoleucine, methionine, leucine, lysine, phenylalanine, proline, serine,
tryptophan, tyrosine, valine, and threonine. Among these, alanine residue at position 100
is preferably substituted with an amino acid having a hydroxyl group in its side chain(threonine, serine, tyrosine), and is particularly preferably substituted with threonine
residue. Examples of such mutation include a mutation in which G at position 1734 issubstituted with A in SEQ ID NO: 5.
The nucleotide sequence of the mutant-type yggB gene into which this mutation is
introduced is shown in SEQ ID NO: 21, and the amino acid sequence of the mutant-type
YggB protein encoded by this mutant-type yggB gene is shown in SEQ ID NO: 22.
(c) Mutation in the Fifth Transmembrane Region (L30-Type Mutation, 8-Type Mutation)[0076]... This mutation is a mutation in which alanine residue at position 111 is substituted
with another amino acid in an amino acid sequence shown in any of SEQ ID NOS: 6, 62,
68, 84, and 85. "Another amino acid" may be any amino acid residue other than alanine,and means any of arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine,
glycine, histidine, isoleucine, methionine, leucine, lysine, phenylalanine, proline, serine,
tryptophan, tyrosine, valine, and threonine. Among these, alanine residue at position 111
is preferably substituted with a branched amino acid (valine, isoleucine, leucine),
particularly preferably valine residue, or with an amino acid having hydroxyl group in its
side chain (threonine, serine, tyrosine), particularly preferably threonine residue.
Examples of this mutation include a mutation in which C at position 1768 is substituted
with T in SEQ ID NO: 5 (L30-type mutation) or a mutation in which G at position 837 issubstituted with A in SEQ ID NO: 61 (8-type mutation). The nucleotide sequence of themutant-type yggB gene into which L30-type mutation is introduced is shown in SEQ ID
NO: 23, and the amino acid sequence of the mutant-type YggB protein encoded by thismutant-type yggB gene is shown in SEQ ID NO: 24. The nucleotide sequence of themutant-type yggB gene into which 8-type mutation is introduced is shown in SEQ ID
NO: 63, and the amino acid sequence of the mutant-type YggB protein encoded by thismutant-type yggB gene is shown in SEQ ID NO: 64.
C.
[0077]
The above-mentioned "mutant-type yggB gene," so far as it has a function capable ofenhancing L-glutamic acid-producing ability under a condition in which an excess of232biotin is present, may be a functionally equivalent gene containing the mutation describedabove, for example, a gene that is substantially homologous to a mutant-type gene, such
as DNA containing a sequence of nucleotide numbers 1437 to 2705 of SEQ ID NO: 7,
DNA containing a sequence of nucleotide numbers 1437 to 3044 of SEQ ID NO: 19,
DNA containing a sequence of nucleotide numbers 1437 to 3035 of SEQ ID NO: 21,
DNA containing a sequence of nucleotide numbers 1437 to 3035 of SEQ ID NO: 23,
DNA containing a sequence of nucleotide numbers 507 to 2093 of SEQ ID NO: 63, DNA
containing a sequence of nucleotide numbers 403 to 2001 of SEQ ID NO: 69, or DNA
containing a sequence of nucleotide numbers 548 to 2146 of SEQ ID NO: 73, that is
capable of hybridizing to a polynucleotide containing a nucleotide sequence
complementary to such sequence or to a probe having a part of that nucleotide sequence
under stringent conditions. "Stringent conditions" as used herein are conditions underwhich a so-called specified hybrid is formed, and a non-specific hybrid is not formed.
Examples of stringent conditions include typical Southern hybridization washingconditions, i.e. washing once or preferably two to three times at a salt concentration andtemperature equivalent to 0.1×SSC, 0.1%SDS at 60°C, more preferably 0.1×SSC,
0.1%SDS at 68°C.
[0078]As a protein which is encoded by the mutant-type yggB gene, so far as an activity ofthe protein has a function capable of enhancing L-glutamic acid-producing ability undera condition in which an excess of biotin is present, the protein may be a functionallyequivalent protein which has, for example, an amino acid sequence in which one orseveral amino acids in addition to the amino acids of the above mutation point aresubstituted, deleted, inserted, or added in an amino acid sequence selected from SEQ ID
NOS: 8, 20, 22, 24, 64, 70, and 74. The number of "several" as referred to herein means,for example, 2 to 20, preferably 2 to 10, more preferably 2 to 5. The above substitution ispreferably a conservative substitution (neutral mutation). Examples of the conservative
substitution include: substitution of ala with ser or thr; substitution of arg with gln, his, orlys; substitution of asn with glu, gln, lys, his, or asp; substitution of asp with asn, glu, or
gln; substitution of cys with ser or ala; substitution of gln with asn, glu, lys, his, asp, or
arg; substitution of glu with gly, asn, gln, lys, or asp; substitution of gly with pro;
substitution of his with asn, lys, gln, arg, or tyr; substitution of ile with leu, met, val, orphe; substitution of leu with ile, met, val, or phe; substitution of lys with asn, glu, gln, his,or arg; substitution of met with ile, leu, val, or phe; substitution of phe with trp, tyr, met,ile, or leu; substitution of ser with thr or ala; substitution of thr with ser or ala; substitutionof trp with phe or tyr; substitution of tyr with his, phe, or trp; and substitution of val with233

met, ile, or leu. As mentioned above, the amino acid which is present in Xaa in SEQ ID
NO: 85 may be substituted. The yggB gene of the present invention includes a homologue
which encodes a protein which has at least 80% homology, preferably at least 90%
homology, more preferably at least 95% homology, and particularly preferably at least
97% homology, to the entire amino acid sequence of SEQ ID NOS: 8, 20, 22, 24, 64, 70,
and 74, and which encodes a protein having an activity which is equivalent to that of aprotein encoded by the mutant-type yggB gene in the coryneform bacterium.
[0079]
Especially, the following amino acids may be substituted in the amino acid sequencesof SEQ ID NO: 8, 80, 22, 24, 64, 70, or 74:
Glu at position 48 (preferably replaced by Arg)
Asp at position 275 (preferably replaced by Ser)Glu at position 298 (preferably replaced by Ala)
Ala at position 343 (preferably replaced by Val)Phe at position 396 (preferably replaced by Ile)
Ser at position 438 (preferably replaced by Gly)
Val at position 445 (preferably replaced by Ala)
Ala at position 454 (preferably replaced by Val)Pro at position 457 (preferably replaced by Ser)Ser at position 474 (preferably replaced by Asp)
Val at position 517 (preferably deleted)Glu at position 518 (preferably deleted)
Ala at position 519 (preferably deleted)
Pro at position 520 (preferably deleted)(5) Method of Introducing Mutant-Type yggB Gene into Coryneform Bacterium[0080]... The mutant-type yggB gene having the above mutation can be obtained by, forexample, the site-directed mutagenesis method. More specifically, cloning can be
performed by using an overlap extension PCR method, etc. which amplifies the relevant
locations using a PCR primer having a sequence containing a mutation in thecorresponding region of the yggB gene (Urban, A., Neukirchen, S. and Jaeger, K. E., A
rapid and efficient method for site-directed mutagenesis using one-step overlap extension
PCR. Nucleic Acids Res, 25, pp 2227-8. (1997).).
The coryneform bacterium of the present invention can be obtained by introducing
the obtained mutant-type yggB gene into a coryneform bacterium. Examples of a methodof introduction include a method of substituting a wild-type yggB gene on a chromosome234with the mutant-type yggB gene. The mutant-type yggB gene may be introduced into astrain in which a wild-type yggB gene on a chromosome is disrupted. In this regard, themutant-type yggB gene may be introduced while a wild-type yggB gene is left on achromosome as a single recombinant strain. One copy of the incorporated wild-type geneand one copy of the incorporated mutant-type gene may be present on a chromosome.
The substitution of the yggB gene on the chromosome can be performed by using, forexample, a temperature-sensitive plasmid containing a sacB gene encoding the above
levan sucrase.
(6) Method of Producing L-glutamic Acid of the Present Invention[0085]... L-glutamic acid can be produced by obtaining a coryneform bacterium in the above
manner, then culturing the obtained coryneform bacterium in a medium, to thereby cause
production and accumulation of L-glutamic acid in the medium, and collecting the Lglutamic acid from the medium.
[0086]As the medium for the culture, an ordinary medium can be used where the ordinary
medium contains carbon sources, nitrogen sources, and inorganic salts, and, if necessary,organic micronutrients such as amino acids and vitamins. Both synthetic medium andnatural medium can be used. Any types of carbon sources and nitrogen sources may be
used for the medium so far as such sources can be utilized by a strain to be cultured.
[0087]As the carbon sources, saccharides such as glucose, glycerol, fructose, sucrose,
maltose, mannose, galactose, starch hydrolysate, and molasses can be used. In addition,organic acids such as acetic acid and citric acid, and alcohols such as ethanol may also be
used alone or in combination with other carbon sources. As the nitrogen sources,
ammonia, ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium
chloride, ammonium phosphate, and ammonium acetate, or nitrates, and the like can beused. As the organic micronutrients, amino acids, vitamins, fatty acids, nucleic acids,
peptone containing these substances, casamino acid, yeast extract, and soybean protein
decomposition products can be used. When an auxotrophic mutant strain which requires
an amino acid and the like for growth is used, such a required nutrient is preferably added.
As the inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganesesalts, and the like can be used.
Further, surfactants and penicillin having appropriate concentrations may be added
according to properties of the yggB gene-modified strain to be used. Furthermore, aconcentration of biotin may be adjusted according to properties of the yggB gene235

modified strain to be used.
[0088]As to the culture, aerobic culture is performed by preferably controlling thefermentation temperature to 20 to 45°C and pH to 3 to 9. When the pH decreases during
the culture, neutralization should be performed by, for example, adding calcium carbonate
or neutralizing with alkali such as ammonia gas. Under these conditions, culturing is
performed for preferably about 10 to 120 hours, to thereby accumulate a considerable
amount of L-glutamic acid in the culture solution.
[0089]
Further, the culture can also be performed while precipitating L-glutamic acid in themedium by using a liquid medium adjusted to conditions such that the produced Lglutamic acid precipitates. Examples of the conditions such that L-glutamic acidprecipitates include a pH of 5.0 to 4.0, preferably a pH of 4.5 to 4.0, more preferably a
pH of 4.3 to 4.0, particularly preferably a pH of 4.0 (Specification of European Patent
Application Publication No. 1078989).
[0090]
Collection of L-glutamic acid from the culture solution after completion of the culture
may be performed by conventional methods. For example, L-glutamic acid may becollected by removing bacterial cells from the culture solution, followed by concentrating
and crystallizing L-glutamic acid, or by using ion exchange chromatography. When theculture is performed under conditions such that L-glutamic acid precipitates, the Lglutamic acid which is precipitated in the culture solution can be collected bycentrifugation, filtration, etc. In this case, L-glutamic acid which is dissolved in themedium may also be isolated after crystallization of the dissolved L-glutamic acid.
7. Examples(1) Working Example 1[0092]<Construction of Vector for Gene Disruption Carrying sacB Gene>(A) Construction of pBS3
Construction of a vector for gene disruption carrying sacB gene was performed withreference to the method disclosed in International Publication Nos. WO 2005/113745 andWO 2005/113744. A sacB gene (SEQ ID NO: 11) was obtained by PCR using achromosomal DNA of Bacillus subtilis as a template and SEQ ID NOS: 13 and 14 asprimers. The PCR was performed using LA taq (TaKaRa) as follows: one cycle of heatretention at 94°C for 5 minutes; and 25 cycles of denaturing at 94°C for 30 seconds,
annealing at 49°C for 30 seconds, and elongation at 72°C for 2 minutes. The obtained236

PCR product was purified by a conventional method, and then digested with BglII and
BamHI and blunt-ended. This fragment was digested with AvaII of pHSG299, and thenwas inserted into a blunt-ended site. The obtained DNA was used to transform competentcells of Escherichia coli JM109 (manufactured by Takara Bio Inc.). Then, thetransformed bacterial cells were spread on LB medium containing 25 μg/ml kanamycin(hereinafter, abbreviated as Km) and were incubated overnight. Thereafter, colonies thatappeared were selected, and single colonies were isolated to obtain transformants.
Plasmids were extracted from the obtained transformants, and the plasmid having an
insert of the objective PCR product was named pBS3. Figure 1 shows the procedure forconstructing pBS3.
[0093](B) Construction of pBS4S
The SmaI site in the kanamycin-resistant gene sequence on pBS3 was modified by
nucleotide substitution (a mutation which is not cut by SmaI) without causing amino acidsubstitution using cross-over PCR, to thereby obtain a plasmid in which kanamycinresistant gene was disrupted. First, PCR was performed using pBS3 as a template andsynthetic DNAs of SEQ ID NOS: 15 and 16 as primers, to thereby obtain an N-terminal
amplified product of the kanamycin-resistant gene. On the other hand, in order to obtaina C-terminal side amplified product of the Km-resistant gene, PCR was performed using
pBS3 as a template and synthetic DNAs of SEQ ID NOS: 17 and 18 as templates. PCR
was performed using Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) as
follows: one cycle of heat retention at 98°C for 5 minutes; and 25 cycles of denaturing at
98°C for 10 seconds, annealing at 57°C for 30 seconds, and elongation at 72°C for 1
minute, to obtain the objective PCR product. SEQ ID NOS: 16 and 17 are partiallycomplementary to each other. Further, the SmaI site in these sequences was disrupted by
nucleotide substitution without causing amino acid substitution. Then, in order to obtaina fragment of the mutant-type kanamycin-resistant gene in which the SmaI site wasdisrupted, the above N-terminal and C-terminal gene products were mixed together insubstantially equimolar amounts. Thereafter, PCR was performed using the gene products
as a template and synthetic DNAs of SEQ ID NOS: 15 and 18 as primers to obtain a Kmresistant gene amplified product in which a mutation was introduced. The PCR was
performed using Pyrobest DNA Polymerase (manufactured by Takara Bio Inc.) as
follows: one cycle of heat retention at 98°C for 5 minutes; and 25 cycles of denaturing at
98°C for 10 seconds, annealing at 57°C for 30 seconds, and elongation at 72°C for 1.5
minutes, to thereby obtain the objective PCR product.
[0094]
237

The PCR product was purified by a conventional method, and then digested with
BanII, and thereafter inserted into the above BanII site of pBS3. This DNA was used totransform competent cells of Escherichia coli JM109 (manufactured by Takara Bio Inc.).
Then, the transformed bacterial cells were spread on LB medium containing 25 μg/ml
kanamycin and were incubated overnight. Thereafter, colonies that appeared wereselected, and single colonies were isolated to obtain transformants. Plasmids wereextracted from the obtained transformants, and the plasmid having an insert of theobjective PCR product was named pBS4S. Figure 2 shows the procedure for constructing
pBS4S.
(2) Working Example 2[0095]<Construction of odhA Mutant Strain Derived from C. glutamicum ATCC13869 Strain>
The sequence of the odhA gene encoding the α-ketoglutarate dehydrogenase of acoryneform bacterium has already been identified (Microbiology 142, 3347-3354, (1996),
GenBank accession No. D84102). Based on the published sequence of the odhA gene,
the primers stated in SEQ ID NOS: 1 and 2 were designed, and PCR was performed using
the primers and the chromosomal DNA of ATCC13869 strain as a template to amplify
only the internal sequence of the odhA gene. The amplified PCR fragment wascompletely digested with BamHI and inserted to the BamHI site of pBS4S constructed in
Working Example 1, whereby the plasmid pBS4SΔsucAint was constructed (Figure 3
shows the constructing procedure).
[0096]
pBS4SΔsucAint was introduced into C. glutamicum ATCC13869 strain by theelectric pulse method (Unexamined Patent Application Publication No. 1990-207791)
and was spread over CM-Dex agar medium (5 g/l glucose, 10 g/l polypeptone, 10 g/l
yeast extract, 1 g/l KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O, 0.01 g/l MnSO4·4-5H2O, 3 g/l urea, 1.2 g/l soybean protein hydrolysate solution, and 20 g/l agar, adjusted
to pH 7.5 with NaOH: autoclaved at 120°C for 20 minutes) containing 25 μg/ml
kanamycin. After culturing at 31.5°C, PCR was performed using each of thechromosomes extracted from strains, which appeared to confirm that these strains were
single cross-over recombinants in which pBS4SΔsucAint was incorporated byhomologous recombination into the chromosome. Whether the strain is single cross-overrecombinant can be easily confirmed by performing PCR using the chromosome of thecandidate strain as a template, and a sequence (SEQ ID NO: 3) specific to pBS4S plasmid
and a sequence (SEQ ID NO: 4) complementary to a chromosomal sequence as primers(the sequence of pBS4S is absent on chromosome of a non-recombinant strain, and thus,238

no fragment is amplified from the non-recombinant strain, thereby enabling
determination on whether the strain is single cross-over recombinant).
[0097]The single cross-over recombinant thus obtained was named 2A-1 strain. The wildtype ATCC13869 strain and the 2A-1 strain were inoculated in 20 ml of a flask medium(30 g/l glucose, 15 g/l ammonium sulfate, 1 g/l KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l
FeSO4 ·7H2O, 0.01 g/l MnSO4 ·4-5H2O, 200 μg/l VB1, 300 μg/l biotin, and 0.48 g/lsoybean hydrolysates (T-N), adjusted to pH 8.0 with KOH: autoclaved at 115°C for 10
minutes), followed by addition of 1 g of calcium carbonate which had been dry heatsterilized beforehand, and each of the strains was cultured with shaking at 31.5°C. Afterthe sugar was completely consumed, the concentration of L-glutamic acid in the medium
was determined. The results are shown in Table 1 (OD620 is a turbidity diluted to 101
times, and indicates an amount of the bacterial cells; Glu (g/L) indicates an amount ofaccumulated L-glutamic acid). It was confirmed that the 2A-1 strain has an L-glutamic
acid-producing ability under the condition where the parent ATCC13869 strain did not
produce L-glutamic acid at all.
[0098][Table 1]<Table 1 Amount of L-glutamic Acid Produced by 2A-1 Strain>
OD620 (x101)

Glu (g/L)

0.658
0.315
0.002

0.2
17.7
0.4

ATCC13869
2A-1
Blank

(3) Working Example 3[0099]<Construction of odhA Revertant Strain Derived from 2A-1 Strain>
In the obtained 2A-1 strain, the odhA gene on the chromosome was disrupted by
pBS4SΔT¥sucAint. By removing the plasmid on the chromosome from this strain, theodhA gene was reverted to the wild-type strain. On the other hand, the odhA-deleted
strain grows very slowly in a medium containing no sugar. However, it is presumed thatthe odhA-revertant strain in which the odhA reverted to the wild-type grows well in a
medium containing no sugar such as CM2B medium (10 g/l polypeptone, 10 g/l yeast
extract, 5 g/l NaCl, 10 μg/L biotin, 20 g/l agar, adjusted to pH 7.0 with KOH). To obtainsuch a revertant strain, the 2A-1 strain was spread over a CM2B plate to select growthimproved strains. The growth-improved 2A-1R strain which thus appeared was purified
239

on the CM2B plate, and the kanamycin-sensitivity of the 2A-1R strain was examined. As
a result, it was found that all of the selected strains were kanamycin-sensitive and sucroseresistant.
[0100]
Since the pBS4SΔsucAint contains a kanamycin-resistant gene and the sacB geneencoding levan sucrase, strains harboring pBS4SΔsucAint exhibit kanamycin-resistanceand sucrose-sensitivity, while strains from which pBS4SΔsucAint was removed exhibit
kanamycin-sensitivity and sucrose-resistance. From these results, it was considered thatthe odhA had reverted to the wild-type one in the 2A-1R strain. Further, the sequence ofthe odhA gene was confirmed. As a result, it was confirmed that the odhA gene had no
mutations. Therefore, it was concluded that the odhA had reverted to the wild-type [one]in the 2A-1R strain. The L-glutamic acid-producing ability of the 2A-1R strain was
confirmed by a method similar to the method stated in Working Example 2. The results
are shown in Table 2 (OD620 indicates an amount of the bacterial cells diluted to 101
times; Glu (g/L) indicates an amount of accumulated L-glutamic acid). Although theaccumulation of L-glutamic acid by the 2A-1R strain was inferior to that by the 2A-1
strain, the 2A-1R strain accumulated a much higher amount of L-glutamic acid than thewild-type ATCC13869 strain (Table 2). In addition, when the shaking culture wascontinued after the sugar was completely consumed, decomposition of L-glutamic acidwas observed in the 2A-1R strain, which has proved that the odhA gene had reverted to
the wild-type in this strain (Figure 4).
[0101][Table 2]<Table 2 Amount of L-glutamic Acid Produced by odhA Revertant Strain>
OD620 (x101)

Glu (g/L)

0.696
0.332
0.327
0.002

0.5
17.1
14.3
0.6

ATCC13869
2A-1
2A-1R
Blank
(4) Working Example 4[0102]<Isolation of Gene which is Involved in L-glutamic Acid Production by 2A-1R Strain>
On the CM2B plate medium, 2A-1R strain could form colonies at substantially thesame rate as that of the wild-type strain ATCC13869. However, on the minimum medium(20 g/l glucose, 2.64 g/L ammonium sulfate, 0.5 g/L KH2PO4, 0.5 g/L K2HPO4, 0.25 g/L
240

MgSO4·7H2O, 0.01 g/L FeSO4·7H2O, 0.01 g/L MnSO4·7H2O, 0.01 g/L CaCl2, 0.02
mg/L CuSO4, 40 g/L MOPS, 30 mg/L protocatechuic acid, 200 μg/L VB1·HCl, 300 μg/L
biotin, 20 g/L agar, adjusted to pH 6.7 with NaOH) plate, 2A-1R strain was found to show
a considerably decreased colony-forming rate as compared to the wild-type strain
ATCC13869. Accordingly, a gene which can recover the growth of the 2A-1R strain in
the minimum medium was searched.
[0103]
The chromosomal DNA of ATCC13869 strain was partially digested with Sau3AI
and ligated to the shuttle vector pVK9 which had been digested with BamHI. The
obtained plasmid was precipitated with ethanol and used to transform electrocompetentcell of E. coli DH5α (Takara Bio Inc.) by the electric pulse method. pVK9 is a shuttle
vector obtained by blunt-ending the AvaII site of pHSG299 (Takara Bio Inc.) andinserting therein a fragment comprising a sequence automatically replicable incoryneform bacteria excised with BamHI and KpnI from pHK4 (Unexamined Patent
Application Publication No. 1993-007491). The transformed bacterial cells were spreadover an LB plate medium (10 g/l polypeptone, 5 g/l yeast extract, 5 g/l NaCl, 20 g/l agar,
adjusted to pH 7.0 with NaOH) containing 25 μg/ml kanamycin, and cultured at 37°C
overnight. On the next day, all of the colonies which had appeared were collected fromthe plate with a platinum loop and plasmids were extracted to construct a library of
ATCC13869. This library was transformed to the 2A-1R strain obtained in Working
Example 3 by the electric pulse method, and the transformed cells were spread over aminimum medium plate containing 25 μg/ml kanamycin. The strains which showed anincreased colony-forming rate were screened. By extracting a plasmid from the selectedstrains showing the increased colony-forming rate, it was found that the fragment having
a sequence shown in SEQ ID NO: 5 was inserted into the BamHI site of pVK9. The
obtained plasmid was named pL5k.
[0104]
Comparison of the nucleotide sequence inserted in the pL5k with the alreadypublished genome sequence of Corynebacterium glutamicum ATCC13032 (Acc. No.
NC_003450) showed that pL5k contained only one ORF having the amino acid sequence
shown in SEQ ID NO: 6 as a full-length ORF. Whether the ORF is a membrane protein
can be predicted by the program "SOSUI" available on the internet (linked tohttp://sosui.proteome.bio.tuat.ac.jp/sosuiframe0E.html as of October 7, 2004). Results ofanalysis of the ORF by using "SOSUI" suggested that five transmembrane regions arepresent. In the amino acid sequence of SEQ ID NO: 6, the transmembrane regions
correspond to the regions of amino acid numbers 1 to 23, amino acid numbers 25 to 47,241amino acid numbers 62 to 84, amino acid numbers 86 to 108, and amino acid numbers
110 to 132. DNA sequences encoding these regions correspond to the nucleotide numbers
1437 to 1505, nucleotide numbers 1509 to 1577, nucleotide numbers 1620 to 1688,
nucleotide numbers 1692 to 1760, and nucleotide numbers 1764 to 1832 of SEQ ID NO:
5. Each of the amino acid sequences of these regions is shown in SEQ ID NOS: 25 to 29
and Table 3.
[0105][Table 3]<Table 3 Predicted Transmembrane Regions of YggB Protein>(5) Working Example 5[0107]<Identification of Mutation Point of yggB Gene of 2A-1R Strain>
pL5K complemented the growth of the 2A-1R strain in the minimum medium, whichsuggested the possibility that the yggB gene of the 2A-1R strain has some mutations.
Accordingly, a nucleotide sequence of the yggB gene of the 2A-1R strain was determined.
As a result, it was found that in the 2A-1R strain, an IS was inserted into the C-terminalside region of the yggB gene (Figure 5). The nucleotide sequence of the mutant-type yggB
gene of the 2A-1R strain is shown in SEQ ID NO: 7 and the corresponding amino acidsequence is shown in SEQ ID NO: 8. This suggested the possibility that the L-glutamic
acid-producing ability of the 2A-1R strain was maintained by the mutation in the yggB
gene. In this regard, this mutation was present not only in the 2A-1R strain, but also in
the 2A-1 strain. This mutation is presumed to have occurred as a suppressor mutation tostably excrete L-glutamic acid out of the cell when the mutation was introduced into the
odhA gene. The mutation in which an IS was inserted was named 2A-1-type mutation.
(6) Working Example 6242

[0108]<Construction of 2A-1-Type yggB Mutant Strain and Evaluation of L-glutamic AcidProducing Ability>(6-1) Introduction of 2A-1-Type Mutation into Wild-Type Strain, and Evaluation Thereof(Single Cross-Over Recombinant Strain)
PCR was performed using the chromosomal DNA of the 2A-1 strain as a templateand synthetic DNAs shown in SEQ ID NOS: 9 and 10 as primers to amplify a fragmentof yggB gene having the 2A-1-type mutation. The amplified product was digested with
SacI and inserted into the SacI site of pBS3 of Working Example 1. A plasmid in whicha yggB fragment having 2A-1-type mutation was cloned was named pBS3yggB2A.
The pBS3yggB2A was introduced into C. glutamicum ATCC13869 strain by theelectric pulse method and was spread over a CM-Dex agar medium containing 25 μg/ml
kanamycin. The strains that appeared after culturing at 31.5°C were evaluated by PCR to
confirm that they are single cross-over recombinants in which pBS3yggB2A wasincorporated into the chromosome by homologous recombination.
The obtained single cross-over recombinant strain was named 13869-2A strain. In
this strain, the wild-type yggB gene and the mutant-type yggB gene were both expressed.
[0109]The L-glutamic acid-producing ability of the obtained yggB mutation-introduced
strain 13869-2A was confirmed by the method stated in Working Example 2. The results
are shown in Table 4 (OD620 indicates an amount of the bacterial cells diluted to 101
times; Glu (g/L) indicates an amount of accumulated L-glutamic acid). It could be
confirmed that 13869-2A strain clearly had an L-glutamic acid-producing ability evenunder the condition in which ATCC13869 strain did not produce L-glutamic acid. Fromthese results, it has been shown that the mutation in the yggB gene can induce L-glutamic
acid production.
[0110][Table 4]<Table 4 Amount of L-glutamic Acid Produced by Mutant-Type yggB Gene-Introduced
Strain>OD620 (x101)

Glu (g/L)

0.625
0.334
0.582
0.002

0.3
15.5
3.6
0.6

ATCC13869
2A-1R
13869-2A
Blank

243

[0111](6-2) Introduction of 2A-1-Type Mutation into Wild-Type Strain, and Evaluation Thereof(Double Cross-Over Recombinant Strain)
In order to construct a strain having only the mutant-type gene, 13869-2A strain was
cultured on a CM-Dex liquid medium overnight and the obtained suspension was spreadover an S10 agar medium (100 g/l sucrose, 10 g/l polypeptone, 10 g/l yeast extract, 1 g/l
KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O, 0.01 g/l MnSO4·4-5H2O, 3 g/lurea, 1.2 g/l soybean protein hydrolysate solution, 20 g/l agar, adjusted to pH 7.5 with
NaOH: autoclaved at 120°C for 20 minutes) and cultured at 31.5°C. Among the colonies
that appeared, the strain exhibiting sensitivity to kanamycin was purified on an s2B agar
medium. Chromosomal DNAs were prepared from these strains. Then, PCR was
performed using synthetic DNAs shown in SEQ ID NOS: 9 and 10 as primers to confirm
the mutation. The strain in which an IS-like sequence was inserted into the yggB gene
was named 13869-2A-7 strain.
[0112]The L-glutamic acid-producing ability of the obtained 13869-2A-7 strain was
confirmed by the method stated in Working Example 2. The results are shown in Table 5(OD620 indicates an amount of the bacterial cells diluted to 101 times; Glu (g/L) indicates
an amount of accumulated L-glutamic acid). It was shown that the 13869-2A-7 strain has
the L-glutamic acid-producing ability which was equivalent to or higher than that of the
2A-1R strain, from which it could be confirmed that L-glutamic acid production in thepresence of an excess of biotin was caused by the mutation in the yggB gene.
[0113][Table 5]<Table 5 Amount of L-glutamic Acid Produced by Mutant-Type yggB Gene
Chromosome-Introduced Strain>
OD620 (x101)

Glu (g/L)

0.648
0.420
0.414
0.002

0.4
13.8
16.1
0.7

ATCC13869
2A-1R
13869-2A-7
Blank(7) Working Example 7[0114]<Construction of A1-Type YggB Mutant Strain, and Evaluation Thereof>
The above L-glutamic acid-producing odhA mutant strains were analyzed. As a result,244

five types of mutations were identified on the yggB gene in addition to the above 2A-1
mutation. Hereinafter, these mutations are called A1-type mutation, 19-type mutation,
L30-type mutation, 8-type mutation, and 66-type mutation, respectively. Each of theformer three mutations, i.e., the A1-type mutation, 19-type mutation, and L30-type
mutation, was introduced into the chromosome of ATCC13869 strain, and effects of thesemutations were confirmed. In addition, the 8-type mutation was introduced into thechromosome of the ATCC14067 strain, and the effect of the mutation was confirmed.
Further, the 66-type mutation was introduced into the chromosome of the C. melassecola
ATCC17965 strain, and the effect of the mutation was confirmed.
[0115]The A1-type mutation is a mutation in which TTCATTGTG is inserted next to G at
position 1480 in SEQ ID NO: 5, and a mutation in which cysteine-serine-leucine residues
are inserted between leucine residue at position 14 and tryptophan residue at position 15
in SEQ ID NO: 6. The nucleotide sequence of the mutant-type yggB gene into which thismutation is introduced is shown in SEQ ID NO: 19, and the amino acid sequence of themutant-type YggB protein encoded by this mutant-type yggB gene is shown in SEQ ID
NO: 20.
[0116]The A1-type mutant gene can be obtained as follows. PCR is performed by using thesynthetic DNAs shown in SEQ ID NOS: 30 and 31 as primers and the chromosomal DNA
of ATCC13869 strain as a template to prepare an N-terminal side fragment. Similarly,
PCR is performed by using the synthetic DNAs shown in SEQ ID NOS: 32 and 33 asprimers to prepare a C-terminal side fragment. Subsequently, PCR can be performed byusing a mixture of an equal amount of the N-terminal side fragment and the C-terminalside fragment as a template and the synthetic DNAs shown in SEQ ID NOS: 9 and 34 asprimers to obtain a partial fragment of the A1-type mutant yggB gene. The obtained YggB
fragment can be digested with SacI and inserted into the SacI site of pBS4S to constructa plasmid for introducing this type of mutation. pBS4 YggBA1 thus obtained is
introduced into the chromosome of ATCC13869 strain by a method similar to the method
stated in Working Example 6, and then removed. The sequence of the yggB gene of theobtained kanamycin-sensitive strain can be determined and the strain having the A1-type
substituted yggB gene is selected, to thereby construct a strain having A1-type mutation.
The A1-type mutant strain having these mutations was named ATCC13869-A1 strain.
[0117]
ATCC13869-A1 strain and the parent ATCC13869 strain were cultured by a method
similar to the method stated in Working Example 2. After completion of the culture, the245amount of L-glutamic acid contained in the culture solution was measured by a known
method.
The ATCC13869-A1 strain in which the A1-type mutation was introduced on thechromosome greatly enhanced L-glutamic acid accumulation as compared to the parent
ATCC13869 strain.
[0118][Table 6]<Table 6 Amount of L-glutamic Acid Produced by Mutant-Type yggB Gene (A1)
Chromosome-Introduced Strain>
OD620 (x101)

Glu (g/L)

0.650
0.548

0.5
8.6

ATCC13869
ATCC13869-A1(8) Working Example 8[0119]<Construction of 19-Type YggB Mutant Strain, and Evaluation Thereof>
The 19-type mutation is a mutation in which G at position 1734 is substituted with A
in SEQ ID NO: 5, and a mutation in which alanine at position 100 is substituted withthreonine in the amino acid sequence of SEQ ID NO: 6. The nucleotide sequence of themutant-type yggB gene into which this mutation is introduced is shown in SEQ ID NO:
21, and the amino acid sequence of the mutant-type YggB protein encoded by this geneis shown in SEQ ID NO: 22. By a method similar to the method stated in Working
Example 7, the 19-type mutation-introduced yggB mutant strain is constructed.
Specifically, PCR is performed by using the synthetic DNAs shown in SEQ ID NOS: 30
and 35 as primers and the chromosomal DNA of ATCC13869 strain as a template toprepare an N-terminal side fragment. Similarly, PCR is performed by using the synthetic
DNAs shown in SEQ ID NOS: 33 and 36 as primers to prepare a C-terminal side fragment.
Subsequently, PCR can be performed by using a mixture of an equal amount of the Nterminal side fragment and the C-terminal side fragment as a template and the synthetic
DNAs shown in SEQ ID NOS: 9 and 34 as primers to obtain a partial fragment of the 19type yggB gene. The obtained yggB fragment can be digested with SacI and inserted into
the SacI site of pBS4S to construct a plasmid for introducing this type of mutation. pBS4
YggB19 thus obtained is introduced into the chromosome of ATCC13869 strain by amethod similar to the method stated in Working Example 6, and then removed. The
sequence of the yggB gene of the obtained kanamycin-sensitive strain can be determined
and the strain having the 19-type substituted yggB gene is selected, to thereby construct246

a strain having 19-type mutation. The 19-type mutant strain having these mutations wasnamed ATCC13869-19 strain.
[0120]
ATCC13869-19 strain and the parent ATCC13869 strain were cultured by a method
similar to the method stated in Working Example 2. After completion of the culture, theamount of L-glutamic acid contained in the culture solution was measured by a known
method. The ATCC13869-19 strain in which the 19-type mutation was introduced on thechromosome greatly enhanced L-glutamic acid accumulation as compared to the parent
ATCC13869 strain.
[0121][Table 7]<Table 7 Amount of L-glutamic Acid Produced by Mutant-Type yggB Gene (19Mutation) Chromosome-Introduced Strain>
OD620 (x101)

Glu (g/L)

0.650
0.614

0.5
0.7

ATCC13869
ATCC13869-19(9) Working Example 9[0122]<Construction of L30-Type YggB Mutant Strain >The L30-type mutation is a mutation in which C at position 1768 is substituted withT in SEQ ID NO: 5, and a mutation in which alanine at position 111 is substituted withvaline in SEQ ID NO: 6. The nucleotide sequence of the mutant-type yggB gene in which
this mutation is introduced is shown in SEQ ID NO: 23, and the amino acid sequence ofthe mutant-type YggB protein encoded by this gene is shown in SEQ ID NO: 24.
By a method similar to the method stated in Working Example 7, the L30-type
mutation-introduced yggB mutant strain is constructed. Specifically, PCR is performedby using the synthetic DNAs shown in SEQ ID NOS: 30 and 37 as primers and thechromosomal DNA of ATCC13869 strain as a template to prepare an N-terminal sidefragment. Similarly, PCR is performed by using the synthetic DNAs shown in SEQ ID
NOS: 34 and 38 as primers to prepare a C-terminal side fragment. Subsequently, PCR
was performed by using a mixture of an equal amount of the N-terminal side fragment
and the C-terminal side fragment as a template and the synthetic DNAs shown in SEQ ID
NOS: 9 and 34 as primers to obtain a partial fragment of the L30-type yggB gene. The
obtained mutant-type yggB fragment is digested with SacI and inserted into the SacI siteof pBS4S to construct a plasmid for introducing this type of mutation. pBS4 YggB-L thus247

obtained was introduced into the chromosome of ATCC13869 strain by a method similar
to the method stated in Working Example 6, and then removed. The sequence of the yggB
gene of the obtained kanamycin-sensitive strain can be determined and the strain having
the L30-type substituted yggB gene is selected, to thereby construct a strain having L30type mutation. The L30-type mutant strain having these mutations was named
ATCC13869-L strain.
[0123]
ATCC13869 strain and ATCC13869-L strain were cultured by a method similar to
the method stated in Working Example 2. After completion of the culture, the amount of
L-glutamic acid contained in the culture solution was measured by a known method. The
results are shown in Table 8 (OD620 indicates an amount of the bacterial cells diluted to
101 times; Glu (g/L) indicates an amount of accumulated L-glutamic acid). ATCC13869L30 strain into which L-30 mutation was introduced greatly enhanced L-glutamic acidaccumulation as compared to the parent ATCC13869 strain.
[0124][Table 8]<Table 8 Amount of L-glutamic Acid Produced by Mutant-Type yggB Gene (L-30
Mutation) Chromosome-Introduced Strain>
OD620 (x101)

Glu (g/L)

ATCC13869

0.650

0.5

ATCC13869-L30

0.389

15.9

(10) Working Example 10[0125]<Culture of the Mutant-Type yggB Gene-Introduced Strains Under L-glutamic AcidProducing Conditions, and Evaluation Thereof>
L-glutamic acid production of a coryneform bacterium can be induced by adding fatty
acid-based surfactants such as Tween 40 or by limiting biotin. Accordingly, theATCC13869 strain and the ATCC13869-19 strain were also cultured under Tween40added condition and biotin-limited condition, respectively. For seed culture, each of thestrains was inoculated into 20 ml of flask medium (80 g/l glucose, 30 g/l ammonium
sulfate, 1 g/l KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O, 0.01 g/l MnSO4·45H2O, 200 μg/l VB1, 60 μg/l biotin, 0.48 g/l soybean hydrolysates (T-N), adjusted to pH
8.0 with KOH: autoclaved at 115°C for 10 minutes), and then 1 g of calcium carbonate
which had been dry heat-sterilized beforehand was added thereto, followed by a shaking
culture at 31.5°C. The culture solution obtained after complete consumption of sugars
248was used as a seed culture solution. For Tween40-added culture, 2 ml of the seed culture
solution was inoculated into 20 ml of the flask medium (80 g/l glucose, 30 g/l ammonium
sulfate, 1 g/l KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O, 0.01 g/l MnSO4·45H2O, 200 μg/l VB1, 60 μg/l biotin, 0.48 g/l soybean hydrolysates (T-N), adjusted to pH
8.0 with KOH: autoclaved at 115°C for 10 minutes), and then 1 g of calcium carbonate
which had been dry heat-sterilized beforehand was added thereto, followed by a shaking
culture at 31.5°C. At the time when OD620 (× 101) reached 0.2 after the start of culture,
Tween 40 was added in order to attain a final concentration of 5 g/L, and then the culture
was continued. For biotin-limited culture, 1 ml of the seed culture solution was inoculated
into 20 ml of the flask medium (80 g/l glucose, 30 g/l ammonium sulfate, 1 g/l KH2PO4,
0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O, 0.01 g/l MnSO4·4-5H2O, 200 μg/l VB1,
0.48 g/l soybean hydrolysates (T-N), adjusted to pH 8.0 with KOH: autoclaved at 115°C
for 10 minutes), and then 1 g of calcium carbonate which had been dry heat-sterilized
beforehand was added thereto, followed by a shaking culture at 31.5°C. Under theseculture conditions, the final concentration of biotin became about 2.9 μg/L.
After 40 hours from the start of culture, the concentrations of L-glutamic acid in themedium were measured for both of the Tween40-added culture and the biotin-limited
culture. The results are shown in Table 9 (OD620 indicates an amount of the bacterial
cells diluted to 101 times; Glu (g/L) indicates an amount of accumulated L-glutamic acid).
It has been found that ATCC13869-19 strain increased L-glutamic acid accumulationeven under L-glutamic acid-producing condition.
[0126][Table 9]<Table 9 Amount of L-glutamic Acid Produced by Mutant-Type yggB Gene-Introduced
Strain under the Glutamic Acid Production-Inducing Condition>
OD620 (x101)

Glu (g/L)

0.538
0.395

25.6
28.6

0.462

36.0

0.431

40.0

Tween40-added
ATCC13869
ATCC13869-19
biotin-limited
ATCC13869
ATCC13869-19[0127]The ATCC13869 strain, the ATCC13869-L30 strain, the ATCC13869-A1 strain, theATCC13869-19 strain, and the strain in which yggB was amplified by plasmid were also249

cultured with Tween 40 added. Each of these strains were cultured on a CM-Dex platemedium overnight, and cells collected from 1/6 area of the plate were inoculated in 20 mlof a flask medium (80 g/l glucose, 30 g/l ammonium sulfate, 1 g/l KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O, 0.01 g/l MnSO4·4-5H2O, 200 μg/l VB1, 60 μg/l biotin,
and 0.48 g/l soybean hydrolysates (T-N), adjusted to pH 8.0 with KOH: autoclaved at
115°C for 10 minutes), followed by addition of 1 g of calcium carbonate which had been
dry heat-sterilized beforehand, and each of the strains was cultured with shaking at 31.5°C.
After 5 hours from the start of culture, Tween 40 was added in order to attain a final
concentration of 1 g/L. After 24 hours from the start of culture, the amount of bacterial
cells and the yield of L-glutamic acid were each analyzed. As a result, as shown in Table
10, it is shown that all of the ATCC13869-19 strain, the ATCC13869-L30 strain, theATCC13869-A1 strain, and the wild-type yggB gene-amplified strain(ATCC13869/pL5k-1) enhanced the yield of L-glutamic acid under L-glutamic acidproducing condition.
[0128][Table 10]<Table 10 Amount of L-glutamic Acid Produced by Each Strain in Tween40-Added
Culture>OD620 (x101)

Glu (g/L)

ATCC13869

0.887

12.8

ATCC13869/pVK9
ATCC13869/pL5k-1
ATCC13869-19
ATCC13869-A1
ATCC13869-L30
Blank

0.748
0.711
0.786
0.629
0.649
0.001

12.4
19.2
21.0
34.9
28.3
0.5

(11) Working Example 11[0129]<Construction of 8-Type YggB Mutant Strain>
The 8-type mutation is a mutation in which G at position 837 is substituted with A in
SEQ ID NO: 61, and a mutation in which alanine at position 111 is substituted withthreonine in SEQ ID NO: 62. The nucleotide sequence of the mutant-type yggB gene into
which this mutation is introduced is shown in SEQ ID NO: 63, and the amino acidsequence of the mutant-type YggB protein encoded by this gene is shown in SEQ ID NO:
64.
250

By a method similar to the method stated in Working Example 7, the 8-type mutationintroduced yggB mutant strain is constructed. Specifically, PCR is performed by using
the synthetic DNAs shown in SEQ ID NOS: 30 and 65 as primers and the chromosomal
DNA of Brevibacterium flavumATCC14067 strain as a template to prepare an N-terminalside fragment. Similarly, PCR is performed by using the synthetic DNAs shown in SEQ
ID NOS: 34 and 66 as primers to prepare a C-terminal side fragment. Subsequently, PCR
is performed by using a mixture of an equal amount of the N-terminal side fragment andthe C-terminal side fragment as a template and the synthetic DNAs shown in SEQ ID
NOS: 9 and 34 as primers to obtain a partial fragment of the 8-type yggB gene. The
obtained mutant-type yggB fragment was digested with SacI and inserted into the SacIsite of pBS4S to construct a plasmid for introducing this type of mutation. pBS4 YggB8
thus obtained is introduced into the chromosome of ATCC14067 strain by a method
similar to the method stated in Working Example 6, and then removed. The sequence ofthe yggB gene of the obtained kanamycin-sensitive strain is determined and the strain
having the 8-type substituted yggB gene is selected, to thereby construct a strain having
8-type mutation. The 8-type mutant strain having these mutations is named
ATCC14067yggB8 strain.
(12) Working Example 12[0130]<Construction of 66-Type YggB Mutant Strain>
The 66-type mutation is a mutation in which C at position 1673 is substituted with T
in SEQ ID NO: 67, and a mutation in which proline at position 424 is substituted withleucine in SEQ ID NO: 68. The nucleotide sequence of the mutant-type yggB gene inwhich this mutation is introduced is shown in SEQ ID NO: 69, and the amino acidsequence of the mutant-type YggB protein encoded by this gene is shown in SEQ ID NO:
70.
By a method similar to the method stated in Working Example 7, the 66-type
mutation-introduced yggB mutant strain is constructed. Specifically, PCR is performedby using the synthetic DNAs shown in SEQ ID NOS: 30 and 71 as primers and thechromosomal DNA of C. melassecola ATCC17965 strain as a template to prepare an Nterminal side fragment. Similarly, PCR is performed by using the synthetic DNAs shown
in SEQ ID NOS: 34 and 72 as primers to prepare a C-terminal side fragment.
Subsequently, PCR can be performed by using a mixture of an equal amount of the Nterminal side fragment and the C-terminal side fragment as a template and the synthetic
DNAs shown in SEQ ID NOS: 9 and 10 as primers to obtain a partial fragment of the 66type yggB gene. The obtained mutant-type yggB fragment was digested with SacI and251

inserted into the SacI site of pBS4S to construct a plasmid for introducing this type ofmutation. pBS4 YggB66 thus obtained is introduced into the chromosome of
ATCC17965 strain by a method similar to the method stated in Working Example 6, andthen removed. The sequence of the yggB gene of the obtained kanamycin-sensitive strain
can be determined and the strain having the 66-type substituted yggB gene is selected, tothereby construct a strain having 66-type mutation. The 66-type mutant strain having
these mutations is named yggB66 strain.
(13) Working Example 13[0131]<Screening of the Mutant-Type yggB Genes by In Vitro Mutation>(13-1) Construction of yggB-Deleted Strain
Mutant-type yggB genes can also be obtained by introducing a mutation into yggB in
vitro at random, and selecting a clone capable of producing L-glutamic acid without theaddition of surfactants. In order to perform screening for mutant-type genes, a yggB genedeleted strain was constructed first. PCR was performed by using the synthetic DNAs
shown in SEQ ID NOS: 39 and 40 as primers and the chromosomal DNA of ATCC13869
strain as a template to prepare an N-terminal side fragment. Similarly, PCR was
performed by using the synthetic DNAs shown in SEQ ID NOS: 41 and 42 as primers to
prepare a C-terminal side fragment. SEQ ID NO: 40 and SEQ ID NO: 41 arecomplementary to each other. Subsequently, PCR was performed by using a mixture ofan equal amount of the N-terminal side fragment and the C-terminal side fragment as atemplate and the synthetic DNAs shown in SEQ ID NOS: 39 and 42 as primers to obtaina fragment in which an ORF of the yggB gene was deleted.
[0132]The obtained PCR fragment was digested with SacI and inserted into the SacI site ofpBS4S to construct a plasmid for introducing deletion mutation. pBS4ΔYggB thus
obtained is introduced into the chromosome of ATCC13869 strain by the method statedin Working Example 6, and then removed. PCR was performed by using the chromosomal
DNA of the obtained kanamycin-sensitive strain as a template and the synthetic DNAs of
SEQ ID NOS: 39 and 42 as primers to confirm that the yggB gene was deleted. The
obtained yggB-deleted strain was named ATCC13869ΔyggB strain.
[0133](13-2) In Vitro Screening of Mutant-Type yggB Genes
On the other hand, mutagenesis treatment on the yggB gene was performed as follows.
First, since the plasmid pL5k contains some regions other than the yggB gene, pL5k wastreated with XhoI and SalI, and then self-ligated to obtain the plasmid pL5kXS. A SalI
252

recognition site is not present in the nucleotide sequence of SEQ ID NO: 5, but is present
in the multi-cloning site of pBS3. About 10 μg of the obtained pL5kXS was dissolved in
500 mM phosphate buffer, 400 mM hydroxylamine, and 1 mM EDTA (pH 6.0), and thenheated at 75°C for 30 to 90 minutes to introduce a mutation. The plasmid after mutation
introduction was desalted using SUPREC-02 (manufactured by Takara Bio Inc.), and thenintroduced into ATCC13869ΔyggB strain by the method stated in Working Example 6.
Thereafter, transformed cells were selected in the CM2B medium containing 25 μg/ml
Km. On the other hand, pL5kXS before mutagenesis treatment was introduced into the
ATCC13869ΔyggB strain as a control. The appeared transformants are inoculated into 2
ml of a liquid CM2BGU2 medium (CM2B medium stated in Working Example 3 further
containing 10 g/L glucose and 15 g/L urea) and cultured at 31.5°C for 5 hours withshaking, followed by determining a concentration of glutamic acid.
The strain obtained by transforming the ATCC13869ΔyggB strain with the mutated
pL5kXS was cultured in the CM2BGU2 medium in vitro. The evaluation results of theculture are shown in Table 11. There were three clones which cause accumulation of 1
g/L or more of L-glutamic acid among the transformants obtained from a plasmid mixture
with a mutagenesis treatment time of 60 minutes and 90 minutes. The amount of Lglutamic acid contained in the starting medium was 0.16 g/L, and the amount of Lglutamic acid which had accumulated by the ATCC13869ΔyggB/pL5kXS (without
mutagenesis treatment) strain was 0.31 g/L.
The strain obtained by transforming the ATCC13869ΔyggB strain with pL5kXS wascultured in the CM2BGU medium (the same composition as CM2BGU2 medium, exceptthat urea concentration is 1.5 g/L) in vitro. The evaluation results of the culture are shownin Table 12. One clone which causes accumulation of 1 g/L or more of L-glutamic acidwas obtained among the transformants obtained from a plasmid mixture with amutagenesis treatment time of 90 minutes.
[0134][Table 11]<Table 11 Amount of L-glutamic Acid Produced by yggB Plasmid Subjected to
Mutagenesis Treatment - 1>
Amountof

Number of Clones

Accumulated
(g/L)

Glu

Mutagenesis Treatment Time
30 min60 min90 minGlu≤0.4

40

36

39

0.4<Glu≤0.6

8

11

6

0.6<Glu≤0.8

0

0

1

253

0.8<Glu≤1

0

0

0

1<Glu

0

1

2

[0135][Table 12]<Table 12 Amount of L-glutamic Acid Produced by yggB Plasmid Subjected to
Mutagenesis Treatment - 2>
Amount of Accumulated
Glu (g/L)

Number of Clones
Mutagenesis Treatment Time60 min90 minGlu≤0.7

45

41

0.7<Glu≤0.9

2

7

0.9<Glu

10[0136]
A plasmid which gained a producing ability to accumulate 1 g/L or more of Lglutamic acid by mutagenesis treatment for 60 minutes as shown in Table 11 was named
pL5kXSm-22. A plasmid which gained a producing ability to accumulate 0.9 g/L or moreof L-glutamic acid by mutagenesis treatment for 90 minutes as shown in Table 12 wasnamed pL5kXSm-27. ATCC13869ΔyggB/pL5kXS strain, ATCC13869ΔyggB/
pL5kXSm-27 strain, and ATCC13869ΔyggB/pL5kXSm-22 strain were cultured under
the conditions stated in Working Example 2, and the amounts of bacterial cells and Lglutamic acid accumulation after 4 hours were analyzed. Table 13 shows the mean valueof three independent experiments. It could be confirmed that the
ATCC13869ΔyggB/pL5kXSm-27 strain and the ATCC13869ΔyggB/pL5kXSm-22
strain significantly enhanced L-glutamic acid accumulation. These results demonstratethat the mutant-type gene which is advantageous to L-glutamic acid can be constructedeven by introducing mutation in vitro at random. The sequence of the yggB gene which
pL5kXSm-22 contains is shown in SEQ ID NO: 73 (22-Type Mutation).
The 22-type mutation is a mutation in which proline at position 437 is substituted withserine and a mutation in which C at position 2745 is substituted with T in SEQ ID NO: 5,
for example. Further, this mutation is accompanied by a mutation in which C at position
3060 is substituted with T. This mutant-type yggB gene is shown in SEQ ID NO: 73, andthe amino acid sequence of the mutant-type YggB protein encoded by this mutant-type
yggB gene is shown in SEQ ID NO: 74.
[0137]
254

[Table 13]<Table 13 Amount of L-glutamic Acid Produced by yggB Plasmid Subjected to
Mutagenesis Treatment - 3>
StrainATCC13869ΔyggB/pL5kXS (Untreated)
ATCC13869ΔyggB/pL5kXSm22
ATCC13869ΔyggB/pL5kXSm27

OD620 (x101)

Glu (g/L)

0.253
0.232
0.245

0.58
1.10
0.82

[0138](13-3) Introduction of Mutant-Type Genes into Coryneform Bacteria, and Confirmationof L-glutamic Acid Production
The novel mutant-type yggB gene obtained in (13-2) is introduced into a coryneformbacterium. Method of introduction is as follows. The mutant-type yggB gene wasintroduced into pBS4S by the method stated in Example 6, and substituted with a wildtype yggB gene on the chromosome of ATCC13869. A strain in which this novel mutanttype yggB gene is introduced, and the parent ATCC13869 strain are cultured by a method
similar to the method stated in Working Example 2. After completion of the culture, theamount of L-glutamic acid in the culture solution is measured by a known method toconfirm that L-glutamic acid accumulation is enhanced due to the mutation-introduced
strain. In this manner, the yggB gene mutant strain of which the L-glutamic acidproducing ability is enhanced can be obtained.
(14) Working Example 14[0139]<Sensitivity to Glutamic Acid Analogue of the Strains Carrying the Mutant-Type yggB
Gene>
(14-1) Sensitivity to 4-Fluoroglutamic Acid on Solid Medium
It was predicted that strains which enhanced the L-glutamic acid-producing ability
due to yggB mutation would have decreased sensitivity to L-glutamic acid analogues.
Accordingly, as an L-glutamic acid analogue due to yggB mutations, a change ofsensitivity to 4-fluoroglutamic acid was investigated and considered. 4-fluoroglutamic
acid which had been adjusted to pH 6.7 with NaOH and sterile-filtered was added to aminimum medium stated in Working Example 4 so as to attain a final concentration of
4-fluoroglutamic acid of 7.5 mM. ATCC13869 strain, ATCC13869-L30 strain, and
ATCC13869-A1 strain were spread over a CM-Dex medium and cultured overnight.
Then, the bacterial cells were collected from the medium, washed with sterilized 0.85%
NaCl solution, and diluted so as to attain the bacterium concentration shown in the top of
255

Figure 6, and spotted onto the plate. The plate was cultured at 31.5°C. The time-course isshown in Figure 6. It was shown that the ATCC13869 strain grew the fastest of threestrains under the condition where 4-fluoroglutamic acid was not added. In contrast, it was
shown that under the condition where 4-fluoroglutamic acid was added, the growth of the
ATCC13869 strain was suppressed, and the growth of the ATCC13869-L strain and the
ATCC13869-A1 strain became better than that of the ATCC13869 strain.
[0140](14-2) Sensitivity to 4-Fluoroglutamic Acid on Liquid Medium
4-fluoroglutamic acid was adjusted to pH 6.7 with NaOH and was sterile-filtered.
This 4-fluoroglutamic acid was added to a minimum medium having the same
composition as stated in Working Example 4 (except for not containing agar) so as toattain a final concentration of 1.25 mM, 2.5 mM, 5 mM, 10 mM, and 20 mM, respectively.
Each of ATCC13869 strain, ATCC13869ΔyggB strain, ATCC13869-L30 strain, and
ATCC13869-A1 strain was spread over a CM-Dex medium and was cultured at 31.5°C
overnight. Then, the bacterial cells were collected, washed with sterilized 0.85% NaClsolution, inoculated into the liquid medium, and cultured at 31.5°C with shaking. In a
medium under the condition where 4-fluoroglutamic acid is not added in each sterilizedstrain, when the OD660 value of the medium reached 1.0, the culture was terminated, andthe obtained culture solution was diluted appropriately and spread over a CM-Dex plate.
The number of colonies which appeared on the next day was measured to be a viable cellnumber at the end of liquid culture. Figure 7 shows the change of the relative viable cellnumber at each concentration of 4-fluoroglutamic acid when the cell number of theculture under the condition where 4-fluoroglutamic acid was not added was set to 1. It
has been found that the ATCC13869-A1 strain and the ATCC13869-L30 strain havedecreased sensitivity to 4-fluoroglutamic acid (i.e., enhanced resistance to 4fluoroglutamic acid).
From these results, it has been shown that strains having a mutant-type yggB strain of
the present invention can also be obtained by screening using sensitivity to glutamic acidstructural analogues such as 4-fluoroglutamic acid.
(15) Working Example 15[0141]<Construction of the Mutant-Type yggB Gene-Introduced Strain in odhA Weakened
Strain, and Evaluation Thereof>[0142]The yggB odhA-double mutant strain was constructed by using the ATCC13869-L30
strain which was constructed in Working Example 9, and the mutant-type odhA gene was256

evaluated.
First, a strain in which the mutation shown in Table 14 was introduced into the odhA
gene of the ATCC13869-L strain is constructed. In Table 14, nucleotide sequences of theregion corresponding to nucleotide numbers 2528 to 2562 of SEQ ID NO: 43 are shown.
In Table 15, amino acid sequences of the region corresponding to amino acid numbers
696 to 707 of SEQ ID NO: 44 are shown.
L30sucA8 strain into which the odhA gene having the nucleotide sequence of SEQ
ID NO: 45 was introduced can be obtained as follows. The odhA gene fragment isprepared by PCR using synthetic DNAs as shown in SEQ ID NOS: 53 and 54 as primers.
The obtained odhA fragment is digested with BamHI and cloned to the BamHI site ofplasmid pKF19m which is attached to Mutan-Super Express Km (manufactured by
Takara Bio Inc.). Then, PCR is performed using a synthetic DNA of SEQ ID NO: 55
having a phosphorylated 5'-end and a selection primer attached to Mutan-Super Express
Km, and the obtained PCR product is used to transform sup0-E. coli strain, for example
MV 1184 strain, thereby to construct a plasmid containing the mutant odhA fragment.
Then, this fragment is excised with BamHI and inserted into the BamHI site of the pBS4S
plasmid, to thereby construct a plasmid for introducing this mutation. The ATCC13869L strain is transformed by a method similar to the method stated in Working Example 1,
to thereby obtain a strain in which the plasmid for introducing this mutation is inserted
into the chromosome. Then, a strain which is resistant to sucrose and is sensitive tokanamycin is isolated from these strains in which the plasmid is inserted into thechromosome. L30sucA8 (odhA8) strain in which the odhA gene is deleted can beconstructed by further confirming the sequence of the odhA gene and selecting the strain
into which an objective frame-shift was introduced.
[0143]In addition, other odhA mutant strains can be obtained by the following methods usinga yggB mutant strain.
L30sucA801 strain into which a mutant odhA gene having a nucleotide sequence of
SEQ ID NO: 47 is introduced can be obtained according to the above method by using a
synthetic DNA of SEQ ID NO: 56 having a phosphorylated 5'-end instead of a synthetic
DNA of SEQ ID NO: 55 having a phosphorylated 5'-end.
L30sucA805 strain into which a mutant odhA gene having a nucleotide sequence of
SEQ ID NO: 49 is introduced can be obtained according to the above method by using a
synthetic DNA of SEQ ID NO: 57 having a phosphorylated 5'-end instead of a synthetic
DNA of SEQ ID NO: 55 having a phosphorylated 5'-end.
L30sucA77 strain into which a mutant odhA gene having a nucleotide sequence of257

SEQ ID NO: 51 is introduced can be obtained according to the above method by using a
synthetic DNA of SEQ ID NO: 58 having a phosphorylated 5'-end instead of a synthetic
DNA of SEQ ID NO: 55 having a phosphorylated 5'-end.
The L30sucA8 strain does not have an α-KGDH activity, because the odhA gene of
the L30sucA8 strain has a frame-shift mutation. On the other hand, in the L30sucA801
strain, the L30sucA805 strain, and the L30sucA77 strain, these strains have mutations inwhich there is deletion in odhA gene but frame-shift will not occur. These strains havethe α-KGDH activity, but their activity was lower than that of a non-modified strain (datanot shown).
[0144][Table 14]<Table 14 Partial Sequences of Mutant-type odhA Gene>
StrainNameodhA Sequence

ATCC13869-L

CTG

GCT

AAG

CTG

CGT

L30sucA8

CTG

GCT

AAG

CTG

sucA801

CTG

GCT

AAG

sucA805

CTG

GCT

sucA77

CTG

GCT

GGC

TAC

GAC

GTC

GGA

GGC

AC

CGT

C

GAC

GTC

GGA

GGC

AC

CTG

CGT

CTC

GAC

GTC

GGA

GGC

AC

AAA

AGC

TGC

GTC

GAC

GTC

GGA

GGC

AC

ATA

AGC

TGC

GTC

GAC

GTC

GGA

GGC

AC

[0145][Table 15]<Table 15 Partial Sequences of Mutant-type α-Ketoglutarate Dehydrogenase>
α-KGDH Sequence

Strain Name
wild

Leu

Ala

Lys

Leu

Arg

Gly

Tyr

Asp

Val

Gly

Gly

Thr

∆sucA (L30sucA8)

Leu

Ala

Lys

Leu

Arg

sucA801

Leu

Ala

Lys

Leu

Arg

---

Leu

Asp

Val

Gly

Gly

Thr

sucA805

Leu

Ala

Lys

Ser

Cys

---

Val

Asp

Val

Gly

Gly

Thr

sucA77

Leu

Ala

Ile

Ser

Cys

---

Val

Asp

Val

Gly

Gly

Thr

[0146]<Culture of odhA Modified Strain, and Production of L-glutamic Acid by the odhA
Modified Strain>
L-glutamic acid-producing ability of these odhA modified strains was evaluated byculturing these strains in a Sakaguchi flask. Each of the strains shown in Table 14 wascultured on a CM-Dex agar medium at 31.5°C for a whole day and night. Then, 1/6 plate258

bacterial cells of this culture were inoculated to 20 ml of a flask medium (60 g/l glucose,
22.5 g/l ammonium sulfate, 1 g/l KH2PO4, 0.4 g/l MgSO4·7H2O, 0.01 g/l FeSO4·7H2O,
0.01 g/l MnSO4 ·4-5H2O, 200 μg/l vitamin B1, 0.48 g/l soybean protein hydrolysate
solution, and 300 μg/l biotin, adjusted to pH 8.0 with KOH). 1 g of calcium carbonate
which had been dry heat-sterilized beforehand was added to this flask medium, and eachof the strains was cultured with shaking at 115 rpm at 31.5°C. The amount of accumulated
L-glutamic acid after 19 hours from the start of culture is shown in Table 16. odhA
modified strains include a strain which exhibited L-glutamic acid accumulationequivalent to that of the ATCC13869-L strain. However, the sucA801 strain, the sucA805
strain, and the sucA77 strain exhibited higher L-glutamic acid accumulation than did thesucA8 strain. From these results, it has been found that the yield of L-glutamic acid isfurther enhanced by introducing mutations into the odhA gene in addition to the yggB
gene.
[0147]<Table 16 Amount of L-glutamic Acid Produced by odhA Modified Strain>
Strain Name

Glu (g/L)

ATCC13869-L

4.9

L30sucA8

19.8

L30sucA801

22.1

L30sucA805

23.8

L30sucA77

21.6(16) Working Example 16[0148]<Construction of odhA Weakened Strain from ATCC14067 Strain and
ATCC14067yggB8 Strain>odhA-deleted strain was constructed from yggB mutant strain having 8-type mutation,and the constructed strain was cultured as well as a strain having only odhA deletion, tocompare the two strains. First, a plasmid for completely deleting odhA was constructed.
PCR was performed using chromosomal DNA of ATCC14067 strain as a template andsynthetic DNAs shown in SEQ ID NOS: 77 and 78 to prepare an upstream side fragmentof the sucA gene. Subsequently, PCR was performed using chromosomal DNA of
ATCC14067 strain as a template and the synthetic DNAs shown in SEQ ID NOS: 79 and
80 to prepare a downstream side fragment of the sucA gene. Then, PCR was performed
using a mixture of equimolar amounts of the upstream side fragment and the downstreamside fragment as a template and synthetic DNAs of SEQ ID NOS: 81 and 82 to prepare a259

gene fragment in which odhA was deleted. The obtained PCR fragment was digested with
BamHI and was cloned in the pBS4S constructed in Working Example 1. The plasmidthus obtained was named pBSΔsucA47.
The obtained pBSΔsucA47 was inserted into the chromosome of ATCC14067 strain
or ATCC14067yggB8 strain by a method similar to the method stated in Working
Example 6, and then was removed. Then, a chromosomal DNA was prepared from theobtained kanamycin-sensitive strain. Thereafter, PCR was performed by using thischromosomal DNA as a template and the synthetic DNAs of SEQ ID NOS: 77 and 80 toselect a strain in which odhA region was deleted. odhA-deleted strains thus constructed
were named ATCC14067ΔodhA strain and ATCC14067ΔodhA yggB8 strain,
respectively.
The constructed ATCC14067ΔodhA strain and the ATCC14067ΔodhA yggB8 strain
were cultured by the method stated in Working Example 3. The results of this culture areshown in Table 17. It has been found that yggB8 mutation can enhance the yield of Lglutamic acid of the odhA mutant strain.
[0149][Table 17]<Table 17 Amount of L-glutamic Acid Produced by 8-Type yggB Gene ChromosomeIntroduced Strain>
OD620 (x101)

Glu (g/L)

0.270
0.242

5.8
22.0

ATCC14067ΔodhA
ATCC14067ΔodhA yggB8(17) Working Example 17[0150]<Construction of symA-Deleted Strain from 2A-1R Strain>
The symA (suppressor of ygg mutation A) gene was deleted from the 2A-1R strain
having a mutation of IS insertion into yggB, which was constructed in Working Example
3, thereby to construct a strain. The obtained strain was cultured as well as the 2A-1R
strain, to compare the two strains. The nucleic acid sequence and the amino acid sequenceof NCgl1867 gene of the ATCC13869 strain is shown in SEQ ID NO: 86 and SEQ ID
NO: 87, respectively. First, a plasmid for the purpose of completely deleting the symA
gene was constructed. PCR was performed using chromosomal DNA of the ATCC13869
strain as a template and the synthetic DNAs shown in SEQ ID NOS: 88 and 89 to prepare
an upstream side fragment of the symA gene. Subsequently, PCR was performed using
chromosomal DNA of ATCC13869 strain as a template and the synthetic DNAs shown260

in SEQ ID NOS: 90 and 91 to prepare a downstream side fragment of the symA gene.
Then, PCR was performed using a mixture of equimolar amounts of the upstream sidefragment and the downstream side fragment as a template and synthetic DNAs of SEQ
ID NOS: 88 and 91 to prepare a gene fragment in which the symA gene was deleted. The
obtained PCR fragment was digested with BglII and was cloned in the pBS4S constructedin Working Example 1. The plasmid thus obtained was named pBSΔsymA.
The obtained pBSΔsymA was inserted into the chromosome of 2A-1R strain by amethod similar to the method stated in Working Example 6, and then was removed. Then,a chromosomal DNA was prepared from the obtained kanamycin-sensitive strain.
Thereafter, PCR was performed by using this chromosomal DNA as a template and thesynthetic DNAs of SEQ ID NOS: 88 and 91 to select a strain in which symA region wasdeleted. The symA-deleted strain thus constructed was named 2A-1RΔsymA strain.
The constructed 2A-1RΔsymA strain and the parent 2A-1R strain were cultured by
the method stated in Working Example 3. The results of these culture are shown in Table
18. It has been found that the deletion of the symA gene can enhance the L-glutamic acidproducing ability of a strain having the mutant -type yggB gene.
[0151][Table 18]<Table 18 Enhancement of L-glutamic Acid-Producing Ability by symA Gene Deletion>
OD620 (x51)Glu (g/L)

2A-1R

0.846

12.4

2A-1R∆symA

0.709

15.8

8. Drawings

261

[Figure 1] A figure showing the procedure for constructing plasmid pBS3
SEQ ID NO: 13

B. subtilis
chromosome
SEQ ID NO: 14

Digestion with BamHI
and BglII and blunting

Digestion with AvaIIand blunting

262

[Figure 2] A figure showing the procedure for constructing plasmid pBS4S
SEQ ID NO: 15
SEQ ID NO: 16

SEQ ID NO: 17

SEQ ID NO: 18

SEQ ID NO: 15 SEQ ID NO: 16 SEQ ID NO: 17 SEQ ID NO: 18

SEQ ID NO: 15

SEQ ID NO: 18[Figure 3] A figure showing the procedure for constructing plasmid pBS4sucAint

SEQ ID NO: 1

SEQ ID NO: 2

BamHI digestion

BamHI digestion263

[Figure 4] A figure showing the accumulation of L-glutamic acid by the mutant-type
yggB gene-introduced strain and the control strain

Culture time (hr)[Figure 5] A figure showing the location for inserting the mutation in a yggB gene having
the 2A-1-type mutation[Figure 6] A figure showing growth of the mutant-type yggB gene-introduced strains ina CM-Dex plate medium containing 4-fluoroglutamic acid (photograph)264

[Figure 7] A figure showing growth of the mutant-type yggB gene-introduced strains ina liquid medium containing 4-fluoroglutamic acid (4-FG).

265

Attachment 14 "Drawings of Cited Documents"
1. Exhibit Otsu 6 Document
Fig. 1266

No. of occurrences

Fig. 5

Table 2Bacterium

Percentage conservation of single nucleotides in the consensus motif of the:-35 region-10 region

T

T

G

A*

C

A

T

A

T

A

A

T

C. glutamicum

48

48

73

54

45

52

73

78

82

68

58

52

54

Bacillus subtilis

87

83

78

64

51

58

Lactobacillus

90

87

60

63

43

60

3
3
5
2
6
0
6
0

5
8
5
9
7
8
8
7

4
2
4
9
7
4
6
3

85

E. coli

7
9
8
2
9
4
8
3

Streptococcus

88

76

88

59

59

47

7
6

10
0

6
5

7
1

6
5

10
0

89
96
83

89
94
90

* In the C. glutamicum -35 consensus sequence the fourth nucleotide is C instead of A.
An A at this position was found in only 7 of the 33 promoters (21% conservation).

267

2. Exhibit Otsu 9 Document
Table 9
Table 9StrainBacterial cellconcentration (OD)

L-glutamic
acid (g/l)∆S∆S/pGDH∆S/pGLTA∆S/pICD∆S/pPPC

0.84
1.01
0.83
0.83
0.75

35
35
37
37
37

∆S/pGDH+GLTA+ICD∆S/pGDH+GLTA+PPC∆S/pSAC4

0.95
0.85
0.83

38
40
35

3. Exhibit Otsu 12 Document
Nucleotide sequence of a DNA fragment shown in the upper right column on page 6268

4. Exhibit Otsu 24 Document
Table 1
Efflux solute

Analytical

Solute retained after dilution to osmolality ofmethodbefore

1860

1060

860

710

540

μmol・mg dm-1 (%)
Gly betaine

sc. count

Prolinesc. count

EctoineNMR

Gly betainea

NMR+ ectoinea

NMR

Alanined

HPLC

GlutamatedenzymeLysined

HPLC

ATPb

luciferase

Na+c,dflame ph.

K+c,dflame ph.

0.92
0.94
0.52
0.35
0.45

0.90 (98)0.91 (97)0.51 (98)
-

0.69
0.63
0.50
-

0.56
0.50
0.46
0.15
0.34

0.43
0.38
0.44
0.11
0.32

0.28 (30)0.27 (29)0.29 (56)0.01 (3)0.23 (51)

0.32
0.46
0.20
0.007

0.32 (100)0.42 (91)0.18 (90)
0.007
(100)
0.22 (39)0.95 (92)

0.22
0.40
0.15
0.007

0.20
0.41
0.16
0.007

0.20
0.15
0.007

0.15 (47)0.37 (80)0.15 (75)0.007 (99)

-

0.21
0.86

0.17
0.79

0.16 (28)0.67 (65)

0.57
1.03

a

Loading with both glycine betaine (3 mM) and ectoine (3 mM) at the same time.
Efflux of ATP never exceeded 2% of the total ATP content in the cytoplasm.
c
The case of Na+ and K+, the steps in osmolality were 2100, 1140, 820, 420 mOsrn.
bdLoading in the presence of 2 mM glycine betaine, in order to create a competitive
situation (see text).
5. Exhibit Otsu 35 Document
Table 2
Specific activity of GDH1)

Strain8012)
801(pAG50)
801(pAG1001)

0.68
0.65
3.71

269

Table 10
Specific activity of CS1)

Strain8012)
801(pAG50)
801(pAG4001)
801(pAG4002)
801(pAG4003)

0.50
0.52
2.06
2.07
2.10

Table 11StrainCS1)

ICDH2)

GDH3)

8014)
801(pCI31)
801(pCG5)801(pCIG231)

0.33
1.6
1.7
1.1

0.82
3.4
0.75
3.9

0.85
0.85
3.2
2.5

270

Table 13
StrainGlutamate concentration(g/dl)1)

Yield based on sugar
(%)2)

8013)
801(pAG1001)
801(pAG3001)
801(pAG4003)
801(pAG5001)
801(pIG101)
801(pAIG321)

9.1
9.2
9.1
9.2
9.2
10.9
10.6

52
54
54
53
53
57
58

801(pCI31)
801(pCG5)801(pCIG231)

9.1
9.3
11.2

54
56
60

801(pCAIG4)

11.2

61

271

Attachment 15 "Calculation Table of the Value of Damages under Article 102, Paragraph(2) of the Patent Act for the Portion Sold by the Defendant, etc."(omitted)

272

Attachment 16 "Situation of Import of Monosodium Glutamate During the Period of Useof the Defendant's Production Processes"(omitted)

273

Attachment 17 "List of Products Subject to Injunction"
Monosodium glutamate with the product name "MI-POONG"
which satisfies the following requirements:
1. One produced by using Corynebacterium glutamicum into which a yggB gene, wherein
alanine at position 100 in the amino acid sequence indicated in the sequence listing
attached to this list is substituted with threonine, is introduced.
2. One produced by using Corynebacterium glutamicum into which a yggB gene, wherein
alanine at position 98 in the amino acid sequence encoded by the yggB gene derived from
Corynebacterium callunae (DSM20147 strain) is substituted with threonine, and valine
at position 241 in that amino acid sequence is substituted with isoleucine, is introduced.
End

274

Sequence listing attached to Attachment 17
Met Ile1
Trp IleLeu Gly Val Pro
5
Val Asp Thr Gly
20
Phe Leu Ile Pro Arg Ile
35
Arg Val Glu Ser Ala Ala
50

Ile

Gln Tyr Leu Leu Tyr Ser Leu Trp Asn
1015
Phe Asp Val Ala Ile Ile Leu Val Leu Ala
25
30
Gly Arg Leu Ala Met Arg Ile Ile Lys Gln
40
45
Asp Ala Asp Thr Thr Lys Asn Gln Leu Ala
55
60

Phe Ala Gly Val Gly Val Tyr Ile
6570
Leu Ala Val Ser Ala Met Gln Ala
85
Ala Ile Pro Ala Thr Ile Ala Ser
100
Ser Ile Val Ala Asp Phe Leu Ala
115
120
Gln Phe Gly Val Gly Asp Trp Val
130
135
Val Glu Gly Thr Val
145
Thr Ile Ala Gln Glu
165
Cys Ile Asn Asn Ser
180

Ala Gln Ile
75
Phe Gly Phe
90
Ala Ala Ile
105
Gly Phe Phe

Val Ala Phe Phe Met
80
Ser Leu Ala Gly Ala
95
Gly Leu Gly Ala Gln
110
Ile Leu Thr Glu Lys
125
Arg Phe Glu Gly Asn Gly Ile Val
140

Ile Glu Ile150
Thr Val Ile

Thr Met Arg Ala Thr Lys Ile Arg
155
160
Ile Pro Asn Ser Thr Ala Lys Val
170
175
Asn Asn Trp Ser Arg Ala Val Val Val Ile Pro
185
190

275

Ile
Glu
Ile
225
Thr
Val
Ile

Pro Met Leu Gly Ser Glu
195
Ala Ala Thr Arg Arg Ala
210
215
Leu Gly Glu Leu Asp Val
230
Val Val Gly Met Pro Trp
245
Thr Ala Gly Asn Gln Trp
260
Ile

Asn Glu Phe Trp275
Gly Thr Leu Ile Asp Ser
290
Ser Leu Ile Asp Ala Ser
305
310
Ala Ala Ala Thr Val Ala
325
Asp Asn Ala Asp Ala Ser
340

Asn Ile Thr Asp Val Ile200
205
Leu Gly Gln Glu Lys Ile
220
His Pro Ala Thr Glu Val
235
Met Val Thr Met Arg Phe
250
Leu Val Glu Arg Ala Ile
265

Glu Glu Tyr Gly Ser
280
Leu His Val Glu His
295
Pro Gln Ala Leu Lys
315
Ser Leu Ala Ala Ser
330
Ala Ile Asn Ala Gly
345

Leu Asp Ser Asp Val Leu Glu
355
Glu Thr Ala Lys Pro Asp His
370
375
Tyr Tyr Pro Asn Arg Trp Gln
385
390

Ala Arg Ser
Ala Pro Glu
Thr Pro Pro
240
Leu Val Gln
255
Arg Thr Glu
270

Ala Thr Thr Thr Ser
285
Glu Glu Pro Lys Thr
300
Glu Pro Lys Pro Glu
320
Ser Asn Asp Asp Ala
335
Asn Pro Glu Lys Glu
350

Gln Glu Leu Ser Ser Glu Glu Pro Glu
360
365
Ser Leu Arg Gly Phe Phe Arg Thr Asp
380
Lys Ile Leu Ser Phe Gly Gly Arg Val
395
400

276

Arg Met Ser Thr Ser
405
Phe Lys Val Met Thr
420
Gly Trp Leu Ser Pro
435
Glu Thr Ser Ala Pro
450
Val Glu Glu Thr Pro
465

Leu Leu Leu Gly Ala
410
Val Glu Pro Ser Glu
425
Ser Thr Ala Thr Ser
440
Ala Ser Thr Pro Ser
455
Thr Met Glu Ser Ser
470

Thr Ser Thr Pro Ala Thr Ala Thr
485
Pro Thr Glu Glu Ala Thr Ser Gln
500
Gln Ser Pro Ala Val Glu Ala Pro
515
520
Pro Thr Ser Thr Pro
530

Leu Leu Leu Leu Ser
415
Asn Trp Gln Asn Ser
430
Thr Ala Val Thr Thr
445
Met Thr Val Pro Thr
460
Val Glu Thr Gln Gln
475

Pro Gln Arg Ala Asp Thr
490
Glu Glu Thr Thr Ala Ser
505
510
Thr Ala Val Gln Glu Thr
525

End

277

Leu
Ser
Ser
Thr
Glu
480

Ile Glu
495
Gln Thr
Val Ala