Process for the production of L-threonine by fermentation

A process for the production of L-threonine, comprising cultivating a mutant bacterial species belonging to the genus Brevibacterium from which dihydrodipicolinate synthase has been deleted or removed in a liquid medium; accumulating the L-threonine as a product of cultivation; and harvesting the L-threonine from the culture medium.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a process for the production of 
L-threonine (hereinafter referred to as threonine) by a fermentation 
technique. 
2. Description of the Background 
Threonine is an important amino acid which is used in animal feeds and 
medicines. One process which is known for its production is a fermentation 
method. In this technique a mutant of the genus Brevibacterium having 
resistance to bacterial .alpha.-amino-.beta.-hydroxyvaleric acid 
(hereinafter referred to as AHV) which is a threonine analog (Agric. Biol. 
Chem., 34 (3) 448-456 (1970), Japanese Patent Publication No. 26708/70) is 
cultured. This process employs as an AHV-resistant mutant, a strain having 
homoserine dehydrogenase (hereinafter referred to as HD) in which feedback 
inhibition due to threonine has been removed (J. Biochem., 68, 859-866 
(1970)). The yield of threonin produced by this process is low, and 
therefore, it is not an economical method for the production of threonine 
which is added to animal feeds. Accordingly, in order to enhance the yield 
of threonine, a mutant which is frequently used is one which is a 
lysine-producing strain. However, in this case, lysine is frequently 
produced as a by-product, which adversely affects the yield of threonine. 
Further, the separation of lysine from the medium is a complicating 
factor. A need therefore continues to exist for a method of producing 
threonine in improved yields by a fermentation technique. 
SUMMARY OF THE INVENTION 
Accordingly, one object of the present invention is to provide a 
fermentation technique of producing L-threonine in enhanced quantities. 
Briefly, this object and other objects of the present invention as 
hereinafter will become more readily apparent can be obtained by a method 
for the production of L-threonine by cultivating a mutant bacterial 
species belonging to the genus Brevibacterium from which 
dihydrodipicolinate synthase has been deleted or removed in a liquid 
medium; accumulating said L-threonine as a product of cultivation; and 
harvesting said L-threonine from said culture medium. The mutant strains 
are distinguished from other known bacteria by the fact that the feedback 
inhibition of HD because of threonine has not been removed. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As will be evident from the description below, the yield of threonine 
obtained upon fermentation of the mutants used in the present process is 
more or less the same as the yields obtained upon fermentation of 
conventional threonine producing strains. Further, since DPS is the 
primary enzyme in the lysine biosynthetic pathway, if a mutant is produced 
in which it has been deleted or reduced, the amount of the lysine produced 
as a by-product may be decreased. Furthermore, since the mutant of the 
present invention is by nature completely different from conventional 
mutants employed, a possible way of enhancing the yield of threonine is to 
overlap it with a conventional strain. 
The microorganism used in the process for the production of threonine in 
the present invention is a strain which belongs to the genus 
Brevibacterium in which DPS has been deleted or reduced. In addition to 
this feature, if the mutant is imparted with HD feedback inhibition 
removal, L-methionine auxothropy, L-isoleucine auxothropy, ethionine 
resistance, lysine analog resistance, pyruvate kinase deletion, and the 
like, the productivity of threonine may further be enhanced. The parent 
strain of the mutant of the present invention is a microorganism belonging 
to the genus Brevibacterium known as the so-called L-glutamic acid 
producing bacteria, and may be exemplified by the following strains: 
______________________________________ 
Brevibacterium flavum 
ATCC 14067 
Brevibacterium divarictum 
ATCC 14020 
Brevibacterium lactofermentum 
ATCC 13869 
Brevibacterium roseum 
ATCC 13825 
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The mutant used in the present invention may be obtained by carrying out a 
mutation operation on the above-described parent strains, thereby 
effecting the deletion or reduction of DPS. The mutation operation may be 
conducted by a conventional method such as, for example, ultraviolet light 
irradiation, or chemical treatment using 
N-methyl-N'-nitro-N-nitrosoquanidine (hereinafter referred to as NG), 
nitrous acid, or the like. After the mutation operation, the DPS deleted 
or reduced mutant may be separated by selecting strains resistant to AHV 
which also produce threonine, and measuring the DPS activity of these 
strains. Especially, when a lysine-producing strain of a type in which 
synergistic inhibition of aspartokinase (hereinafter referred to as AK) 
due to threonine and lysine has been removed is used as the parent strain, 
by selecting the strain which produces less lysine by-product in 
comparison to the threonine yield among the AHV-resistant, 
threonine-producing strains obtained, it is possible to obtain the DPS 
deleted strain more efficiently. 
A specific method for deriving Brevibacterium flavum AJ 12360 (FERM 
P-9821), which is an example of a strain useful in the present invention, 
is described below. The parent strain in the derivation process is the 
aspartic acid-producing strain AJ 11955 (FERM P-6665, Japanese Patent 
Application Laid-open No. 45895/84) which in turn is derived from 
Brevibacterium flavum AJ 14067. Brevibacterium flavum AJ 11955 was treated 
with 150 .mu.g/ml of NG at 30.degree. C. for 15 minutes. Then, a glucose 
minimum agar plate medium (9 cm in diameter) shown in Table 1 and also 
containing 10 g/l of AHV and 5 g/l of lysine was inoculated with the 
bacterium to give about 10.sup.5 cells per plate, and the colonies 
appearing after cultivation for 5 days were transferred to a complete agar 
plate medium shown in Table 2 as the resistant strains. Thereafter, a 
medium for threonine production shown in Table 3 was dispensed in 3 ml 
amounts into test tubes. (The medium contained 70 g/l of ammonium sulfate 
and 30 ml/l of soybean hydrolysate and is simply referred to as N70S30.) 
The media were inoculated with these strains, and the resulting cultures 
were agitated at 30.degree. C. for 72 hours. Strains producing threonine 
were then selected. Of the mutant strains, strain AJ 12360, which 
exhibited the maximum productivity, was analyzed by the method described 
hereinbelow. It was found that the HD feedback inhibition of the strain 
was the same as the parent strain, but the DPS activity had been deleted 
(Table 4). 
The following is a specific method for the derivation of DPS deleted or 
reduced strains AJ 12361-2 (FERM P-9822-3) obtained from lysine-producing 
Brevibacterium flavum AC 664 as the parent strain having AK in which 
synergistic inhibition due to threonine and lysine is removed. The AC 664 
strain was treated with 600 .mu.g/ml of NG at 30.degree. C. for 15 
minutes. Then, an acetic acid-pyruvic acid agar plate medium shown in 
Table 3 and containing 2-5 g/l of AHV was inoculated with the NG treated 
cells so as to give about 10.sup.7 cells per plate. Thereafter, the 
colonies appearing after cultivation for 12 days were transferred as the 
resistant strains. The threonine and lysine productivity of these strains 
was examined by the above-described cultivation method, except that a 
composition identified as N25S35 was used as the medium for threonine 
production. Five strains which yielded small amounts of lysine by-product 
in comparison to the yield of threonine produced were selected. Of the 
strains, as shown in Table 6, three identified as AJ 12361 and 12362 and 
DA 110, exhibited a feedback inhibition of HD which was the same as that 
of the parent strain, but the DPS activity had been deleted or reduced. 
The reason why the strains giving lower amounts of lysine by-product were 
selected is that since DPS is an enzyme which catalyzes the reaction of 
diaminopimelic acid (hereinafter referred to as DAP) in the lysine 
synthetic pathway, the amount of lysine produced is believed to be greatly 
reduced by using DPS deleted strains. In fact, with these 3 strains, as 
shown in Table 6, the lysine/threonine ratios were lower than the ratios 
obtained from AJ 12363 (FERM P-9824) which is a representative strain of 
the known threonine-producing strains derived from the same parent strain 
and having HD in which feedback inhibition due to threonine has been 
removed. 
The amount of threonine produced in a given fermentation experiment was 
measured by the method described below, and the amount of lysine produced 
was measured by an acidic ninhydrin method. Further, the DPS activity was 
measured as follows. A medium obtained by adding 1 g/l of DAP to a medium 
useful for threonine production shown in Table 3 was dispensed in 20 ml 
amounts into 500 ml flasks which could be shaken. The flasks were 
sterilized by autoclaving. The medium compositions used were N50S30 for 
the AJ 12360 strain and N25S35 for the AJ 12361-2 and DA 110 strains. Each 
medium was inoculated with one loop of the strain which had been 
previously cultivated in a complete agar plate medium shown in Table 2. 
Each medium was supplemented with 1 g/l of DAP and 300 mg/l of L-histidine 
hydrochloride at 30.degree. C. for 24 hours, and culturing was conducted 
at 30.degree. C. for 40 hours in the shaking flasks. The cells were 
collected and washed with a 0.2% potassium chloride solution. These washed 
cells were suspended in a 50 mM potassium phosphate buffer (pH 7.0), and 
then were centrifugally separated after ultrasonic treatment in a 
supernatant. The supernatant was gel filtered through a Sephadex G25 
column using the same buffer to prepare a crude enzyme solution. The 
activity was measured by carrying out the reaction at 30.degree. C. for 
10 minutes, terminating the reaction by adding 1 ml of 0.05 N hydrochloric 
acid thereto, and then measuring the change in absorbance at 340 nm by 
using a reaction mixture shown in Table 8 to determine the amount of 
pyruvic acid consumed. The control for this reaction was a 
DL-aspartate-.beta.-semialdehyde-free system. The HD activity was measured 
as follows. The crude enzyme solution was prepared in a manner similar to 
that employed in the above-described DPS activity measurement, except that 
a 0.1 M potassium phosphate buffer containing 0.5 M potassium chloride (pH 
7.0) was used as the buffer instead of the 50 mM potassium phosphate 
buffer. The activity measurement was conducted by measuring the reduction 
in absorbance at 340 nm in the reaction system shown in Table 9. The 
control for this reaction was a DL-aspartate-.beta.-semialdehyde-free 
system. 
Thereafter, the degrees of AHV resistance of DPS deleted strains AJ 12360 
and AJ 12361 were compared against their parent strains. The degree of AHV 
resistance was determined by cultivating the strain in the complete agar 
plate medium shown in Table 2 which also contained 1 g/l of DAP, 50 ml/l 
of dipicolinic acid and further, in the case of AJ 12361 and its parent 
strain AC 664, 300 mg/l of L-histidine hydrochloride as a growth promoting 
substance, at 30.degree. C. for 24 hours. The cells were then washed with 
the glucose minimum liquid medium shown in Table 10. The same medium 
dispensed in 3 ml into test tubes, was inoculated with the cells, and the 
inoculated media were cultured while being shaked for 24 hours. The 
absorbance of each medium was measured at 566 nm under conditions in which 
AHV was added or was not added to a given medium. To each glucose minimum 
liquid medium prepared were added, as growth promoting substances, 200 
mg/l of L-histidine hydrochloride, in the case of AJ 12361 and AC 664 
strains, and 500 mg/l each of DAP and lysine in the case of AJ 12360 and 
AJ 11955 strains. Especially, since DAP or DAP and lysine often promoted 
the growth of the DPS deleted or reduced strain, DAP was added to the 
liquid and plate media in the experiments described so far as needed. As 
shown in Table 11, growth inhibition of both DPS deleted strains was 
difficult to achieve because of AHV in comparison to the respective parent 
strains, and thus the strains acquired AHV resistance. 
The culture medium used for threonine production is not particularly 
critical in achieving the results of the invention. Conventional media 
containing carbon sources, nitrogen sources, inorganic salts, and minor 
organic nutrients, if needed can be employed. Suitable carbon sources 
include carbohydrates such as glucose, fructose, or hydrolysates of 
starch, cellulose and the like, molasses, and the like; organic acids such 
as acetic acid, citric acid and the like; alcohols such as glycerin, 
ethanol and the like, and hydrocarbons such as normal paraffins, and the 
like. Suitable nitrogen sources include ammonium sulfate, urea, ammonium 
nitrate, ammonium phosphate, ammonium chloride, ammonia gas, and the like. 
Suitable inorganic salts include phosphates, magnesium salts, calcium 
salts, iron salts, manganese salts, other minor metal salts and the like 
as needed. The minor organic nutrients which may be present in appropriate 
amounts include amino acids, if there is auxotrophy, vitamins, fatty 
acids, organic basic substances, and the like. Further, as there is need, 
growth promoting substances such as amino acids, vitamins, Ajieki 
(registered trademark, soybean hydrolysate), yeast extract, peptone, 
casamino acids, and the like may be added. Especially, in the case of the 
DPS deleted or reduced strains, the addition of DAP or DAP and lysine 
often promote growth, to improve the results obtained. 
The conditions of cultivation may be conventional with cultivation being 
conducted at a pH of 5-9 at a temperature of 20.degree.-40.degree. C. 
under aerobic conditions for 24-72 hours. During cultivation, if the pH 
drops, calcium carbonate, separately sterilized is added to the medium or 
a base such as ammonia water, ammonia gas, or the like is used to 
neutralize the medium. Further, where an organic acid is used as the 
carbon source, any increase in pH is neutralized with a mineral acid or an 
organic acid. 
The separation and harvesting of threonine may be carried out in a 
conventional manner. The production of threonine is combined by 
measurement of the appropriate Rf value on thin layer chromatography. The 
biological activity value obtained by microbiological assay agrees that 
threonine is the authentic product. Quantitative analysis for threonine 
was conducted by a microbiological assay technique using Leuconostoc 
mesenteroides (ATCC 8042). 
Brevibacterium flavum FERM-BP 2178 (FERM-P 6665) (AJ 11955) was originally 
deposited on Aug. 14, 1982 at the Fermentation Research Institute, Agency 
of Industrial Sciences and Technology, Ministry of International Trade and 
Industry (FRI), 1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken 305, Japan, 
and was accorded the FERM-P number indicated above. Brevibacterium flavum 
FERM-BP 2179 (FERM-P 9821) (AJ 12360), FERM-BP 2180 (FERM-P 9823) (AJ 
12362) and FERM-BP 2181 (FERM-P 9824) (AJ 12363) were originally deposited 
on Jan. 18, 1988 at FRI, and were accorded the FERM-P numbers indicated 
above. The strains were then converted into deposits under the Budapest 
Treaty on Dec. 7, 1988, and were accorded the corresponding FERM-BP 
numbers. 
Brevibacterium flavum FERM-BP 2186 (FERM-P 9822) (AJ 12361) was originally 
deposited on Jan. 18, 1988 at FRI, and was accorded the FERM-P number 
indicated above. The strain was then converted into a deposit under the 
Budapest Treaty on December 14, 1988 and was accorded the corresponding 
FERM-BP number.