# Patent

## Title
Coryneform bacterium transformant and process for producing phenol using the same

## Abstract

Provided is a phenol-producing transformant constructed by transferring a gene which encodes an enzyme having chorismate-pyruvate lyase activity and a gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity into a coryneform bacterium as a host. Also provided is a process for producing phenol, which comprises a step of allowing the transformant to react in a reaction mixture containing a saccharide under reducing conditions, and a step of collecting phenol from the reaction mixture.

## Background
TECHNICAL FIELD
The present invention relates to a technique for producing phenol. In more detail, the present invention relates to a coryneform bacterium transformant constructed by specific gene recombination and thereby provided with a phenol-producing function, and relates to an efficient phenol-producing process using the transformant.
BACKGROUND ART
Against the backdrop of global warming and exhaustion of fossil resources, production of chemical products using renewable resources, along with production of biofuels, is recognized as an emerging industry, biorefinery, which is an important means for realizing a low-carbon society, and has attracted keen attention.
However, production of biophenol using renewable resources is less productive as compared to production of lactic acid or ethanol because the metabolic reaction from a raw material saccharide consists of a great many steps. In addition, for the reasons that produced phenol inhibits bacterial proliferation and that phenol is cytotoxic, industrial production of phenol has been considered to be impossible.
Important use of phenol is phenol resins. A phenol resin, which is produced by addition condensation of phenol and aldehyde, is one of the oldest plastics, and with its properties including excellent heat resistance and durability, is used for various purposes, such as an alternative automotive material to metal, a semiconductor seal material, and a circuit board even today. Due to extremely high reactivity of phenol and aldehyde as raw materials and to the complicated three-dimensional network structure of resulting phenol resin polymers, precise structural designing and development into nanomaterials thereof had been considered difficult and so had been application to high-value-added use. However, in recent years, the theory of physical-properties of polymers and the simulation thereof have rapidly developed, and therefore it has gradually become possible to create highly functional materials from phenol resins by refining the network structure. Under the circumstances, the phenol resin production in Japan is also increasing year by year.
The currently employed industrial production process of phenol (cumene process) is a typical energy-consumptive process in the chemical industry using petroleum-derived benzene and propylene as raw materials, and requiring great amounts of solvent and thermal energy. Therefore, in the light of global environment conservation and greenhouse gas reduction, there is an urgent need to develop an environment-conscious, energy saving process that allows production of phenol from renewable resources and can reduce carbon dioxide emissions and waste products, that is, to establish biophenol production technologies.
There have not been reported phenol-producing bacteria in nature so far.
Also, there have not been known recombinant bacteria-based phenol-producing technologies to achieve a practically sufficient phenol productivity.
SUMMARY OF INVENTION
Technical Problem
An object of the present invention is to provide a microorganism capable of efficiently producing phenol from a saccharide, and a process for efficiently producing phenol from a saccharide.
Solution to Problem
The present inventors have wholeheartedly carried out investigations in order to achieve the object described above and obtained the following findings.
(i) A transformant constructed by transferring a chorismate-pyruvate lyase gene and a 4-hydroxybenzoate decarboxylase gene into a coryneform bacterium can efficiently produce phenol.
(ii) The transformant can further efficiently produce phenol in the case where the 4-hydroxybenzoate hydroxylase gene on the chromosome of the coryneform bacterium as the host has a disruption or deletion.
(iii) The transformant further efficiently produces phenol in the case where the 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthetase gene is more highly expressed as compared to the gene expression level before transformation.
(iv) The transformant has a particularly higher phenol productivity when proliferation is substantially inhibited in a reaction mixture under reducing conditions.
The present invention, which has been completed based on the above-mentioned findings, provides the following transformant and process for producing phenol.
[1] A phenol-producing transformant constructed by transferring a gene which encodes an enzyme having chorismate-pyruvate lyase activity and a gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity into a coryneform bacterium as a host.

[2] The transformant of the above [1], wherein the gene which encodes an enzyme having chorismate-pyruvate lyase activity is a gene derived from Escherichia coli; a gene derived from Pseudomonas putida; a gene derived from Acinetobacter baumannii; a gene derived from Azotobacter vinelandii; a gene derived from Chromohalobacter salexigens; a gene derived from members of the genus Citrobacter, such as Citrobacter koseri and Citrobacter youngae; a gene derived from Enterobacter cloacae; a gene derived from Marinobacter aquaeolei; a gene derived from Marinomonas mediterranea; a gene derived from Pantoea ananatis; a gene derived from Pseudoalteromonas haloplanktis; a gene derived from Ralstonia eutropha; a gene derived from Shewanella putrefaciens; or a gene derived from Thiobacillus denitrificans. 

[3] The transformant of the above [1], wherein the gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity is a gene derived from Bacillus subtilis, a gene derived from Bacillus atrophaeus, a gene derived from Bacillus subtilis subsp. spizizenii, a gene derived from Citrobacter koseri, a gene derived from Enterobacter aerogenes, a gene derived from Enterobacter cloacae, a gene derived from Enterobacter hormaechei, a gene derived from Enterobacter sakazakii, a gene derived from Escherichia coli, a gene derived from Escherichia fergusonii, a gene derived from Paenibacillus polymyxa, or a gene derived from Pantoea ananatis. 

[4] The transformant of the above [1], wherein the gene which encodes an enzyme having chorismate-pyruvate lyase activity is the DNA of the following (a) or (b).

(a) a DNA consisting of the base sequence of SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, or SEQ ID NO: 93

(b) a DNA which hybridizes to a DNA consisting of a complementary base sequence of any of the DNAs of (a) under stringent conditions and which encodes a polypeptide having chorismate-pyruvate lyase activity

[5] The transformant of the above [1], wherein the gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity is the DNA of the following (c) or (d).

(c) a DNA consisting of the base sequence of SEQ ID NO: 37, SEQ ID NO: 44, SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 65, NO: 68, SEQ ID NO: 71, or SEQ ID NO: 74

(d) a DNA which hybridizes to a DNA consisting of a complementary base sequence of any of the DNAs of (c) under stringent conditions and which encodes a polypeptide having 4-hydroxybenzoate decarboxylase activity

[6] The transformant of any one of the above [1] to [5], wherein the coryneform bacterium as the host is a coryneform bacterium in which a gene which encodes an enzyme having 4-hydroxybenzoate hydroxylase activity on the chromosome is disrupted or deleted.

[7] The transformant of any one of the above [1] to [6], wherein the coryneform bacterium as the host is a coryneform bacterium in which a gene which encodes an enzyme having phenol 2-monooxygenase activity on the chromosome is disrupted or deleted.

[8] The transformant of any one of the above [1] to [7], wherein a gene which encodes an enzyme having 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase activity is highly expressed in the coryneform bacterium as the host.

[9] The transformant of any one of the above [1] to [8], wherein the coryneform bacterium as the host is Corynebacterium glutamicum. 

[10] The transformant of any one of the above [1] to [5], wherein the coryneform bacterium as the host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869.

[11] The transformant of any one of the above [1] to [5], wherein the coryneform bacterium as the host is a strain of Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869 in which a gene which encodes an enzyme having 4-hydroxybenzoate hydroxylase activity on the chromosome is disrupted or deleted.

[12] The transformant of any one of the above [1] to [5], wherein the coryneform bacterium as the host is a strain of Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869 in which a gene which encodes an enzyme having phenol 2-monooxygenase activity on the chromosome is disrupted or deleted.

[13] The transformant of anyone of the above [1] to [5], wherein the coryneform bacterium as the host is a strain of Corynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869 in which a gene which encodes an enzyme having 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase activity is highly expressed.

[14] Corynebacterium glutamicum transformant PHE18 (Accession Number: NITE BP-995), PHE11, PHE12, PHE13, PHE14, PHE15, PHE16, PHE17, PHE19-1, PHE19-2, PHE19-3, PHE19-4, PHE19-5, PHE19-6, PHE19-7, PHE19-8, PHE19-9, PHE19-10, PHE19-11, PHE19-12, PHE20-1, PHE20-2, PHE20-3, PHE20-4, PHE20-5, PHE20-6, PHE20-7, PHE20-8, PHE20-9, PHE20-10, PHE20-11, PHE20-12, PHE20-13, or PHE20-14.

[15] A process for producing phenol, which comprises a step of allowing the transformant of any one of the above [1] to [14] to react in a reaction mixture containing a saccharide under reducing conditions, and a step of collecting phenol from the reaction mixture.

[16] The process of the above [15], wherein the transformant does not substantially proliferate in the reaction step.

[17] The process of the above [15] or [16], wherein the oxidation-reduction potential of the reaction mixture under reducing conditions is −200 mV to −500 mV.

[18] The process of any one of the above [15] to [17], wherein the saccharide is selected from the group consisting of glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol.

Advantageous Effects of Invention
With the use of the transformant of the present invention, phenol can be efficiently produced from a saccharide.
Generally, growth of microorganisms is inhibited by a solvent, such as a phenol, because of its cytotoxicity, and therefore phenol production with the use of microorganisms was difficult. According to the process of the present invention, however, phenol production with the use of microorganisms can be achieved with a practically sufficient efficiency.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the construct of various plasmids used in Examples.
FIG. 2 shows the construct of various plasmids used in Examples.
FIG. 3 shows the construct of various plasmids used in Examples.
FIG. 4 shows the influence of phenol on proliferation of various microorganisms under aerobic conditions.
FIG. 5 shows the influence of phenol on the saccharide consumption by Corynebacterium under reducing conditions.



DESCRIPTION OF EMBODIMENTS
Hereinafter, the present invention will be described in detail.
(I) Phenol-Producing Transformant
The transformant of the present invention capable of producing phenol is a transformant constructed by transferring a gene which encodes an enzyme having chorismate-pyruvate lyase activity and a gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity into a coryneform bacterium as a host.
Host
The coryneform bacterium is a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), and is not particularly limited as long as it proliferates under normal aerobic conditions.
The specific examples include Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium and Micrococcus. Among the coryneform bacteria, Corynebacterium is preferred.
Examples of the Corynebacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum. 
Inter alia, Corynebacterium glutamicum is preferred for safety and high phenol production. Examples of preferred strains include Corynebacterium glutamicum R (FERM P-18976), ATCC13032, ATCC13869, ATCC13058, ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13655, ATCC13745, ATCC13746, ATCC13761, ATCC14020, ATCC31831, MJ-233 (FERM BP-1497), and MJ-233AB-41 (FERM BP-1498). Inter alia, strains R (FERM P-18976), ATCC13032, and ATCC13869 are preferred.
According to molecular biological classification, names of some species of coryneform bacteria, such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, and Corynebacterium lilium are standardized to Corynebacterium glutamicum (Liebl, W. et al., Transfer of Brevibacterium divaricatum DSM 20297T, “Brevibacterium flavum” DSM 20411, “Brevibacterium lactofermentum” DSM 20412 and DSM 1412, and Corynebacterium glutamicum and their distinction by rRNA gene restriction patterns. Int. J. Syst. Bacteriol. 41: 255-260. (1991); and Kazuo Komagata et al., “Classification of the coryneform group of bacteria”, Fermentation and industry, 45: 944-963 (1987)).
Brevibacterium lactofermentum ATCC13869, Brevibacterium flavum MJ-233 (FERM BP-1497) and MJ-233AB-41 (FERM BP-1498), etc. of the old classification are also suitable as Corynebacterium glutamicum. 
Examples of the Brevibacterium include Brevibacterium ammoniagenes (for example, ATCC6872).
Examples of the Arthrobacter include Arthrobacter globiformis (for example, ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738 and ATCC35698).
Examples of the Mycobacterium include Mycobacterium bovis (for example, ATCC19210 and ATCC27289).
Examples of the Micrococcus include Micrococcus freudenreichii (for example, NO. 239 (FERM P-13221)), Micrococcus leuteus (for example, NO. 240 (FERM P-13222)), Micrococcus ureae (for example, IAM1010), and Micrococcus roseus (for example, IF03764).
The coryneform bacteria may be, let alone a wild strain, a mutant thereof or an artificial recombinant thereof. Examples thereof include disruptants in which a gene of lactate dehydrogenase, phosphoenolpyruvate carboxylase, or malate dehydrogenase is disrupted. Using such a disruptant as a host can improve phenol productivity and reduce production of by-products.
Inter alia, preferred is a disruptant in which a lactate dehydrogenase gene is disrupted. In the disruptant, the lactate dehydrogenase gene is disrupted and the metabolic pathway from pyruvic acid to lactic acid is blocked. Inter alia, especially preferred is a disruptant of Corynebacterium glutamicum R (FERM P-18976) strain in which the lactate dehydrogenase gene is disrupted.
Such a disruptant can be prepared based on a conventional gene engineering process. Such a lactate dehydrogenase disruptant and the preparation process thereof are described in WO 2005/010182 A1.
Chorismate-Pyruvate Lyase Gene (ubiC)
Chorismate-pyruvate lyase is an enzyme that catalyzes a reaction in which 4-hydroxybenzoate is produced by elimination of pyruvic acid from chorismate.
The gene which encodes an enzyme having chorismate-pyruvate lyase activity may be of any origin without particular limitation, and preferred examples thereof include a gene derived from Escherichia coli; a gene derived from Pseudomonas putida; a gene derived from Acinetobacter baumannii; a gene derived from Azotobacter vinelandii; a gene derived from Chromohalobacter salexigens; a gene derived from members of the genus Citrobacter, such as Citrobacter koseri and Citrobacter youngae; a gene derived from Enterobacter cloacae; a gene derived from Marinobacter aquaeolei; a gene derived from Marinomonas mediterranea; a gene derived from Pantoea ananatis; a gene derived from Pseudoalteromonas haloplanktis; a gene derived from Ralstonia eutropha; a gene derived from Shewanella putrefaciens; and a gene derived from Thiobacillus denitrificans. More preferred is a gene derived from Pseudomonas putida. 
Examples of the chorismate-pyruvate lyase gene derived from Escherichia coli include the DNA consisting of the base sequence of SEQ ID NO: 31, examples of the chorismate-pyruvate lyase gene derived from Pseudomonas putida include the DNA consisting of the base sequence of SEQ ID NO: 34, examples of the chorismate-pyruvate lyase gene derived from Acinetobacter baumannii include the DNA consisting of the base sequence of SEQ ID NO: 81, examples of the chorismate-pyruvate lyase gene derived from Azotobacter vinelandii include the DNA consisting of the base sequence of SEQ ID NO: 82, examples of the chorismate-pyruvate lyase gene derived from Chromohalobacter salexigens include the DNA consisting of the base sequence of SEQ ID NO: 83, examples of the chorismate-pyruvate lyase gene derived from Citrobacter koseri include the DNA consisting of the base sequence of SEQ ID NO: 84, examples of the chorismate-pyruvate lyase gene derived from Citrobacter youngae include the DNA consisting of the base sequence of SEQ ID NO: 85, examples of the chorismate-pyruvate lyase gene derived from Enterobacter cloacae include the DNA consisting of the base sequence of SEQ ID NO: 86, examples of the chorismate-pyruvate lyase gene derived from Marinobacter aquaeolei include the DNA consisting of the base sequence of SEQ ID NO: 87, examples of the chorismate-pyruvate lyase gene derived from Marinomonas mediterranea include the DNA consisting of the base sequence of SEQ ID NO: 88, examples of the chorismate-pyruvate lyase gene derived from Pantoea ananatis include the DNA consisting of the base sequence of SEQ ID NO: 89, examples of the chorismate-pyruvate lyase gene derived from Pseudoalteromonas haloplanktis include the DNA consisting of the base sequence of SEQ ID NO: 90, examples of the chorismate-pyruvate lyase gene derived from Ralstonia eutropha include the DNA consisting of the base sequence of SEQ ID NO: 91, examples of the chorismate-pyruvate lyase gene derived from Shewanella putrefaciens include the DNA consisting of the base sequence of SEQ ID NO: 92, and examples of the chorismate-pyruvate lyase gene derived from Thiobacillus denitrificans include the DNA consisting of the base sequence of SEQ ID NO: 93.
In the present invention, a DNA which hybridizes to a DNA consisting of a complementary base sequence of the base sequence of SEQ ID NO: 31, 34, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 under stringent conditions and which encodes a polypeptide having chorismate-pyruvate lyase activity can also be used.
The “stringent conditions” as used herein means general conditions, for example, the conditions described in Molecular Cloning, A Laboratory Manual, Second edition, 1989, Vol. 2, p. 11. 45. It means, in particular, conditions where hybridization occurs at a temperature 5 to 10° C. below the melting temperature (Tm) of a perfect hybrid.
The chorismate-pyruvate lyase activity can be measured by the method described in Journal of Bacteriology, 174, 5309-5316 (1992) “Materials and Methods”. Briefly, by adding a test enzyme to a liquid for testing, a reaction mixture containing 50 mM tris-HCL (pH 7.5), 5 mM EDTA, 10 mM β-mercaptoethanol, 60 μM chorismic acid, and the enzyme is prepared, and then the slope of the absorbance at 240 nm (initial rate) is determined. The same measurement is performed using a system without the addition of chorismic acid to obtain a background value. The difference between the two measured values will be regarded as the chorismate-pyruvate lyase activity.
In the present invention, a DNA consisting of a base sequence which has 90% or more, preferably 95% or more, more preferably 98% or more homology with the base sequence of SEQ ID NO: 31, 34, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 and which encodes a polypeptide having chorismate-pyruvate lyase activity can also be used.
The base sequence homology was calculated using GENETYX Ver. 8 (made by Genetyx).
The homologue of the DNA consisting of the base sequence of SEQ ID NO: 31, 34, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93 can be selected from a DNA library of a different species by, for example, PCR or hybridization using a primer or a probe designed based on these base sequences, according to a conventional method, and as a result, a DNA which encodes a polypeptide having chorismate-pyruvate lyase activity can be obtained with a high probability.
4-Hydroxybenzoate Decarboxylase Gene (bsdBCD or Dca)
4-Hydroxybenzoate decarboxylase is an enzyme that catalyzes a phenol-producing reaction in which 4-hydroxybenzoate is decarboxylated.
The gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity may be of any origin without particular limitation, and examples thereof include genes derived from members of the genus Bacillus, such as Bacillus subtilis, Bacillus megaterium, Bacillus licheniformis, Bacillus atrophaeus, and Bacillus subtilis subsp. spizizenii; Citrobacter koseri; members of the genus Enterobacter, such as Enterobacter aerogenes, Enterobacter cloacae, Enterobacter hormaechei, and Enterobacter sakazakii; members of the genus Escherichia, such as Escherichia coli and Escherichia fergusonii; Paenibacillus polymyxa; and Pantoea ananatis. Inter alia, preferred is a gene derived from members of the genus Bacillus, in particular Bacillus subtilis; members of the genus Enterobacter, in particular Enterobacter cloacae; or members of the genus Escherichia, in particular Escherichia coli. 
A gene which encodes an enzyme having 4-hydroxybenzoate decarboxylase activity is called by an abbreviated name that varies according to the origin. For example, a 4-hydroxybenzoate decarboxylase gene derived from Bacillus subtilis is called by an abbreviation bsdBCD. Herein, a 4-hydroxybenzoate decarboxylase gene may be called by an abbreviation “dca” regardless of the origin.
Examples of the 4-hydroxybenzoate decarboxylase gene derived from Bacillus subtilis include the DNA consisting of the base sequence of SEQ ID NO: 37, examples of the 4-hydroxybenzoate decarboxylase gene derived from Bacillus atrophaeus include the DNA consisting of the base sequence of SEQ ID NO: 44, examples of the 4-hydroxybenzoate decarboxylase gene derived from Bacillus subtilis subsp. spizizenii include the DNA consisting of the base sequence of SEQ ID NO: 47, examples of the 4-hydroxybenzoate decarboxylase gene derived from Citrobacter koseri include the DNA consisting of the base sequence of SEQ ID NO: 50, examples of the 4-hydroxybenzoate decarboxylase gene derived from Enterobacter aerogenes include the DNA consisting of the base sequence of SEQ ID NO: 53, examples of the 4-hydroxybenzoate decarboxylase gene derived from Enterobacter cloacae include the DNA consisting of the base sequence of SEQ ID NO: 56, examples of the 4-hydroxybenzoate decarboxylase gene derived from Enterobacter hormaechei include the DNA consisting of the base sequence of SEQ ID NO: 59, examples of the 4-hydroxybenzoate decarboxylase gene derived from Enterobacter sakazakii include the DNA consisting of the base sequence of SEQ ID NO: 62, examples of the 4-hydroxybenzoate decarboxylase gene derived from Escherichia coli include the DNA consisting of the base sequence of SEQ ID NO: 65, examples of the 4-hydroxybenzoate decarboxylase gene derived from Escherichia fergusonii include the DNA consisting of the base sequence of SEQ ID NO: 68, examples of the 4-hydroxybenzoate decarboxylase gene derived from Paenibacillus polymyxa include the DNA consisting of the base sequence of SEQ ID NO: 71, and examples of the 4-hydroxybenzoate decarboxylase gene derived from Pantoea ananatis include the DNA consisting of the base sequence of SEQ ID NO: 74.
In the present invention, a DNA which hybridizes to a DNA consisting of a complementary base sequence of the base sequence of SEQ ID NO: 37, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, or 74 under stringent conditions and which encodes a polypeptide having 4-hydroxybenzoate decarboxylase activity can also be used.
The 4-hydroxybenzoate decarboxylase activity can be measured by the method described in Genomics, 86, 342-351 (2005) “Materials and Methods”. Briefly, by adding a test enzyme to a liquid for testing, a reaction mixture containing 100 mM MES (pH 6.0), 1 mM DTT, 5 mM 4-hydroxybenzoate, and the enzyme is prepared, and then the slope of the absorbance at 270 nm (initial rate) is determined. The same measurement is performed using a system without the addition of 4-hydroxybenzoate to obtain a background value. The difference between the two measured values will be regarded as the 4-hydroxybenzoate decarboxylase activity.
In the present invention, a DNA consisting of a base sequence which has 90% or more, preferably 95% or more, more preferably 98% or more homology with the base sequence of SEQ ID NO: 37, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, or 74 and which encodes a polypeptide having 4-hydroxybenzoate decarboxylase activity can also be used.
The homologue of the DNA consisting of the base sequence of SEQ ID NO: 37, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, or 74 can be obtained by the method described above.
Construction of Vector for Transformation
The DNA which encodes chorismate-pyruvate lyase and the DNA which encodes 4-hydroxybenzoate decarboxylase are separately amplified by PCR and then cloned into a suitable vector which is replicable in a host.
The plasmid vector may be any plasmid vector as long as it comprises a gene responsible for autonomously replicating function in a coryneform bacterium. Specific examples of the plasmid vector include pAM330 derived from Brevibacterium lactofermentum 2256 (JP 58-67699 A; Miwa, K. et al., Cryptic plasmids in glutamic acid-producing bacteria. Agric. Biol. Chem. 48:2901-2903 (1984); and Yamaguchi, R. et al., Determination of the complete nucleotide sequence of the Brevibacterium lactofermentum plasmid pAM330 and the analysis of its genetic information. Nucleic Acids Symp. Ser. 16:265-267 (1985)); pHM1519 derived from Corynebacterium glutamicum ATCC13058 (Miwa, K. et al., Cryptic plasmids in glutamic acid-producing bacteria. Agric. Biol. Chem. 48:2901-2903 (1984)) and pCRY30 derived from the same (Kurusu, Y. et al., Identification of plasmid partition function in coryneform bacteria. Appl. Environ. Microbiol. 57:759-764 (1991)); pCG4 derived from Corynebacterium glutamicum T250 (JP 57-183799 A; and Katsumata, R. et al., Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J. Bacteriol., 159:306-311 (1984)), pAG1, pAG3, pAG14 and pAG50 derived from the same (JP 62-166890 A), and pEK0, pEC5 and pEKEx1 derived from the same (Eikmanns, B. J. et al., A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene, 102:93-98 (1991)); etc.
Examples of a preferred promoter include promoter PgapA as a promoter of the glyceraldehyde-3-phosphate dehydrogenase A gene (gapA), promoter Pmdh as a promoter of the malate dehydrogenase gene (mdh), and promoter PldhA as a promoter of lactate dehydrogenase A gene (ldhA), all of which are derived from Corynebacterium glutamicum R, and inter alia, PgapA is preferred.
Examples of a preferred terminator include terminator rrnB T1T2 of Escherichia coli rRNA operon, terminator trpA of Escherichia coli, and terminator trp of Brevibacterium lactofermentum, and inter alia, terminator rrnB T1T2 is preferred.
Transformation
As a method of transformation, any publicly known method can be used without limitation. Examples of such a known method include the calcium chloride/rubidium chloride method, the calcium phosphate method, DEAE-dextran transfection, and electroporation. Inter alia, preferred for a coryneform bacterium is electroporation, which can be performed by a known method (Kurusu, Y. et al., Electroporation-transformation system for Coryneform bacteria by auxotrophic complementation., Agric. Biol. Chem. 54:443-447 (1990); and Vertes A. A. et al., Presence of mrr- and mcr-like restriction systems in Coryneform bacteria. Res. Microbiol. 144:181-185 (1993)).
The transformant is cultured using a culture medium usually used for culture of microorganisms. The culture medium may be a natural or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, other nutritional substances, etc.
Examples of the carbon source include carbohydrates and sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol and glycerol; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid and gluconic acid; and alcohols such as ethanol and propanol. Hydrocarbons, such as normal paraffin, etc. may also be used as desired. These carbon sources may be used alone or as a mixture of two or more thereof. The concentration of these carbon sources in the culture medium is usually about 0.1 to 10 w/v %.
Examples of the nitrogen source include inorganic or organic ammonium compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate; urea; aqueous ammonia; sodium nitrate; and potassium nitrate. Nitrogen-containing organic compounds, such as corn steep liquor, meat extract, peptone, N—Z-amine, protein hydrolysate, amino acid, etc. may also be used. These nitrogen sources may be used alone or as a mixture of two or more thereof. The concentration of these nitrogen sources in the culture medium varies depending on the kind of the nitrogen compound, but is usually about 0.1 to 10 w/v %.
Examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride, iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts may be used alone or as a mixture of two or more thereof. The concentration of the inorganic salts in the culture medium varies depending on the kind of the inorganic salts, but is usually about 0.01 to 1 w/v %
Examples of the nutritional substances include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soybean hydrochloric acid hydrolysate, and extract from animals, plants or microorganisms, and degradation products thereof. The concentration of the nutritional substances in the culture medium varies depending on the kind of the nutritional substances, but is usually about 0.1 to 10 w/v %. Further, vitamins may be added as needed. Examples of the vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.
The pH of the culture medium is preferably about 5 to 8.
Examples of the preferable microbial culture medium include A medium (Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)), BT medium (Omumasaba, C. A. et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)), etc.
The culture temperature is about 15 to 45° C., and the culture period is about 1 to 7 days.
Disruption or Deletion in Host Chromosomal Gene
In the coryneform bacterium as a host, the gene which encodes an enzyme having 4-hydroxybenzoate hydroxylase activity (pobA) on the chromosome preferably has a disruption or deletion for further efficient phenol production. In addition, in the coryneform bacterium as a host, the gene which encodes an enzyme having phenol 2-monooxygenase activity (poxF) on the chromosome preferably has a disruption or deletion for further efficient phenol production.
Particularly preferred is that both of pobA and poxF have a disruption or deletion.
Replacement of a gene on the chromosome with the corresponding gene having an disruption or deletion can be achieved by creating a gene with deletion mutation for not allowing production of a normally functioning enzyme protein, and transforming a bacterium with a DNA comprising the mutated gene for recombination in which the gene on the chromosome and the mutated gene are exchanged. An enzyme protein encoded by a gene having a disruption or deletion, even when produced, has a conformation different from that of the wild type, and has no or reduced function. The gene deletion or gene disruption by way of gene replacement through such homologous recombination has already been established, and examples thereof include a method using a plasmid containing a temperature sensitive replication origin or a plasmid capable of conjugal transfer, and a method using a suicide vector not having a replication origin that works in a host (U.S. Pat. No. 6,303,383 and JP 05-007491 A).
Specifically, by the method described in Example 1, a coryneform bacterium in which the 4-hydroxybenzoate hydroxylase gene (pobA) is disrupted or deleted can be obtained. In addition, in a similar way, a coryneform bacterium in which the phenol 2-monooxygenase gene (poxF) is disrupted or deleted can be obtained.
High Expression of Metabolic Gene
It is preferred that the 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase gene (aroG) is expressed at a higher level in the coryneform bacterium as a host as compared with the original level in the host, i.e., the level in the wild type host. Such high expression is achieved by transformation via gene transfer or by increase in the number of copies of the desired gene(s) on the chromosome of the host.
Regarding the transformation, the DAHP synthetase gene may be the same or substantially same as that of the host, or of a different type. Preferably, the DAHP synthetase gene may be the same or substantially same as that of the host.
Examples of the DAHP synthetase gene derived from Corynebacterium glutamicum include the DNA consisting of the base sequence of SEQ ID NO: 28.
Examples of the DAHP synthetase gene derived from a different type of coryneform bacteria include a gene derived from Corynebacterium efficiens (SEQ ID NO: 120, DNA Data Bank of Japan: CE2073), a gene derived from Mycobacterium smegmatis (SEQ ID NO: 121, DNA Data Bank of Japan: MSMEG_4244), and a gene derived from Rhodococcus opacus (SEQ ID NO: 122, DNA Data Bank of Japan: ROP_08400).
Regarding the DAHP synthetase gene, examples of the “substantially same gene” include a DNA which encodes a polypeptide having 90% or more, preferably 95% or more, and more preferably 98% or more homology with the amino acid sequence of a polypeptide encoded by the gene, and having a DAHP synthetase activity. Regarding the DAHP synthetase gene, examples of the “substantially same gene” include a DNA which has 90% or more, preferably 95% or more, and more preferably 98% or more homology with the gene, and which encodes a polypeptide having a DAHP synthetase activity.
The DAHP synthetase activity can be determined by the reaction of phosphoenolpyruvic acid and erythrose-4-phosphate as substrates followed by quantification of produced 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) by a chromogenic method with the use of thiobarbituric acid (Appl. Environ. Microbiol., 74: 5497-5503 (2008)).
To increase the number of copies of the DAHP synthetase gene on the chromosome of the host, multiple copies of the gene may be transferred onto the chromosomal DNA. To transfer multiple copies of a gene onto the chromosomal DNA of a microorganism, homologous recombination (Experiments in Molecular Genetics, Cold Spring Harbor Lab. (1972)) may be performed using, as a target, a sequence that exists as multiple copies on the chromosomal DNA. As the sequence that exists as multiple copies on the chromosomal DNA, a repetitive DNA or an inverted repeat that exists at the end of a transposon may be used. Also, as disclosed in JP 02-109985 A, it is feasible to transfer multiple copies of the desired gene with a transposon onto the chromosomal DNA. Alternatively, by a method using Mu phage (JP 02-109985 A), the desired gene may be transferred onto a host chromosome.
Substitution of an expression control sequence, such as a promoter, of the DAHP synthetase gene with a stronger one can also increase the expression of such a gene. For example, a tac promoter, a lac promoter, a trc promoter, a trp promoter, etc. are known as a strong promoter. Further, as disclosed in WO00/18935, it is also feasible to alter a promoter to a stronger one by substitution of a few bases in the promoter region of the gene. Examples of the evaluation method of the strength of a promoter and examples of such a promoter are described in a paper by Goldstein et al. “Prokaryotic promoters in biotechnology”. Biotechnol. Annu. Rev., 1995, 1, 105-128, etc. Substitution of an expression control sequence can be performed in a similar way to the gene substitution with the use of a temperature sensitive plasmid, for example.
Further, it is known that substitution of a spacer between a ribosomal binding site (RBS) and an initiator codon, in particular substitution of a few nucleotides in a sequence immediately upstream of the initiator codon has a great influence on the efficiency of mRNA translation. Therefore, the alteration thereof can improve the amount of translation.
Examples of the method for the above-mentioned gene substitution include a method using a plasmid containing a temperature sensitive replication origin or a plasmid capable of conjugal transfer, and a method using a suicide vector not having a replication origin that works in a host (U.S. Pat. No. 6,303,383 and JP 05-007491 A).
(II) Process for Producing Phenol
Phenol can be produced by a process comprising a step of allowing the above-described transformant of the present invention to react in a reaction mixture containing a saccharide, and a step of collecting phenol from the reaction mixture.
Proliferation of Microorganism
Before the reaction, the transformant is preferably cultured and proliferated under aerobic conditions at about 25 to 38° C. for about 12 to 48 hours.
The culture medium used for aerobic culture of the transformant before the reaction may be a natural or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, other nutritional substances, etc.
Examples of the carbon source that can be used include saccharides (monosaccharides such as glucose, fructose, mannose, xylose, arabinose, and galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, and trehalose; polysaccharides such as starch; and molasses); sugar alcohols such as mannitol, sorbitol, xylitol, and glycerol; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid and gluconic acid; alcohols such as ethanol and propanol; and hydrocarbons such as normal paraffin.
These carbon sources may be used alone or as a mixture of two or more thereof.
Examples of the nitrogen source that can be used include inorganic or organic ammonium compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate; urea; aqueous ammonia; sodium nitrate; and potassium nitrate. Nitrogen-containing organic compounds, such as corn steep liquor, meat extract, peptone, N—Z-amine, protein hydrolysate, amino acid, etc. may also be used. These nitrogen sources may be used alone or as a mixture of two or more thereof. The concentration of these nitrogen sources in the culture medium varies depending on the kind of the nitrogen compound, but is usually about 0.1 to 10 w/v %.
Examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride, iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts may be used alone or as a mixture of two or more thereof. The concentration of the inorganic salts in the culture medium varies depending on the kind of the inorganic salts, but is usually about 0.01 to 1 w/v %
Examples of the nutritional substances include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soybean hydrochloric acid hydrolysate, and extract from animals, plants or microorganisms, and degradation products thereof. The concentration of the nutritional substances in the culture medium varies depending on the kind of the nutritional substances, but is usually about 0.1 to 10 w/v %.
Further, vitamins may be added as needed. Examples of the vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.
The pH of the culture medium is preferably about 6 to 8.
Specific examples of the preferable culture medium for coryneform bacteria include A medium (Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)), BT medium (Omumasaba, C. A. et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)), etc. Such a culture medium can be used after prepared so as to contain a saccharide at a concentration in the above-mentioned range.
Reaction Mixture
The reaction mixture may be a natural or synthetic medium containing a carbon source, a nitrogen source, inorganic salts, other nutritional substances, etc.
As the carbon source, a saccharide is used. Examples of the saccharide include monosaccharides such as glucose, fructose, mannose, xylose, arabinose, and galactose; disaccharides such as sucrose, maltose, lactose, cellobiose, xylobiose, and trehalose; polysaccharides such as starch; and molasses. Inter alia, a monosaccharide is preferred, and glucose is more preferred.
As the carbon source, besides saccharides, sugar alcohols such as mannitol, sorbitol, xylitol, and glycerol; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid and gluconic acid; alcohols such as ethanol and propanol; and hydrocarbons such as normal paraffin can also be used.
These carbon sources may be used alone or as a mixture of two or more thereof.
The concentration of the saccharide in the reaction mixture is preferably about 1 to 20 w/v %, more preferably about 2 to 10 w/v %, and still more preferably about 2 to 5 w/v %.
The total concentration of the carbon sources including the saccharide in the reaction mixture is usually about 2 to 5 w/v %.
Examples of the nitrogen source that can be used include inorganic or organic ammonium compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate; urea; aqueous ammonia; sodium nitrate; and potassium nitrate. Nitrogen-containing organic compounds, such as corn steep liquor, meat extract, peptone, N—Z-amine, protein hydrolysate, amino acid, etc. may also be used. These nitrogen sources may be used alone or as a mixture of two or more thereof. The concentration of these nitrogen sources in the reaction mixture varies depending on the kind of the nitrogen compound, but is usually about 0.1 to 10 w/v %.
Examples of the inorganic salts include potassium dihydrogen phosphate, dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride, iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts may be used alone or as a mixture of two or more thereof. The concentration of the inorganic salts in the reaction mixture varies depending on the kind of the inorganic salts, but is usually about 0.01 to 1 w/v %
Examples of the nutritional substances include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soybean hydrochloric acid hydrolysate, and extract from animals, plants or microorganisms, and degradation products thereof. The concentration of the nutritional substances in the reaction mixture varies depending on the kind of the nutritional substances, but is usually about 0.1 to 10 w/v %.
Further, vitamins may be added as needed. Examples of the vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc.
The pH of the reaction mixture is preferably about 6 to 8.
Specific examples of the preferable culture medium for coryneform bacteria include A medium and BT medium as described above. Such a culture medium can be used after prepared so as to contain a saccharide at a concentration in the above-mentioned range.
Reaction Conditions
The reaction temperature, that is, the temperature for keeping the transformant alive during the reaction is preferably about 20 to 50° C., and more preferably about 25 to 40° C. When the temperature is in the above range, phenol can be efficiently produced.
The reaction period is preferably about 1 to 7 days, and more preferably about 1 to 3 days.
The culture may be a batch process, a fed-batch process, or a continuous process. Inter alia, a batch process is preferred.
<Reducing Conditions>
The reaction may be performed under aerobic conditions or reducing conditions, but preferably is performed under reducing conditions. Under reducing conditions, coryneform bacteria do not substantially proliferate and can further efficiently produce phenol.
The “reducing conditions” is defined based on the oxidation-reduction potential of the reaction mixture. The oxidation-reduction potential of the reaction mixture is preferably about −200 mV to −500 mV, and more preferably about −250 mV to −500 mV.
The reducing conditions of the reaction mixture can be simply estimated with the use of resazurin indicator (in reducing conditions, decolorization from blue to colorless is observed). However, for precise measurement, a redox-potential meter (for example, ORP Electrodes made by BROADLEY JAMES) is used.
As a method of preparing a reaction mixture under reducing conditions, any publicly known method can be used without limitation. For example, as a liquid medium for preparation of the reaction mixture, an aqueous solution for a reaction mixture may be used instead of distillated water or the like. As reference for preparation of the aqueous solution for a reaction mixture, for example, the method for preparing a culture medium for strictly anaerobic microorganisms, such as sulfate-reducing microorganisms (Pfennig, N. et al.: The dissimilatory sulfate-reducing bacteria, In The Prokaryotes, A Handbook on Habitats, Isolation and Identification of Bacteria, Ed. by Starr, M. P. et al. Berlin, Springer Verlag, 926-940, 1981, or Nogeikagaku Jikkensho, Ed. by Kyoto Daigaku Nogakubu Nogeikagaku Kyoshitsu, Vol. 3, Sangyo Tosho, 1990, Issue 26) may be used, and such a method provides an aqueous solution under desired reducing conditions.
Specifically, by treating distillated water or the like with heat or under reduced pressure for removal of dissolved gases, an aqueous solution for a reaction mixture under reducing conditions can be obtained. In this case, for removal of dissolved gases, especially dissolved oxygen, distillated water or the like may be treated under reduced pressure of about 10 mmHg or less, preferably about 5 mmHg or less, more preferably about 3 mmHg or less, for about 1 to 60 minutes, preferably for about 5 to 40 minutes.
Alternatively, by adding a suitable reducing agent (for example, thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoacetic acid, thiol acetic acid, glutathione, sodium sulfide, etc.), an aqueous solution for a reaction mixture under reducing conditions can be prepared.
These methods may be suitably combined to prepare an effective aqueous solution for a reaction mixture under reducing conditions.
It is preferred to maintain the reducing conditions of the reaction mixture during the reaction. For maintenance of reducing conditions, it is preferred that oxygen from the outside of the reaction system is prevented to the utmost extent from entering the system. Specific examples of the method employed for this purpose include a method comprising encapsulating the reaction system with inert gas, such as nitrogen gas, carbon dioxide gas, etc. In some cases, for allowing the metabolic functions in the cells of the aerobic bacterium of the present invention to work effectively during the reaction, addition of a solution of various nutrients or a reagent solution for adjusting and maintaining the pH of the reaction system may be needed. In such a case, for more effective prevention of oxygen incorporation, it is effective to remove oxygen in the solutions to be added, in advance.
Collection of Phenol
Through the culture performed in the above manner, phenol is produced in the reaction mixture. Phenol can be collected by collecting the reaction mixture, and it is also feasible to isolate phenol from the reaction mixture by a known method. Examples of such a known method include distillation, the membrane permeation method, and the organic solvent extraction method.
EXAMPLES
Example 1
Cloning and Expression of Phenol-Producing Genes
(1) Extraction of Chromosomal DNA from Microorganisms
To extract chromosomal DNA from Corynebacterium glutamicum R (FERM P-18976), the bacterium was inoculated, with the use of a platinum loop, into A medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, and 7 g of vitamin assay casamino acid were dissolved in 1 L of distilled water), which was supplemented with 50% (w/v) glucose as a carbon source to a final concentration of 4%, and cultured with shaking at 33° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Escherichia coli (K-12 MG1655), the bacterium was inoculated into LB Medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Pseudomonas putida (KT2440) ATCC 47054, the bacterium was inoculated into LB Medium (10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Acinetobacter baumannii (JCM 6841), the bacterium was inoculated into JCM Medium No. 12 (5 g of peptone, 3 g of beef extract, and 5 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Azotobacter vinelandii (ATCC 9104), the bacterium was inoculated into NBRC Medium No. 805 (1 g of east extract, 5 g of mannitol, 0.7 g of K2HPO4, 0.1 g of KH2PO4, 0.2 g of MgSO4.7H2O, and 1 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Chromohalobacter salexigens (ATCC BAA-138), the bacterium was inoculated into Nutrient Broth with NaCl Medium (8 g of Nutrient Broth (made by Becton, Dickinson and Company, catalog No. 234000) and 100 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Citrobacter youngae (ATCC 29220), the bacterium was inoculated into Nutrient Broth Medium (8 g of Nutrient Broth (made by Becton, Dickinson and Company, catalog No. 234000) was dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
The chromosomal DNA of Marinobacter aquaeolei ATCC 700491 (catalog No. 700491D-5) was obtained from American Type Culture Collection (ATCC).
To extract chromosomal DNA from Marinomonas mediterranea (NBRC 103028), the bacterium was inoculated into NBRC Medium No. 340 (37.4 g of Bacto Marine Broth 2216 (made by Becton, Dickinson and Company, catalog No. 279110) was dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 25° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Pseudoalteromonas haloplanktis (NBRC 102225), the bacterium was inoculated into NBRC Medium No. 340 (37.4 g of Bacto Marine Broth 2216 (made by Becton, Dickinson and Company, catalog No. 279110) was dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 25° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Ralstonia eutropha (IAM 12368), the bacterium was inoculated into Nutrient Broth Medium (8 g of Nutrient Broth (made by Becton, Dickinson and Company, catalog No. 234000) was dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 26° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Shewanella putrefaciens (JCM 20190), the bacterium was inoculated into Nutrient Broth Medium (8 g of Nutrient Broth (made by Becton, Dickinson and Company, catalog No. 234000) was dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 25° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Thiobacillus denitrificans (ATCC 25259, JCM 20190), the bacterium was inoculated into JCM Medium No. 91 (5 g of KNO3 and 0.5 g of NaHCO3 were dissolved in 1 L of S6 medium (1.8 g of KH2PO4, 1.2 g of Na2HPO4, 0.1 g of (NH4)2SO4, 0.1 g of MgSO4.7H2O, 30 mg of FeCl3.6H2O, 30 mg of MnSO4.xH2O, 40 mg of CaCl2.2H2O, and 100 mL of 10% Na2S2O3 solution were dissolved in 900 mL of distilled water)) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Bacillus subtilis NBRC 14144, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Bacillus atrophaeus JCM 9070, the bacterium was inoculated into JCM Medium No. 22 (10 g of polypeptone, 10 g of beef extract, and 5 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Bacillus subtilis subsp. spizizenii NBRC 101239, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
The chromosomal DNA of Citrobacter koseri ATCC BAA-895 (catalog No. BAA-895D-5) was obtained from American Type Culture Collection (ATCC).
To extract chromosomal DNA from Enterobacter aerogenes NBRC 13534, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Enterobacter cloacae NBRC 13535, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 37° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Enterobacter hormaechei ATCC 49162, the bacterium was inoculated into Tryptic Soy Broth Medium (30 g of Tryptic Soy Broth (made by Becton Dickinson and Company, catalog No. 211825) was dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
The chromosomal DNA of Enterobacter sakazakii ATCC BAA-894 (catalog No. BAA-894D-5) was obtained from American Type Culture Collection (ATCC).
To extract chromosomal DNA from Escherichia coli W NBRC 13500, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Escherichia fergusonii NBRC 102419, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Paenibacillus polymyxa NBRC 15309, the bacterium was inoculated into NBRC Medium No. 802 (10 g of polypeptone, 2 g of yeast extract, and 1 g of MgSO4.7H2O were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
To extract chromosomal DNA from Pantoea ananatis LMG 20103, the bacterium was inoculated into BCCM/LMG BateriCulture Medium No. 1 (1 g of beef extract, 2 g of yeast extract, 5 g of peptone, and 5 g of NaCl were dissolved in 1 L of distilled water) with the use of a platinum loop, and cultured with shaking at 30° C. until the logarithmic growth phase. After the bacterial cells were collected, chromosomal DNA was recovered from the collected cells with the use of a DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNA Isolation Kit, made by Amersham) according to the instruction manual.
(2) Construction of Cloning Vectors Construction of Cloning Vector pCRB22
A DNA fragment comprising a DNA replication origin sequence of pCASE1, a plasmid derived from Corynebacterium casei JCM12072 (hereinafter abbreviated as pCASE1-ori) and a DNA fragment comprising a cloning vector pHSG298 (made by Takara Bio, Inc.) were amplified by the following PCR method.
In the PCR, the following sets of primers were synthesized based on SEQ ID NO: 1 (pCASE1-ori sequence) and SEQ ID NO: 2 (cloning vector pHSG298) for cloning of the pCASE1-ori sequence and the cloning vector pHSG298, and were used.
Primers for pCASE1-Ori Sequence Amplification









(SEQ ID NO: 3)



(a-1); 5′-AT AGATCT AGAACGTCCGTAGGAGC-3′



 



(SEQ ID NO: 4)



(b-1); 5′-AT AGATCT GACTTGGTTACGATGGAC-3′






Primers (a-1) and (b-1) each have a BglII restriction enzyme site added thereto.
Primers for Cloning Vector pHSG298 Amplification









(SEQ ID NO: 5)



(a-2): 5′-AT AGATCT AGGTTTCCCGACTGGAAAG-3′



 



(SEQ ID NO: 6)



(b-2): 5′-AT AGATCT CGTGCCAGCTGCATTAATGA-3′






Primers (a-2) and (b-2) each have a BglII restriction enzyme site added thereto.
As the template DNA, total DNA extracted from Corynebacterium casei JCM12072 obtained from Japan Collection of Microorganisms (JCM) and cloning vector pHSG298 (made by Takara Bio, Inc.) were used.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the pCASE1-ori sequence, a combination of primers (a-1) and (b-1), and for amplification of the cloning vector pHSG298, a combination of primers (a-2) and (b-2) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C.

    
    
        pCASE1-ori sequence: 150 seconds
        Cloning vector pHSG298: 180 seconds
    
    


A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed. In the case of the pCASE1-ori sequence, an about 1.4-kb DNA fragment was detected. In the case of the cloning vector pHSG298, an about 2.7-kb DNA fragment was detected.
10 μL of the about 1.4-kb DNA fragment comprising the pCASE1-ori sequence derived from Corynebacterium casei, and 10 μL of the about 2.7-kb DNA fragment comprising the cloning vector pHSG298, both amplified by the above PCR, were each cut with the use of restriction enzyme BglII and processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid A.
With the use of the Ligation Liquid A, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzyme BglII to confirm the inserted fragment. As a result, in addition to an about 2.7-kb DNA fragment of the cloning vector pHSG298, an about 1.4-kb DNA fragment of the pCASE-ori sequence was confirmed.
The cloning vector comprising the pCASE1-ori sequence was named pCRB22.
Construction of Cloning Vector pCRB11
A DNA fragment comprising a DNA replication origin sequence of pCG1 (JP 57-134500 A), which is a plasmid replicable in Corynebacterium glutamicum (hereinafter abbreviated as pCG1-ori) and a DNA fragment comprising a cloning vector pHSG398 (made by Takara Bio, Inc.) were amplified by the following PCR method.
In the PCR, the following sets of primers were synthesized based on SEQ ID NO: 7 (pCG1-ori sequence) and SEQ ID NO: 8 (cloning vector pHSG398) for cloning of the pCG1-ori sequence and the cloning vector pHSG398, and were used.
Primers for pCG1-Ori Sequence Amplification









(SEQ ID NO: 9)



(a-3): 5′-AT AGATCT AGCATGGTCGTCACAGAG-3′



 



(SEQ ID NO: 10)



(b-3): 5′-AT AGATCT GGAACCGTTATCTGCCTATG-3′






Primers (a-3) and (b-3) each have a BglII restriction enzyme site added thereto.
Primers for Cloning Vector pHSG398 Amplification









(SEQ ID NO: 11)



(a-4): 5′-AT AGATCT GTCGAACGGAAGATCACTTC-3′



 



(SEQ ID NO: 12)



(b-4): 5′-AT AGATCT AGTTCCACTGAGCGTCAG-3′






Primers (a-4) and (b-4) each have a BglII restriction enzyme site added thereto.
As the template DNA, pCG1 (JP 57-134500 A) and cloning vector pHSG398 (made by Takara Bio, Inc.) were used.
Actual PCR was performed with use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water:
25.5
μL





The above ingredients were mixed, and 50 μL, of the reaction mixture was subjected to PCR.


*)For amplification of the pCG1-ori sequence, a combination of primers (a-3) and (b-3), and for amplification of the cloning vector pHSG398, a combination of primers (a-4) and (b-4) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C.

    
    
        pCG1-ori sequence: 120 seconds
        Cloning vector pHSG398: 150 seconds
    
    


A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed. In the case of the pCG1-ori sequence, an about 1.9-kb DNA fragment was detected. In the case of the cloning vector pHSG398, an about 2.2-kb DNA fragment was detected.
10 μL of the about 1.9-kb DNA fragment comprising the pCG1-ori gene, which is derived from a plasmid pCG1, and 10 μL of the about 2.2-kb DNA fragment comprising the cloning vector pHSG398, both amplified by the above PCR, were each cut with use of a restriction enzyme BglII and processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total amount was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid B.
With use of the Ligation Liquid B, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% of polypeptone, 0.5% of yeast extract, 0.5% of sodium chloride, and 1.5% of agar) containing 50 μg/mL of chloramphenicol.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture medium and cut with use of a restriction enzyme BglII to confirm the inserted fragment. As a result, in addition to an about 2.2-kb DNA fragment of the cloning vector pHSG398, an about 1.9-kb DNA fragment of pCG1-ori sequence was confirmed.
The plasmid comprising the pCG1-ori sequence was named pCRB11.
Construction of Cloning Vector pCRB15
A DNA fragment comprising a cloning vector pCRB11 and a DNA fragment comprising a zeocin resistance gene derived from pSELECT-zeo-mcs (made by Invitrogen Corp.) were amplified by the following PCR method.
In the PCR, the following sets of primers were synthesized based on SEQ ID NO: 13 (pCRB11) and SEQ ID NO: 14 (zeocin resistance gene) for cloning of the cloning vector pCRB11 and the zeocin resistance gene, and were used.
Primers for Cloning Vector pCRB11 Amplification









(SEQ ID NO: 15)



(a-5): 5′-AT GATATC CGAAGTGATCTTCCGTTCGA-3′



 



(SEQ ID NO: 16)



(b-5): 5′-AT GATATC AAGGCAGTTATTGGTGCCCT-3′






Primers (a-5) and (b-5) each have an EcoRV restriction enzyme site added thereto.
Primers for Zeocin Resistance Gene Amplification









(SEQ ID NO: 17)



(a-6): 5′-AT GATATC TAGCTTATCCTCAGTCCTGC-3′



 



(SEQ ID NO: 18)



(b-6): 5′-AT GATATC CCATCCACGCTGTTTTGACA-3′






Primers (a-6) and (b-6) each have an EcoRV restriction enzyme site added thereto.
As the template DNA, cloning vector pCRB11 and pSELECT-zeo-mcs (made by Invitrogen Corp.) were used.
Actual PCR was performed with use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Tag (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water:
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the cloning vector pCRB11 sequence, a combination of primers (a-5) and (b-5), and for amplification of the zeocin resistance gene, a combination of primers (a-6) and (b-6) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C.

    
    
        pCRB11 sequence: 200 seconds
        Zeocin resistance gene: 45 seconds
    
    


A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed. In the case of the cloning vector pCRB11 sequence, an about 3.3-kb DNA fragment was detected. In the case of the zeocin resistance gene, an about 0.5-kb DNA fragment was detected.
10 μL of the about 3.3-kb DNA fragment comprising the cloning vector pCRB11 and 10 μL of the about 0.5-kb DNA fragment comprising the zeocin resistance gene derived from a plasmid pSELECT-zeo-mcs, both amplified by the above PCR, were each cut with use of a restriction enzyme EcoRV and processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total amount was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid C.
Construction of Cloning Vector pCRB207
A DNA fragment comprising a promoter sequence of the gapA gene encoding the glyceraldehyde-3-phosphate dehydrogenase (hereinafter abbreviated as PgapA) derived from Corynebacterium glutamicum R, and a DNA fragment comprising an rrnBT1T2 bidirectional terminator sequence (hereinafter abbreviated as terminator sequence) derived from a cloning vector pKK223-3 (made by Pharmacia) were amplified by the following method.
In the PCR, the following sets of primers were synthesized based on SEQ ID NO: 19 (PgapA sequence) and SEQ ID NO: 20 (terminator sequence) for cloning of the PgapA sequence and the terminator sequence, and were used.
Primers for PgapA Sequence Amplification









(a-7);



(SEQ ID NO: 21)



5′-CTCT GTCGAC CCGAAGATCTGAAGATTCCTG-3′



 



(b-7);



(SEQ ID NO: 22)



5′-CTCT GTCGAC GGATCC CCATGG



 



TGTGTCTCCTCTAAAGATTGTAGG-3′






Primer (a-7) has a SalI restriction enzyme site added thereto, and primer (b-7) has SalI, BamHI, and NcoI restriction enzyme sites added thereto.
Primers for Terminator Sequence Amplification









(a-8);



(SEQ ID NO: 23)



5′-CTCT GCATGC CCATGG CTGTTTTGGCGGATGAGAGA-3′



 



(b-8);



(SEQ ID NO: 24)



5′-CTCT GCATGC TCATGA



 



AAGAGTTTGTAGAAACGCAAAAAGG-3′






Primer (a-8) has SphI and NcoI restriction enzyme sites added thereto, and primer (b-8) has SphI and BspHI restriction enzyme sites added thereto.
As the template DNA, the chromosomal DNA extracted from Corynebacterium glutamicum R (FERM P-18976) and the plasmid pKK223-3 (made by Pharmacia) were used.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the PgapA sequence, a combination of primers (a-7) and (b-7), and for amplification of the terminator sequence, a combination of primers (a-8) and (b-8) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C.

    
    
        PgapA sequence: 45 seconds
        Terminator sequence: 30 seconds
    
    


A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed. In the case of the PgapA sequence, an about 0.6-kb DNA fragment was detected. In the case of the terminator sequence, an about 0.4-kb DNA fragment was detected.
10 μL of the about 0.6-kb DNA fragment comprising the PgapA sequence derived from Corynebacterium glutamicum R, which was amplified by the above PCR, and the about 4.1-kb cloning vector pCRB22 were each cut with the use of restriction enzyme SalI and processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid D.
With the use of the Ligation Liquid D, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzyme SalI to confirm the inserted fragment. As a result, in addition to an about 4.1-kb DNA fragment of the cloning vector pCRB22, an about 0.6-kb DNA fragment of the PgapA sequence was confirmed.
The cloning vector comprising the PgapA sequence was named pCRB206.
10 μL of the about 0.4-kb DNA fragment comprising the terminator sequence derived from the plasmid pKK223-3, which was amplified by the above PCR, was cut with the use of restriction enzymes NcoI and BspHI, 2 μL of the above cloning vector pCRB206 was cut with the use of restriction enzyme NcoI, and both were processed at 70° C. for 10 minutes for deactivation of the restriction enzymes. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid E.
With the use of the Ligation Liquid E, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of the restriction enzyme to confirm the inserted fragment. As a result, in addition to an about 4.7-kb DNA fragment of the cloning vector pCRB206, an about 0.4-kb DNA fragment of the terminator sequence was confirmed.
The cloning vector comprising the rrnBT1T2 terminator sequence was named pCRB207.
Construction of Cloning Vector pCRB209
A DNA fragment comprising a promoter sequence of the gapA (glyceraldehyde 3-phosphate dehydrogenase A) gene (hereinafter abbreviated as PgapA) derived from Corynebacterium glutamicum R was amplified by the following method.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 25 (pCRB207) for cloning of the pCRB207 sequence, and was used.
Primers for pCRB207 Sequence Amplification







(SEQ ID NO: 26)


(a-9); 5′-CTCT CATATG CTGTTTTGGCGGATGAGAG-3′


 


(SEQ ID NO: 27)


(b-9); 5′-CTCT CATATG GTGTCTCCTCTAAAGATTGTAGG-3′






Primers (a-9) and (b-9) each have an NdeI restriction enzyme site added thereto.
As the template DNA, the cloning vector pCRB207 comprising a gapA promoter and a rrnBT1T2 terminator sequence was used.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara SHUZO) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the pCRB207 sequence, a combination of primers (a-9) and (b-9) was used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C., 307 seconds
A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed, and an about 5.1-kb DNA fragment comprising the cloning vector pCRB207 was detected.
10 μL of the about 5.1-kb DNA fragment comprising the gene derived from pCRB207, which was amplified by the above PCR, was cut with the use of restriction enzyme NdeI and processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. To this, 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara SHUZO) were added. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid F.
With the use of the Ligation Liquid F, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzyme NdeI to confirm the inserted restriction enzyme site.
The cloning vector comprising the PgapA sequence and the rrnBT1T2 terminator sequence was named pCRB209.
(3) Cloning of Phenol-Producing Genes
(3-1) DAHP Synthetase Gene (aroG)
Cloning of Phenol-Producing Gene Derived from Corynebacterium glutamicum 
A DNA fragment comprising the aroG gene which encodes DAHP synthetase derived from Corynebacterium glutamicum was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 28 (the aroG gene of Corynebacterium glutamicum) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the aroG gene and was used.
Primers for aroG Gene Amplification









(a-10);



(SEQ ID NO: 29)



5′-CTCT CATATG AATAGGGGTGTGAGTTGG-3′



 



(b-10);



(SEQ ID NO: 30)



5′-CTCT CATATG TTAATTACGCAGCATTTCTGCAACG-3′






Primers (a-10) and (b-10) each have an NdeI restriction enzyme site added thereto.
(3-2) Chorismate-Pyruvate Lyase Gene (ubiC)
Cloning of Phenol-Producing Gene Derived from Escherichia coli 
A DNA fragment comprising the ubiC gene which is derived from Escherichia coli and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 31 (the ubiC gene of Escherichia coli) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(SEQ ID NO: 32)



(a-11); 5′-CTCT CATATG TCACACCCCGCGTTAA-3′



 



(SEQ ID NO: 33)



(b-11); 5′-CTCT CATATG TTAGTACAACGGTGACGCC-3′






Primers (a-11) and (b-11) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Pseudomonas putida 
A DNA fragment comprising the ubiC gene which is derived from Pseudomonas putida and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 34 (the ubiC gene of Pseudomonas putida) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-12);



(SEQ ID NO: 35)



5′-CTCT CATATG TCGTACGAATCCCCG-3′



 



(b-12);



(SEQ ID NO: 36)



5′-CTCT CATATG TCAGCGGTTTTCCTCCTTG-3′






Primers (a-12) and (b-12) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Acinetobacter baumannii 
A DNA fragment comprising the ubiC gene which is derived from Acinetobacter baumannii and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 81 (the ubiC gene of Acinetobacter baumannii) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-29);



(SEQ ID NO: 94)



5′-CTCT CATATG CGTAAACGACAACCAGTAC-3′



 



(b-29);



(SEQ ID NO: 95)



5′-CTCT CATATG TCATAGTAATTCCTTGTCGTGCTG-3′






Primers (a-29) and (b-29) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Azotobacter vinelandii 
A DNA fragment comprising the ubiC gene which is derived from Azotobacter vinelandii and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 82 (the ubiC gene of Azotobacter vinelandii) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-30);



(SEQ ID NO: 96)



5′-CTCT CATATG ACCGCTGCTCCCG-3′



 



(b-30);



(SEQ ID NO: 97)



5′-CTCT CATATG TTATAGGGTGTCCGGGTC-3′






Primers (a-30) and (b-30) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Chromohalobacter salexigens 
A DNA fragment comprising the ubiC gene which is derived from Chromohalobacter salexigens and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 83 (the ubiC gene of Chromohalobacter salexigens) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-31);



(SEQ ID NO: 98)



5′-CTCT CATATG TCTCCTGACCGCTTC-3′



 



(b-31);



(SEQ ID NO: 99)



5′-CTCT CATATG TTAGCGCGATGGCAGCG-3′






Primers (a-31) and (b-31) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Citrobacter koseri 
A DNA fragment comprising the ubiC gene which is derived from Citrobacter koseri and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 84 (the ubiC gene of Citrobacter koseri) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-32);



(SEQ ID NO: 100)



5′-CTCT CATATG TCACACCCTGCGTTAAC-3′



 



(b-32);



(SEQ ID NO: 101)



5′-CTCT CATATG TTAATACAACGGTGATGCGGG-3′






Primers (a-32) and (b-32) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Citrobacter youngae 
A DNA fragment comprising the ubiC gene which is derived from Citrobacter youngae and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 85 (the ubiC gene of Citrobacter youngae) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-33);



(SEQ ID NO: 102)



5′-CTCT CATATG CCACACCCTGCGTTAA-3′



 



(b-33);



(SEQ ID NO: 103)



5′-CTCT CATATG TCAGTACAACGGCGATGCA-3′






Primers (a-33) and (b-33) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Enterobacter cloacae 
A DNA fragment comprising the ubiC gene which is derived from Enterobacter cloacae and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 86 (the ubiC gene of Enterobacter cloacae) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-34);



(SEQ ID NO: 104)



5′-CTCT CATATG TCACACCCTGCGCTAA-3′



 



(b-34);



(SEQ ID NO: 105)



5′-CTCT CATATG TCAGTACAACGGCGATGC-3′






Primers (a-34) and (b-34) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Marinobacter aquaeolei 
A DNA fragment comprising the ubiC gene which is derived from Marinobacter aquaeolei and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 87 (the ubiC gene of Marinobacter aquaeolei) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-35);



(SEQ ID NO: 106)



5′-CTCT CATATG CCGTTAAAGGACTGTGAC-3′



 



(b-35);



(SEQ ID NO: 107)



5′-CTCT CATATG TTAACCCCGGTTGGGC-3′






Primers (a-35) and (b-35) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Marinomonas mediterranea 
A DNA fragment comprising the ubiC gene which is derived from Marinomonas mediterranea and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 88 (the ubiC gene of Marinomonas mediterranea) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-36);



(SEQ ID NO: 108)



5′-CTCT CATATG ACGTTACTCAATAAAAACGCTG-3′ 



 



(b-36);



(SEQ ID NO: 109)



5′-CTCT CATATG CTACAGCTGGCCTATGGTA-3′






Primers (a-36) and (b-36) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Pantoea ananatis 
A DNA fragment comprising the ubiC gene which is derived from Pantoea ananatis and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 89 (the ubiC gene of Pantoea ananatis) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-37);



(SEQ ID NO: 110)



5′-CTCT CATATG ACGCAAGACCCGCT-3′



 



(b-37);



(SEQ ID NO: 111)



5′-CTCT CATATG TTAACCTTGATCACGATAGAGCG-3′






Primers (a-37) and (b-37) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Pseudoalteromonas haloplanktis 
A DNA fragment comprising the ubiC gene which is derived from Pseudoalteromonas haloplanktis and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 90 (the ubiC gene of Pseudoalteromonas haloplanktis) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-38);



(SEQ ID NO: 112)



5′-CTCT CATATG ATTACTTTCCCTGTTTCATTATCTGC-3′



 



(b-38);



(SEQ ID NO: 113)



5′-CTCT CATATG TCATGAGTACAAATACGCTCCTG-3′






Primers (a-38) and (b-38) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Ralstonia eutropha 
A DNA fragment comprising the ubiC gene which is derived from Ralstonia eutropha and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 91 (the ubiC gene of Ralstonia eutropha) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-39);



(SEQ ID NO: 114)



5′-CTCT CATATG AGCGCGCAGTCCG-3′



 



(b-39);



(SEQ ID NO: 115)



5′-CTCT CATATG TCATCTCGTGGTCTCTTTCTTG-3′






Primers (a-39) and (b-39) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Shewanella putrefaciens 
A DNA fragment comprising the ubiC gene which is derived from Shewanella putrefaciens and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 92 (the ubiC gene of Shewanella putrefaciens) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-40);



(SEQ ID NO: 116)



5′-CTCT CATATG AATGTGACTAGCTTAAGCTTCC-3′



 



(b-40);



(SEQ ID NO: 117)



5′-CTCT CATATG TCACTGGCAAATTGCTCGC-3′






Primers (a-40) and (b-40) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Thiobacillus denitrificans 
A DNA fragment comprising the ubiC gene which is derived from Thiobacillus denitrificans and which encodes a gene having chorismate-pyruvate lyase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 93 (the ubiC gene of Thiobacillus denitrificans) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the ubiC gene, and was used.
Primers for ubiC Gene Amplification









(a-41);



(SEQ ID NO: 118)



5′-CTCT CATATG ATCGCCACGCGCG-3′



 



(b-41);



(SEQ ID NO: 119)



5′-CTCT CATATG TCATGGCGTTAATAGGGCG-3′






Primers (a-41) and (b-41) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Bacillus subtilis 
A DNA fragment comprising the bsdBCD gene which is derived from Bacillus subtilis and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 37 (the bsdBCD gene of Bacillus subtilis) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the bsdBCD gene, and was used.
Primers for bsdBCD Gene Amplification









(a-13);



(SEQ ID NO: 38)



5′-CTCT CATATG AAAGCAGAATTCAAGCGTAAAG-3′



 



(b-13);



(SEQ ID NO: 39)



5′-CTCT CATATG GATCAAGCCTTTCGTTCCG-3′






Primers (a-13) and (b-13) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Bacillus atrophaeus 
A DNA fragment comprising the dca gene which is derived from Bacillus atrophaeus and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 44 (the dca gene of Bacillus atrophaeus) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 45)



(a-16); 5′-CTCT CATATG AAACTCGTTGTCGGGATG-3′



 



(SEQ ID NO: 46)



(b-16); 5′-CTCT CATATG TCAGGCCTTTCTTTCC-3′






Primers (a-16) and (b-16) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Bacillus subtilis subsp. spizizenii 
A DNA fragment comprising the dca gene which is derived from Bacillus subtilis subsp. spizizenii and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 47 (the dca gene of Bacillus subtilis subsp. spizizenii) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification







(SEQ ID NO: 48)


(a-17); 5′-CTCT CATATG AAAGCAGAATTCAAGCGTAAAG-3′


 


(SEQ ID NO: 49)


(b-17); 5′-CTCT CATATG TCAAGCCTTTCGTTCCGG-3′






Primers (a-17) and (b-17) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Citrobacter koseri 
A DNA fragment comprising the dca gene which is derived from Citrobacter koseri and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 50 (the dca gene of Citrobacter koseri) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 51)



(a-18); 5′-CTCT CATATG AAACTCGTTGTCGGGATG-3′



 



(SEQ ID NO: 52)



(b-18); 5′-CTCT CATATG TCAGGCCTTTCTTTCC-3′






Primers (a-18) and (b-18) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Enterobacter aerogenes 
A DNA fragment comprising the dca gene which is derived from Enterobacter aerogenes and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 53 (the dca gene of Enterobacter aerogenes) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification







(SEQ ID NO: 54)


(a-19); 5′-CTCT CATATG AAACTGATTATTGGGATGACCG-3′


 


(SEQ ID NO: 55)


(b-19); 5′-CTCT CATATG TTAACGCTTATCTGCCGCC-3′






Primers (a-19) and (b-19) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Enterobacter cloacae 
A DNA fragment comprising the dca gene which is derived from Enterobacter cloacae and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 56 (the dca gene of Enterobacter cloacae) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 57)



(a-20); 5′-CTCT CATATG AGATTGATCGTGGGAATGAC-3′



 



(SEQ ID NO: 58)



(b-20); 5′-CTCT CATATG TTACAGCAATGGCGGAATGG-3′






Primers (a-20) and (b-20) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Enterobacter hormaechei 
A DNA fragment comprising the dca gene which is derived from Enterobacter hormaechei and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 59 (the dca gene of Enterobacter hormaechei) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification







(SEQ ID NO: 60)


(a-21); 5′-CTCT CATATG AGATTGATTGTGGGAATGAC-3′


 


(SEQ ID NO: 61)


(b-21); 5′-CTCT CATATG GAGTCTGGTTTAGTTCTCTGC-3′






Primers (a-21) and (b-21) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Enterobacter sakazakii 
A DNA fragment comprising the dca gene which is derived from Enterobacter sakazakii and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 62 (the dca gene of Enterobacter sakazakii) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 63)



(a-22); 5′-CTCT CATATG AGGCTAATTGTCGGAATGAC-3′



 



(SEQ ID NO: 64)



(b-22); 5′-CTCT CATATG TTAACGCTTACCATCCGCC-3′






Primers (a-22) and (b-22) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Escherichia coli 
A DNA fragment comprising the dca gene which is derived from Escherichia coli and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 65 (the dca gene of Escherichia coli) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 66)



(a-23); 5′-CTCT CATATG AAACTGATCGTCGGGATG-3′



 



(SEQ ID NO: 67)



(b-23); 5′-CTCT CATATG TTAGCGCTTACCTTCCGC-3′






Primers (a-23) and (b-23) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Escherichia fergusonii 
A DNA fragment comprising the dca gene which is derived from Escherichia fergusonii and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 68 (the dca gene of Escherichia fergusonii) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 69)



(a-24); 5′-CTCT CATATG AGACTGATCGTCGGGAT-3′



 



(SEQ ID NO: 70)



(b-24); 5′-CTCT CATATG TTAGCGCTTATCTGCCGC-3′






Primers (a-24) and (b-24) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Paenibacillus polymyxa 
A DNA fragment comprising the dca gene which is derived from Paenibacillus polymyxa and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 71 (the dca gene of Paenibacillus polymyxa) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification







(SEQ ID NO: 72)


(a-25); 5′-CTCT CATATG AAGAAAATCATTGTAGGAATATCGG-3′


 


(SEQ ID NO: 73)


(b-25); 5′-CTCT CATATG CTATATCCGCTCTGGAATAGG-3′






Primers (a-25) and (b-25) each have an NdeI restriction enzyme site added thereto.
Cloning of Phenol-Producing Gene Derived from Pantoea ananatis 
A DNA fragment comprising the dca gene which is derived from Pantoea ananatis and which encodes a gene having 4-hydroxybenzoate decarboxylase activity was amplified by the PCR method as described below.
In the PCR, the following set of primers was synthesized based on SEQ ID NO: 74 (the dca gene of Pantoea ananatis) with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems for cloning of the dca gene, and was used.
Primers for dca Gene Amplification









(SEQ ID NO: 75)



(a-26); 



5′-CTCT CATATG AGTAGATTACTGTTAATTTCATTCGTAC-3′



 



(SEQ ID NO: 76)



(b-26); 



5′-CTCT CATATG TTACTTAGCTAACAGAGGAGGG-3′






Primers (a-26) and (b-26) each have an NdeI restriction enzyme site added thereto.
(3-4) Conditions
As the template DNA for Corynebacterium glutamicum, the chromosomal DNA extracted from Corynebacterium glutamicum R was used.
For Escherichia coli, the chromosomal DNA extracted from Escherichia coli K12 MG1655 was used.
For Pseudomonas putida, the chromosomal DNA extracted from Pseudomonas putida ATCC 47054 obtained from American Type Culture Collection (ATCC) was used.
For Acinetobacter baumannii, the chromosomal DNA extracted from Acinetobacter baumannii JCM 6841 obtained from Japan Collection of Microorganisms (JCM) was used.
For Azotobacter vinelandii, the chromosomal DNA extracted from Azotobacter vinelandii ATCC 9104 obtained from American Type Culture Collection (ATCC) was used.
For Chromohalobacter salexigens, the chromosomal DNA extracted from Chromohalobacter salexigens ATCC BAA-138 obtained from American Type Culture Collection (ATCC) was used.
For Citrobacter youngae, the chromosomal DNA extracted from Citrobacter youngae ATCC 29220 obtained from American Type Culture Collection (ATCC) was used.
For Marinobacter aquaeolei, the Marinobacter aquaeolei chromosomal DNA obtained from American Type Culture Collection (ATCC) (catalog No. 700491D-5) was used.
For Marinomonas mediterranea, the chromosomal DNA extracted from Marinomonas mediterranea NBRC 103028 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Pseudoalteromonas haloplanktis, the chromosomal DNA extracted from Pseudoalteromonas haloplanktis NBRC 102225 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Ralstonia eutropha, the chromosomal DNA extracted from Ralstonia eutropha IAM 12368 obtained from Institute of Applied Microbiology Culture Collection (IAM) was used.
For Shewanella putrefaciens, the chromosomal DNA extracted from Shewanella putrefaciens JCM 20190 obtained from Japan Collection of Microorganisms (JCM) was used.
For Thiobacillus denitrificans, the chromosomal DNA extracted from Thiobacillus denitrificans ATCC 25259 obtained from American Type Culture Collection (ATCC) was used.
For Bacillus subtilis, the chromosomal DNA extracted from Bacillus subtilis NBRC 14144 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Bacillus atrophaeus, the chromosomal DNA extracted from Bacillus atrophaeus JCM 9070 obtained from Japan Collection of Microorganisms (JCM) was used.
For Bacillus subtilis subsp. spizizenii, the chromosomal DNA extracted from Bacillus subtilis subsp. spizizenii NBRC 101239 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Citrobacter koseri, the Citrobacter koseri chromosomal DNA obtained from American Type Culture Collection (ATCC) (catalog No. BAA-895D-5) was used.
For Enterobacter aerogenes, the chromosomal DNA extracted from Enterobacter aerogenes NBRC 13534 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Enterobacter cloacae, the chromosomal DNA extracted from Enterobacter cloacae NBRC 13535 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Enterobacter hormaechei, the chromosomal DNA extracted from Enterobacter hormaechei ATCC 49162 obtained from American Type Culture Collection (ATCC) was used.
For Enterobacter sakazakii, the Enterobacter sakazakii chromosomal DNA obtained from American Type Culture Collection (ATCC) (catalog No. BAA-894D-5) was used.
For Escherichia coli W, the chromosomal DNA extracted from Escherichia coli W NBRC 13500 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Escherichia fergusonii, the chromosomal DNA extracted from Escherichia fergusonii NBRC 102419 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Paenibacillus polymyxa, the chromosomal DNA extracted from Paenibacillus polymyxa NBRC 15309 obtained from NITE (National Institute of Technology and Evaluation) Biological Resource Center (NBRC) was used.
For Pantoea ananatis, the chromosomal DNA extracted from Pantoea ananatis LMG 20103 obtained from BCCM/LMG (Belgian Coordinated Collections of Microorganisms/Laboratory for Microbiology, University of Gent) was used.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the aroG gene of Corynebacterium glutamicum, a combination of primers (a-10) and (b-10); for amplification of the ubiC gene of Escherichia coli, a combination of primers (a-11) and (b-11); for amplification of the ubiC gene of Pseudomonas putida, a combination of primers (a-12) and (b-12); for amplification of the ubiC gene of Acinetobacter baumannii, a combination of primers (a-29) and (b-29); for amplification of the ubiC gene of Azotobacter vinelandii, a combination of primers (a-30) and (b-30); for amplification of the ubiC gene of Chromohalobacter salexigens, a combination of primers (a-31) and (b-31); for amplification of the ubiC gene of Citrobacter koseri, a combination of primers (a-32) and (b-32); for amplification of the ubiC gene of Citrobacter youngae, a combination of primers (a-33) and (b-33); for amplification of the ubiC gene of Enterobacter cloacae, a combination of primers (a-34) and (b-34); for amplification of the ubiC gene of Marinobacter aquaeolei, a combination of primers (a-35) and (b-35); for amplification of the ubiC gene of Marinomonas mediterranea, a combination of primers (a-36) and (b-36); for amplification of the ubiC gene of Pantoea ananatis, a combination of primers (a-37) and (b-37); for amplification of the ubiC gene of Pseudoalteromonas haloplanktis, a combination of primers (a-38) and (b-38); for amplification of the ubiC gene of Ralstonia eutropha, a combination of primers (a-39) and (b-39); for amplification of the ubiC gene of Shewanella putrefaciens, a combination of primers (a-40) and (b-40); for amplification of the ubiC gene of Thiobacillus denitrificans, a combination of primers (a-41) and (b-41); for amplification of the bsdBCD gene of Bacillus subtilis, a combination of primers (a-13) and (b-13); for amplification of the dca gene of Bacillus atrophaeus, a combination of primers (a-16) and (b-16); for amplification of the dca gene of Bacillus subtilis subsp. spizizenii, a combination of primers (a-17) and (b-17); for amplification of the dca gene of Citrobacter koseri, a combination of primers (a-18) and (b-18); for amplification of the dca gene of Enterobacter aerogenes, a combination of primers (a-19) and (b-19); for amplification of the dca gene of Enterobacter cloacae, a combination of primers (a-20) and (b-20); for amplification of the dca gene of Enterobacter hormaechei, a combination of primers (a-21) and (b-21); for amplification of the dca gene of Enterobacter sakazakii, a combination of primers (a-22) and (b-22); for amplification of the dca gene of Escherichia coli W, a combination of primers (a-23) and (b-23); for amplification of the dca gene of Escherichia fergusonii, a combination of primers (a-24) and (b-24); for amplification of the dca gene of Paenibacillus polymyxa, a combination of primers (a-25) and (b-25); and for amplification of the dca gene of Pantoea ananatis, a combination of primers (a-26) and (b-26) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds

Annealing step: 52° C., 60 seconds

Extension step: 72° C.

















Corynebacterium glutamicum aroG gene
84 seconds



Escherichia coli ubiC gene
30 seconds



Pseudomonas putida ubiC gene
33 seconds



Acinetobacter baumannii ubiC gene
31 seconds



Azotobacter vinelandii ubiC gene
33 seconds



Chromohalobacter salexigens ubiC gene
33 seconds



Citrobacter koseri ubiC gene
30 seconds



Citrobacter youngae ubiC gene
30 seconds



Enterobacter cloacae ubiC gene
30 seconds



Marinobacter aquaeolei ubiC gene
34 seconds



Marinomonas mediterranea ubiC gene
32 seconds



Pantoea ananatis ubiC gene
31 seconds



Pseudoalteromonas haloplanktis ubiC gene
33 seconds



Ralstonia eutropha ubiC gene
40 seconds



Shewanella putrefaciens ubiC gene
34 seconds



Thiobacillus denitrificans ubiC gene
34 seconds



Bacillus subtilis bsdBCD gene
137 seconds 



Bacillus atrophaeus dca gene
135 seconds 



Bacillus subtilis subsp. spizizenii dca gene
137 seconds 



Citrobacter koseri dca gene
136 seconds 



Enterobacter aerogenes dca gene
136 seconds 



Enterobacter cloacae dca gene
135 seconds 



Enterobacter hormaechei dca gene
141 seconds 



Enterobacter sakazakii dca gene
137 seconds 



Escherichia coli W dca gene
136 seconds 



Escherichia fergusonii dca gene
136 seconds 



Paenibacillus polymyxa dca gene
138 seconds 



Pantoea ananatis dca gene
139 seconds 










A cycle consisting of the above 3 steps was repeated 30 times.
With the use of 10 μL of the reaction mixture produced above, 0.8% agarose gel electrophoresis was performed. As a result, detected were an about 1.4-kb DNA fragment in the case of the Corynebacterium glutamicum aroG gene, an about 0.5-kb DNA fragment in the case of the Escherichia coli ubiC gene, an about 0.6-kb DNA fragment in the case of the Pseudomonas putida ubiC gene, an about 0.5-kb DNA fragment in the case of the Acinetobacter baumannii ubiC gene, an about 0.6-kb DNA fragment in the case of the Azotobacter vinelandii ubiC gene, an about 0.6-kb DNA fragment in the case of the Chromohalobacter salexigens ubiC gene, an about 0.5-kb DNA fragment in the case of the Citrobacter koseri ubiC gene, an about 0.5-kb DNA fragment in the case of the Citrobacter youngae ubiC gene, an about 0.5-kb DNA fragment in the case of the Enterobacter cloacae ubiC gene, an about 0.6-kb DNA fragment in the case of the Marinobacter aquaeolei ubiC gene, an about 0.5-kb DNA fragment in the case of the Marinomonas mediterranea ubiC gene, an about 0.5-kb DNA fragment in the case of the Pantoea ananatis ubiC gene, an about 0.5-kb DNA fragment in the case of the Pseudoalteromonas haloplanktis ubiC gene, an about 0.7-kb DNA fragment in the case of the Ralstonia eutropha ubiC gene, an about 0.6-kb DNA fragment in the case of the Shewanella putrefaciens ubiC gene, an about 0.6-kb DNA fragment in the case of the Thiobacillus denitrificans ubiC gene, an about 2.3-kb DNA fragment in the case of the Bacillus subtilis bsdBCD gene, an about 2.3-kb DNA fragment in the case of the Bacillus atrophaeus bsdBCD gene, an about 2.3-kb DNA fragment in the case of the Bacillus subtilis subsp. spizizenii dca gene, an about 2.3-kb DNA fragment in the case of the Citrobacter koseri dca gene, an about 2.3-kb DNA fragment in the case of the Enterobacter aerogenes dca gene, an about 2.3-kb DNA fragment in the case of the Enterobacter cloacae dca gene, an about 2.4-kb DNA fragment in the case of the Enterobacter hormaechei dca gene, an about 2.3-kb DNA fragment in the case of the Enterobacter sakazakii dca gene, an about 2.3-kb DNA fragment in the case of the Escherichia coli W dca gene, an about 2.3-kb DNA fragment in the case of the Escherichia fergusonii dca gene, an about 2.3-kb DNA fragment in the case of the Paenibacillus polymyxa dca gene, and an about 2.3-kb DNA fragment in the case of the Pantoea ananatis dca gene.
(4) Construction of Phenol-Producing Gene Expression Plasmids Cloning of Phenol-Producing Genes to pCRB209
10 μL of the about 1.4-kb DNA fragment comprising the aroG gene derived from Corynebacterium glutamicum, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Escherichia coli, the about 0.6-kb DNA fragment comprising the ubiC gene derived from Pseudomonas putida, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Acinetobacter baumannii, the about 0.6-kb DNA fragment comprising the ubiC gene derived from Azotobacter vinelandii, the about 0.6-kb DNA fragment comprising the ubiC gene derived from Chromohalobacter salexigens, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Citrobacter koseri, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Citrobacter youngae, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Enterobacter cloacae, the about 0.6-kb DNA fragment comprising the ubiC gene derived from Marinobacter aquaeolei, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Marinomonas mediterranea, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Pantoea ananatis, the about 0.5-kb DNA fragment comprising the ubiC gene derived from Pseudoalteromonas haloplanktis, the about 0.7-kb DNA fragment comprising the ubiC gene derived from Ralstonia eutropha, the about 0.6-kb DNA fragment comprising the ubiC gene derived from Shewanella putrefaciens, the about 0.6-kb DNA fragment comprising the ubiC gene derived from Thiobacillus denitrificans, the about 2.3-kb DNA fragment comprising the bsdBCD gene derived from Bacillus subtilis, the about 2.3-kb DNA fragment comprising the dca gene derived from Bacillus atrophaeus, the about 2.3-kb DNA fragment comprising the dca gene derived from Bacillus subtilis subsp. spizizenii, the about 2.3-kb DNA fragment comprising the dca gene derived from Citrobacter koseri, the about 2.3-kb DNA fragment comprising the dca gene derived from Enterobacter aerogenes, the about 2.3-kb DNA fragment comprising the dca gene derived from Enterobacter cloacae, the about 2.4-kb DNA fragment comprising the dca gene derived from Enterobacter hormaechei, the about 2.3-kb DNA fragment comprising the dca gene derived from Enterobacter sakazakii, the about 2.3-kb DNA fragment comprising the dca gene derived from Escherichia coli W, the about 2.3-kb DNA fragment comprising the dca gene derived from Escherichia fergusonii, the about 2.3-kb DNA fragment comprising the dca gene derived from Paenibacillus polymyxa, or the about 2.3-kb DNA fragment comprising the dca gene derived from Pantoea ananatis, each amplified by the PCR in the above (3), and 2 μL of the cloning vector pCRB209 comprising promoter PgapA were each cut with the use of restriction enzyme NdeI, and were processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. The resulting liquid was named Ligation Liquid G, H, I, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, AK, AL, AM, J, O, P, Q, R, S, T, U, V, W, X or Y.
With the use of each of the obtained 28 kinds of Ligation Liquids G, H, I, AA, AB, AC, AD, AE, AF, AG, AH, AI, AJ, AK, AL, AM, J, O, P, Q, R, S, T, U, V, W, X and Y, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzyme to confirm the inserted fragment. As a result, in addition to an about 5.1-kb DNA fragment of the plasmid pCRB209, confirmed were an about 1.4-kb inserted fragment in the case of the aroG gene derived from Corynebacterium glutamicum (Ligation Liquid G), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Escherichia coli (Ligation Liquid H), an about 0.6-kb inserted fragment in the case of the ubiC gene derived from Pseudomonas putida (Ligation Liquid I), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Acinetobacter baumannii (Ligation Liquid AA), an about 0.6-kb inserted fragment in the case of the ubiC gene derived from Azotobacter vinelandii (Ligation Liquid AB), an about 0.6-kb inserted fragment in the case of the ubiC gene derived from Chromohalobacter salexigens (Ligation Liquid AC), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Citrobacter koseri (Ligation Liquid AD), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Citrobacter youngae (Ligation Liquid AE), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Enterobacter cloacae (Ligation Liquid AF), an about 0.6-kb inserted fragment in the case of the ubiC gene derived from Marinobacter aquaeolei (Ligation Liquid AG), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Marinomonas mediterranea (Ligation Liquid AH), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Pantoea ananatis (Ligation Liquid AI), an about 0.5-kb inserted fragment in the case of the ubiC gene derived from Pseudoalteromonas haloplanktis (Ligation Liquid AJ), an about 0.7-kb inserted fragment in the case of the ubiC gene derived from Ralstonia eutropha (Ligation Liquid AK), an about 0.6-kb inserted fragment in the case of the ubiC gene derived from Shewanella putrefaciens (Ligation Liquid AL), an about 0.6-kb inserted fragment in the case of the ubiC gene derived from Thiobacillus denitrificans (Ligation Liquid AM), an about 2.3-kb inserted fragment in the case of the bsdBCD gene derived from Bacillus subtilis (Ligation Liquid J), an about 2.3-kb inserted fragment in the case of the dca gene derived from Bacillus atrophaeus (Ligation Liquid 0), an about 2.3-kb inserted fragment in the case of the dca gene derived from Bacillus subtilis subsp. spizizenii (Ligation Liquid P), an about 2.3-kb inserted fragment in the case of the dca gene derived from Citrobacter koseri (Ligation Liquid Q), an about 2.3-kb inserted fragment in the case of the dca gene derived from Enterobacter aerogenes (Ligation Liquid R), an about 2.3-kb inserted fragment in the case of the dca gene derived from Enterobacter cloacae (Ligation Liquid S), an about 2.4-kb inserted fragment in the case of the dca gene derived from Enterobacter hormaechei (Ligation Liquid T), an about 2.3-kb inserted fragment in the case of the dca gene derived from Enterobacter sakazakii (Ligation Liquid U), an about 2.3-kb inserted fragment in the case of the dca gene derived from Escherichia coli W (Ligation Liquid V), an about 2.3-kb inserted fragment in the case of the dca gene derived from Escherichia fergusonii (Ligation Liquid W), an about 2.3-kb inserted fragment in the case of the dca gene derived from Paenibacillus polymyxa (Ligation Liquid X), and an about 2.3-kb inserted fragment in the case of the dca gene derived from Pantoea ananatis was (Ligation Liquid Y).
The plasmid comprising the aroG gene derived from Corynebacterium glutamicum was named pCRB209-aroG/CG, the plasmid comprising the ubiC gene derived from Escherichia coli was named pCRB209-ubiC/EC, the plasmid comprising the ubiC gene derived from Pseudomonas putida was named pCRB209-ubiC/PP, the plasmid comprising the ubiC gene derived from Acinetobacter baumannii was named pCRB209-ubiC/ACB, the plasmid comprising the ubiC gene derived from Azotobacter vinelandii was named pCRB209-ubiC/AVN, the plasmid comprising the ubiC gene derived from Chromohalobacter salexigens was named pCRB209-ubiC/CSA, the plasmid comprising the ubiC gene derived from Citrobacter koseri was named pCRB209-ubiC/CKO, the plasmid comprising the ubiC gene derived from Citrobacter youngae was named pCRB209-ubiC/CIT, the plasmid comprising the ubiC gene derived from Enterobacter cloacae was named pCRB209-ubiC/ECL, the plasmid comprising the ubiC gene derived from Marinobacter aquaeolei was named pCRB209-ubiC/MAQ, the plasmid comprising the ubiC gene derived from Marinomonas mediterranea was named pCRB209-ubiC/MME, the plasmid comprising the ubiC gene derived from Pantoea ananatis was named pCRB209-ubiC/PAM, the plasmid comprising the ubiC gene derived from Pseudoalteromonas haloplanktis was named pCRB209-ubiC/PHA, the plasmid comprising the ubiC gene derived from Ralstonia eutropha was named pCRB209-ubiC/REH, the plasmid comprising the ubiC gene derived from Shewanella putrefaciens was named pCRB209-ubiC/SPC, the plasmid comprising the ubiC gene derived from Thiobacillus denitrificans was named pCRB209-ubiC/TBD, the plasmid comprising the bsdBCD gene derived from Bacillus subtilis was named pCRB209-bsdBCD/BS, the plasmid comprising the dca gene derived from Bacillus atrophaeus was named pCRB209-dca/BAE, the plasmid comprising the dca gene derived from Bacillus subtilis subsp. spizizenii was named pCRB209-dca/BSS, the plasmid comprising the dca gene derived from Citrobacter koseri was named pCRB209-dca/CKO, the plasmid comprising the dca gene derived from Enterobacter aerogenes was named pCRB209-dca/EAE, the plasmid comprising the dca gene derived from Enterobacter cloacae was named pCRB209-dca/ECL, the plasmid comprising the dca gene derived from Enterobacter hormaechei was named pCRB209-dca/EHO, the plasmid comprising the dca gene derived from Enterobacter sakazakii was named pCRB209-dca/ESA, the plasmid comprising the dca gene derived from Escherichia coli W was named pCRB209-dca/ECK, the plasmid comprising the dca gene derived from Escherichia fergusonii was named pCRB209-dca/EFE, the plasmid comprising the dca gene derived from Paenibacillus polymyxa was named pCRB209-dca/PPY, and the plasmid comprising the dca gene derived from Pantoea ananatis was named pCRB209-dca/PAM.
The construct of the plasmid pCRB209-aroG/CG comprising the aroG gene derived from Corynebacterium glutamicum, the plasmid pCRB209-ubiC/EC comprising the ubiC gene derived from Escherichia coli, the plasmid pCRB209-ubiC/PP comprising the ubiC gene derived from Pseudomonas putida, and the plasmid pCRB209-bsdBCD/BS comprising the bsdBCD gene derived from Bacillus subtilis are shown in FIG. 1.
Cloning of Phenol-Producing Genes to pCRB1
The above plasmids pCRB209-ubiC/EC, pCRB209-ubiC/PP, pCRB209-ubiC/ACB, pCRB209-ubiC/AVN, pCRB209-ubiC/CSA, pCRB209-ubiC/CKO, pCRB209-ubiC/CIT, pCRB209-ubiC/ECL, pCRB209-ubiC/MAQ, pCRB209-ubiC/MME, pCRB209-ubiC/PAM, pCRB209-ubiC/PHA and pCRB209-ubiC/SPC were cut with the use of a restriction enzyme SalI. After agarose gel electrophoresis, an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Escherichia coli, and a terminator sequence were ligated; an about 1.6-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Pseudomonas putida, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Acinetobacter baumannii, and a terminator sequence were ligated; an about 1.6-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Azotobacter vinelandii, and a terminator sequence were ligated; an about 1.6-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Chromohalobacter salexigens, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Citrobacter koseri, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Citrobacter youngae, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Enterobacter cloacae, and a terminator sequence were ligated; an about 1.6-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Marinobacter aquaeolei, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Marinomonas mediterranea, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Pantoea ananatis, and a terminator sequence were ligated; an about 1.5-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Pseudoalteromonas haloplanktis, and a terminator sequence were ligated; or an about 1.6-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Shewanella putrefaciens, and a terminator sequence were ligated; each of which was recovered from the agarose gel with the use of QIAquick Gel Extraction Kit (made by QIAGEN), and an about 4.1-kb DNA fragment obtained by SalI digestion of the cloning vector pCRB1, followed by 10 min-treatment at 70° C. for deactivation of SalI (Nakata, K. et al., Vectors for the genetics engineering of corynebacteria; in Saha, B. C. (ed.): Fermentation Biotechnology, ACS Symposium Series 862. Washington, American Chemical Society: 175-191 (2003)) were mixed. To this, 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. The resulting liquids were named Ligation Liquids K, L, AN, AO, AP, AQ, AR, AS, AT, AU, AV, AW, and AX.
Similarly, the above-mentioned plasmids pCRB209-ubiC/REH and pCRB209-ubiC/TBD were cut with the use of a restriction enzyme BamHI. After agarose gel electrophoresis, an about 1.7-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Ralstonia eutropha, and a terminator sequence were ligated; or an about 1.6-kb DNA fragment in which a gapA promoter, a ubiC gene derived from Thiobacillus denitrificans, and a terminator sequence were ligated; both of which were recovered from the agarose gel with the use of QIAquick Gel Extraction Kit (made by QIAGEN), and an about 4.1-kb DNA fragment obtained by BamHI digestion of the cloning vector pCRB1, followed by 10 min-treatment at 70° C. for deactivation of BamHI (Nakata, K. et al., Vectors for the genetics engineering of corynebacteria; in Saha, B. C. (ed.): Fermentation Biotechnology, ACS Symposium Series 862. Washington, American Chemical Society: 175-191 (2003)) were mixed. To this, 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. The resulting liquids were named Ligation Liquids AY and AZ.
With use of each of the obtained Ligation Liquids K, L, AN, AO, AP, AQ, AR, AS, AT, AU, AV, AW, AX, AY and AZ, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% of polypeptone, 0.5% of yeast extract, 0.5% of sodium chloride, and 1.5% of agar) containing 50 μg/mL of chloramphenicol.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture medium and cut with the use of restriction enzyme SalI or BamHI to confirm inserted fragment. As a result, in addition to an about 4.1-kb DNA fragment of the plasmid pCRB1, confirmed were an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Escherichia coli (Ligation Liquid K), an about 1.6-kb inserted fragment in the case of the ubiC gene derived from Pseudomonas putida (Ligation Liquid L), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Acinetobacter baumannii (Ligation Liquid AN), an about 1.6-kb inserted fragment in the case of the ubiC gene derived from Azotobacter vinelandii (Ligation Liquid AO), an about 1.6-kb inserted fragment in the case of the ubiC gene derived from Chromohalobacter salexigens (Ligation Liquid AP), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Citrobacter koseri (Ligation Liquid AQ), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Citrobacter youngae (Ligation Liquid AR), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Enterobacter cloacae (Ligation Liquid AS), an about 1.6-kb inserted fragment in the case of the ubiC gene derived from Marinobacter aquaeolei (Ligation Liquid AT), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Marinomonas mediterranea (Ligation Liquid AU), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Pantoea ananatis (Ligation Liquid AV), an about 1.5-kb inserted fragment in the case of the ubiC gene derived from Pseudoalteromonas haloplanktis (Ligation Liquid AW), an about 1.7-kb inserted fragment in the case of the ubiC gene derived from Ralstonia eutropha (Ligation Liquid AY), an about 1.6-kb inserted fragment in the case of the ubiC gene derived from Shewanella putrefaciens (Ligation Liquid AX), and an about 1.6-kb inserted fragment in the case of the ubiC gene derived from Thiobacillus denitrificans (Ligation Liquid AZ).
The plasmid comprising the ubiC gene derived from Escherichia coli was named pCRB1-ubiC/EC, and the plasmid comprising the ubiC gene derived from Pseudomonas putida was named pCRB1-ubiC/PP (FIG. 2). The plasmid comprising the ubiC gene derived from Acinetobacter baumannii was named pCRB1-ubiC/ACB, the plasmid comprising the ubiC gene derived from Azotobacter vinelandii was named pCRB1-ubiC/AVN, the plasmid comprising the ubiC gene derived from Chromohalobacter salexigens was named pCRB1-ubiC/CSA, the plasmid comprising the ubiC gene derived from Citrobacter koseri was named pCRB1-ubiC/CKO, the plasmid comprising the ubiC gene derived from Citrobacter youngae was named pCRB1-ubiC/CIT, the plasmid comprising the ubiC gene derived from Enterobacter cloacae was named pCRB1-ubiC/ECL, the plasmid comprising the ubiC gene derived from Marinobacter aquaeolei was named pCRB1-ubiC/MAQ, the plasmid comprising the ubiC gene derived from Marinomonas mediterranea was named pCRB1-ubiC/MME, the plasmid comprising the ubiC gene derived from Pantoea ananatis was named pCRB1-ubiC/PAM, the plasmid comprising the ubiC gene derived from Pseudoalteromonas haloplanktis was named pCRB1-ubiC/PHA, the plasmid comprising the ubiC gene derived from Ralstonia eutropha was named pCRB1-ubiC/REH, the plasmid comprising the ubiC gene derived from Shewanella putrefaciens was named pCRB1-ubiC/SPC, and the plasmid comprising the ubiC gene derived from Thiobacillus denitrificans was named pCRB1-ubiC/TBD.
Cloning of Phenol-Producing Gene to pCRB15
The above plasmid pCRB209-aroG/CG was cut with the use of a restriction enzyme BamHI. After agarose gel electrophoresis, an about 2.4-kb DNA fragment recovered from the agarose gel with the use of QIAquick Gel Extraction Kit (made by QIAGEN), in which fragment a gapA promoter, an aroG gene derived from Corynebacterium glutamicum, and a terminator sequence were ligated, and an about 3.8-kb DNA fragment obtained by BamHI digestion of the plasmid pCRB15, followed by 10 min-treatment at 70° C. for deactivation of BamHI were mixed. To this, 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid M.
With the use of the Ligation Liquid M, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 25 μg/mL of zeocin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzyme BamHI to confirm inserted fragment. As a result, in addition to an about 3.8-kb DNA fragment of the plasmid pCRB15, an about 2.4-kb inserted fragment of the aroG gene derived from Corynebacterium glutamicum (Ligation Liquid M) was confirmed.
The plasmid comprising the aroG gene derived from Corynebacterium glutamicum was named pCRB15-aroG/CG (FIG. 3).
(5) Construction of Plasmids for Corynebacterium glutamicum Chromosomal Gene Disruption
Construction of Plasmid for Corynebacterium glutamicum pobA Gene Disruption
A DNA fragment required for constructing a plasmid for markerless disruption of the pobA gene on the chromosome of Corynebacterium glutamicum was amplified by the PCR method as described below.
In the PCR, the following sets of primers were synthesized based on the sequence of Corynebacterium glutamicum R with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems, and were used.
Primers for Amplification of pobA-1 Region









(SEQ ID NO: 40)



(a-14); 



5′-CTCT TCTAGA GAAACGATCAAGTGCACCAG-3′



 



(SEQ ID NO: 41)



(b-14); 



5′-GACACGAGCGTTTATACCTCTAATTGCCACTGGTACGTGG-3′






Primer (a-14) has an XbaI restriction enzyme site added thereto.
Primers for Amplification of pobA-2 Region







(SEQ ID NO: 42)


(a-15); 5′-GAGGTATAAACGCTCGTGTC-3′


 


(SEQ ID NO: 43)


(b-15); 5′ -CTCT GAGCTC GAGAACACGAACCATACGAG-3′






Primer (b-15) has a Sad restriction enzyme site added thereto.
As the template DNA, the chromosomal DNA extracted from Corynebacterium glutamicum R was used.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the pobA-1 region, a combination of primers (a-14) and (b-14), and for amplification of the pobA-2 region, a combination of primers (a-15) and (b-15) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C.

    
    
        pobA-1 region: 60 seconds
        pobA-2 region: 60 seconds
    
    


A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed. An about 1.0-kb DNA fragment in the case of the Corynebacterium glutamicum pobA-1 region, and an about 1.0-kb DNA fragment in the case of the pobA-2 region were detected.
Subsequently, 1 μL each of the pobA-1 region fragment and the pobA-2 region fragment, which were amplified by the above PCR, were mixed and subjected to PCR for ligation.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:

















TaKaRa LA Taq TM (5 units/μL)
0.5
μL



10× LA PCR TM Buffer II (Mg2+ free)
5
μL



25 mM MgCl2
5
μL



dNTP Mixture (2.5 mM each)
8
μL



The above 2 fragments*)
1
μL each



Sterile distilled water
29.5
μL







The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.



*)pobA-1 region fragment and pobA-2 region fragment were used.






PCR Cycle:

Denaturation step: 95° C., 20 seconds
Annealing step: 52° C., 5 seconds
Extension step: 72° C., 50 seconds
A cycle consisting of the above 3 steps was repeated 30 times.
Further, using, as the template DNA, the obtained fragment in which pobA-1 and pobA-2 were ligated, a pobA deletion fragment was amplified by PCR.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the pobA deletion fragment, a combination of primers (a-14) and (b-15) was used.






PCR Cycle:

Denaturation step: 95° C., 20 seconds
Annealing step: 52° C., 5 seconds
Extension step: 72° C., 97 seconds
A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed, and an about 2.0-kb fragment of the pobA deletion fragment was detected.
10 μL of the about 2.0-kb DNA fragment of the pobA deletion fragment derived from Corynebacterium glutamicum R, which was amplified by the above PCR, and 2 μL of an about 4.4-kb plasmid, pCRA725 for markerless chromosomal gene transfection (J. Mol. Microbiol. Biotechnol., Vol. 8, 243-254, 2004 (JP 2006-124440 A)) were each cut with the use of restriction enzymes XbaI and SacI, and processed at 70° C. for 10 minutes for deactivation of the restriction enzymes. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid N.
With the use of the Ligation Liquid N, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes XbaI and SacI to confirm the inserted fragment. As a result, in addition to an about 4.4-kb DNA fragment of the plasmid pCRA725, an about 2.0-kb inserted fragment of the pobA deletion gene derived from Corynebacterium glutamicum (Ligation Liquid N) was confirmed.
The plasmid comprising the pobA deletion gene derived from Corynebacterium glutamicum was named pCRA725-pobA/CG.
Construction of Plasmid for Corynebacterium glutamicum poxF Gene Disruption
A DNA fragment required for constructing a plasmid for markerless disruption of the poxF gene on the chromosome of Corynebacterium glutamicum was amplified by the PCR method as described below.
In the PCR, the following sets of primers were synthesized based on the sequence of Corynebacterium glutamicum R with the use of “394 DNA/RNA Synthesizer” made by Applied Biosystems, and were used.
Primers for Amplification of poxF-1 Region









(SEQ ID NO: 77)



(a-27); 



5′-CTCT TCTAGA TACGTCCTAAACACCCGAC-3′



 



(SEQ ID NO: 78)



(b-27); 



5′-GACCAACCATTGCTGACTTGCGTATCCATAGTCAGGCTTC-3′






Primer (a-27) has an XbaI restriction enzyme site added thereto.
Primers for Amplification of poxF-2 Region









(SEQ ID NO: 79)



(a-28); 5′-CAAGTCAGCAATGGTTGGTC-3′



 



(SEQ ID NO: 80)



(b-28); 5′-CTCT TCTAGA TGATCAGTACCAAGGGTGAG-3′






Primer (b-28) has an XbaI restriction enzyme site added thereto.
As the template DNA, the chromosomal DNA extracted from Corynebacterium glutamicum R was used.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the poxF-1 region, a combination of primers (a-27) and (b-27), and for amplification of the poxF-2 region, a combination of primers (a-28) and (b-28) were used.






PCR Cycle:

Denaturation step: 94° C., 60 seconds
Annealing step: 52° C., 60 seconds
Extension step: 72° C.

    
    
        poxF-1 region: 50 seconds
        poxF-2 region: 50 seconds
    
    


A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed. An about 0.8-kb DNA fragment in the case of the Corynebacterium glutamicum poxF-1 region, and an about 0.8-kb DNA fragment in the case of the poxF-2 region were detected.
Subsequently, 1 μL each of the poxF-1 region fragment and the poxF-2 region fragment, which were amplified by the above PCR, were mixed and subjected to PCR for ligation.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Taq (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:

















TaKaRa LA Taq TM (5 units/μL)
0.5
μL



10× LA PCR TM Buffer II (Mg2+ free)
5
μL



25 mM MgCl2
5
μL



dNTP Mixture (2.5 mM each)
8
μL



The above 2 fragments*)
1
μL each



Sterile distilled water
29.5
μL







The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.



*)poxF-1 region fragment and poxF-2 region fragment were used.






PCR Cycle:

Denaturation step: 95° C., 20 seconds
Annealing step: 52° C., 5 seconds
Extension step: 72° C., 50 seconds
A cycle consisting of the above 3 steps was repeated 30 times.
Further, using, as the template DNA, the obtained fragment in which poxF-1 and poxF-2 were ligated, a poxF deletion fragment was amplified by PCR.
Actual PCR was performed with the use of a thermal cycler, GeneAmp PCR System 9700 (made by Applied Biosystems) and TaKaRa LA Tag (made by Takara Bio, Inc.) as a reaction reagent under the conditions described below.
Reaction Mixture:














TaKaRa LA Taq TM (5 units/μL)
0.5
μL


10× LA PCR TM Buffer II
5
μL


(Mg2+ free)


25 mM MgCl2
5
μL


dNTP Mixture (2.5 mM each)
8
μL


Template DNA
5
μL (DNA content: 1 μg or less)


The above 2 primers*)
0.5
μL each (final conc.: 1 μM)


Sterile distilled water
25.5
μL





The above ingredients were mixed, and 50 μL of the reaction mixture was subjected to PCR.


*)For amplification of the poxF deletion fragment, a combination of primers (a-27) and (b-28) was used.






PCR Cycle:

Denaturation step: 95° C., 20 seconds
Annealing step: 52° C., 5 seconds
Extension step: 72° C., 50 seconds
A cycle consisting of the above 3 steps was repeated 30 times.
Using 10 μL of the above-produced reaction mixture, 0.8% agarose gel electrophoresis was performed, and an about 1.6-kb fragment of the poxF deletion fragment was detected.
10 μL of the about 1.7-kb DNA fragment of the poxF deletion fragment derived from Corynebacterium glutamicum R, which was amplified by the above PCR, and 2 μL of an about 4.4-kb plasmid, pCRA725 for markerless chromosomal gene transfection (J. Mol. Microbiol. Biotechnol., Vol. 8, 243-254, 2004 (JP 2006-124440 A) were each cut with the use of restriction enzyme XbaI, and processed at 70° C. for 10 minutes for deactivation of the restriction enzyme. Both were mixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made by Takara Bio, Inc.) were added thereto. Sterile distilled water was added thereto so that the total volume was 10 μL, and the mixture was allowed to react at 15° C. for 3 hours for ligation. This was named Ligation Liquid Z.
With the use of the Ligation Liquid Z, Escherichia coli JM109 was transformed by the calcium chloride method (Journal of Molecular Biology, 53, 159 (1970)) and was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar) containing 50 μg/mL of kanamycin.
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzyme XbaI to confirm the inserted fragment. As a result, in addition to an about 4.4-kb DNA fragment of the plasmid pCRA725, an about 1.7-kb inserted fragment of the pheA deletion gene derived from Corynebacterium glutamicum (Ligation Liquid Z) was confirmed.
The plasmid comprising the poxF deletion gene derived from Corynebacterium glutamicum was named pCRA725-poxF/CG.
(6) Construction of Strain in which a Gene Associated with Degradation of 4-Hydroxybenzoate is Disrupted
Vector pCRA725 for markerless chromosomal gene transfection is a plasmid that cannot be replicated within Corynebacterium glutamicum R. With the use of the plasmid pCRA725-pobA/CG, transformation of Corynebacterium glutamicum R was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium (A liquid medium and 1.5% agar) containing 50 μg/mL of kanamycin. The single crossover strain obtained on the above medium was applied to BT agar medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution dissolved in 1 L of distilled water, and 1.5% agar) containing 10% (w/v) sucrose.
In the case of a strain having a single crossover of the plasmid pCRA725-pobA/CG with the homologous region on the chromosome, the strain shows kanamycin resistance resulting from the expression of the kanamycin resistance gene on the pCRA725-pobA/CG and mortality on a culture medium containing sucrose resulting from the expression of the Bacillus subtilis sacR-sacB gene. In the case of a strain having a double crossover of the plasmid pCRA725-pobA/CG, the strain shows kanamycin sensitivity resulting from the loss of the kanamycin resistance gene on the pCRA725-pobA/CG and growing ability on a culture medium containing sucrose resulting from the loss of the sacR-sacB gene. The markerless chromosomal gene disruptant shows kanamycin sensitivity and growing ability on a culture medium containing sucrose. Therefore, a strain that showed kanamycin sensitivity and growing ability on a culture medium containing sucrose was selected.
The obtained markerless pobA gene disruptant of Corynebacterium glutamicum R was named Corynebacterium glutamicum ΔpobA.
Construction of Corynebacterium glutamicum pobA and poxF Gene Disruptant
Vector pCRA725 for markerless chromosomal gene transfection is a plasmid that cannot be replicated within Corynebacterium glutamicum R. With the use of the plasmid pCRA725-poxF/CG, transformation of Corynebacterium glutamicum ΔpobA was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium (A liquid medium and 1.5% agar) containing 50 μg/mL of kanamycin. The single crossover strain obtained on the above medium was applied to BT agar medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution dissolved in 1 L of distilled water, and 1.5% agar) containing 10% (w/v) sucrose.
In the case of a strain having a single crossover of the plasmid pCRA725-poxF/CG with the homologous region on the chromosome, the strain shows kanamycin resistance resulting from the expression of the kanamycin resistance gene on the pCRA725-poxF/CG and mortality on a culture medium containing sucrose resulting from the expression of the Bacillus subtilis sacR-sacB gene. In the case of a strain having a double crossover of the plasmid pCRA725-poxF/CG, the strain shows kanamycin sensitivity resulting from the loss of the kanamycin resistance gene on the pCRA725-poxF/CG and growing ability on a culture medium containing sucrose resulting from the loss of the sacR-sacB gene. The markerless chromosomal gene disruptant shows kanamycin sensitivity and growing ability on a culture medium containing sucrose. Therefore, a strain that showed kanamycin sensitivity and growing ability on a culture medium containing sucrose was selected.
The obtained markerless poxF gene disruptant of Corynebacterium glutamicum ΔpobA was named Corynebacterium glutamicum ΔpobAΔpoxF.
(7) Construction of Transgenic Strains for Phenol Production Gene
(7-1) Transformation of Corynebacterium glutamicum Wild Strain and ΔpobA
With the use of the above-described plasmids pCRB1-ubiC/EC and pCRB209-bsdBCD/BS, transformation of Corynebacterium glutamicum R or Corynebacterium glutamicum ΔpobA was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium containing 5 μg/mL of chloramphenicol and 50 μg/mL of kanamycin. These two kinds of plasmids can coexist in Corynebacterium glutamicum. 
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes to confirm the inserted plasmids. As a result, transfection of the above-constructed plasmids pCRB1-ubiC/EC and pCRB209-bsdBCD/BS was confirmed. The obtained transformant of the R strain was named Corynebacterium glutamicum PHE11, and the obtained transformant of the ΔpobA strain was named Corynebacterium glutamicum PHE13.
With the use of the above-described plasmids pCRB1-ubiC/EC, pCRB209-bsdBCD/BS, and pCRB15-aroG/CG, transformation of Corynebacterium glutamicum R or Corynebacterium glutamicum ΔpobA was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin. These three kinds of plasmids can coexist in Corynebacterium glutamicum. 
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes to confirm the inserted plasmids. As a result, transfection of the above-constructed plasmids pCRB1-ubiC/EC, pCRB209-bsdBCD/BS, and pCRB15-aroG/CG was confirmed. The obtained transformant of the R strain was named Corynebacterium glutamicum PHE12, and the obtained transformant of the ΔpobA strain was named Corynebacterium glutamicum PHE14.
With the use of the above-described plasmids pCRB1-ubiC/PP and pCRB209-bsdBCD/BS, transformation of Corynebacterium glutamicum R or Corynebacterium glutamicum ΔpobA was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium containing 5 μg/mL of chloramphenicol and 50 μg/mL of kanamycin. These two kinds of plasmids can coexist in Corynebacterium glutamicum. 
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes to confirm the inserted plasmids. As a result, transfection of the above-constructed plasmids pCRB1-ubiC/PP and pCRB209-bsdBCD/BS was confirmed. The obtained transformant of the R strain was named Corynebacterium glutamicum PHE15, and the obtained transformant of the ΔpobA strain was named Corynebacterium glutamicum PHE17.
With the use of the above-described plasmids pCRB1-ubiC/PP, pCRB209-bsdBCD/BS, and pCRB15-aroG/CG, transformation of Corynebacterium glutamicum R or Corynebacterium glutamicum ΔpobA was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin. These three kinds of plasmids can coexist in Corynebacterium glutamicum. 
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes to confirm the inserted plasmids. As a result, transfection of the above-constructed plasmids pCRB1-ubiC/PP, pCRB209-bsdBCD/BS, and pCRB15-aroG/CG was confirmed. The obtained transformant of the R strain was named Corynebacterium glutamicum PHE16, and the obtained transformant of the ΔpobA strain was named Corynebacterium glutamicum PHE18.
(7-2) Transformation of Corynebacterium glutamicum ΔpobAΔpoxF
With the use of each of various combinations of the above-described plasmids pCRB1-ubiC/PP, pCRB209-bsdBCD/BS, pCRB209-dca/BAE, pCRB209-dca/BSS, pCRB209-dca/CKO, pCRB209-dca/EAE, pCRB209-dca/ECL, pCRB209-dca/EHO, pCRB209-dca/ESA, pCRB209-dca/ECK, pCRB209-dca/EFE, pCRB209-dca/PPY, pCRB209-dca/PAM, and pCRB15-aroG/CG, transformation of Corynebacterium glutamicum ΔpobAΔpoxF was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin. These three kinds of plasmids (pCRB1, pCRB209, and pCRB15) can coexist in Corynebacterium glutamicum. 
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes to confirm the inserted plasmids. As a result, transfection of the above-constructed plasmids pCRB1-ubiC/PP, pCRB209-bsdBCD/BS, pCRB209-dca/BAE, pCRB209-dca/BSS, pCRB209-dca/CKO, pCRB209-dca/EAE, pCRB209-dca/ECL, pCRB209-dca/EHO, pCRB209-dca/ESA, pCRB209-dca/ECK, pCRB209-dca/EFE, pCRB209-dca/PPY, pCRB209-dca/PAM, and pCRB15-aroG/CG was confirmed.
Among the ΔpobAΔpoxF strains, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-bsdBCD/BS, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-1, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/BAE, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-2, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/BSS, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-3, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/CKO, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-4, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/EAE, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-5, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-6, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/EHO, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-7, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/ESA, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-8, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/ECK, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-9, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/EFE, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-10, the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/PPY, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-11, and the strain in which transfection of pCRB1-ubiC/PP, pCRB209-dca/PAM, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE19-12. With the use of each of various combinations of the above-described plasmids pCRB1-ubiC/EC, pCRB1-ubiC/ACB, pCRB1-ubiC/AVN, pCRB1-ubiC/CSA, pCRB1-ubiC/CKO, pCRB1-ubiC/CIT, pCRB1-ubiC/ECL, pCRB1-ubiC/MAQ, pCRB1-ubiC/MME, pCRB1-ubiC/PAM, pCRB1-ubiC/PHA, pCRB1-ubiC/REH, pCRB1-ubiC/SPC, pCRB1-ubiC/TBD, pCRB209-dca/ECL and pCRB15-aroG/CG, transformation of Corynebacterium glutamicum ΔpobAΔpoxF was performed by electroporation (Agric. Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144, 181-185 (1993)), and the strain was applied to A agar medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin. These three kinds of plasmids (pCRB1, pCRB209, and pCRB15) can coexist in Corynebacterium glutamicum. 
A growing strain on the culture medium was subjected to liquid culture in the usual manner. Plasmid DNA was extracted from the culture and cut with the use of restriction enzymes to confirm the inserted plasmids. As a result, transfection of the above-constructed plasmids pCRB1-ubiC/EC, pCRB1-ubiC/ACB, pCRB1-ubiC/AVN, pCRB1-ubiC/CSA, pCRB1-ubiC/CKO, pCRB1-ubiC/CIT, pCRB1-ubiC/ECL, pCRB1-ubiC/MAQ, pCRB1-ubiC/MME, pCRB1-ubiC/PAM, pCRB1-ubiC/PHA, pCRB1-ubiC/REH, pCRB1-ubiC/SPC, pCRB1-ubiC/TBD, pCRB209-dca/ECL and pCRB15-aroG/CG was confirmed.
Among the ΔpobAΔpoxF strains, the strain in which transfection of pCRB1-ubiC/EC, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-1, the strain in which transfection of pCRB1-ubiC/ACB, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-2, the strain in which transfection of pCRB1-ubiC/AVN, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-3, the strain in which transfection of pCRB1-ubiC/CSA, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-4, the strain in which transfection of pCRB1-ubiC/CKO, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-5, the strain in which transfection of pCRB1-ubiC/CIT, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-6, the strain in which transfection of pCRB1-ubiC/ECL, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-7, the strain in which transfection of pCRB1-ubiC/MAQ, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-8, the strain in which transfection of pCRB1-ubiC/MME, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-9, the strain in which transfection of pCRB1-ubiC/PAM, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-10, the strain in which transfection of pCRB1-ubiC/PHA, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-11, the strain in which transfection of pCRB1-ubiC/REH, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-12, the strain in which transfection of pCRB1-ubiC/SPC, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-13, and the strain in which transfection of pCRB1-ubiC/TBD, pCRB209-dca/ECL, and pCRB15-aroG/CG had been confirmed was named Corynebacterium glutamicum PHE20-14.
The outline of gene recombination in the above-described strains is shown in Table 1 below.
Corynebacterium glutamicum PHE18 was deposited in Incorporated Administrative Agency National Institute of Technology and Evaluation, NITE Patent Microorganisms Depositary (2-5-8 Kazusakamatari, Kisarazu-shi, Chiba 292-0818 Japan) under Accession Number NITE BP-995 on Oct. 21, 2010.







TABLE 1







Phenol-producing gene transgenic strains










Host strain




Corynebacterium
Transfected gene


Strain
glutamicum
(gene name/origin)














PHE11
Wild strain
ubiC/EC
bsdBCD/BS
—


PHE12

ubiC/EC
bsdBCD/BS
aroG/CG


PHE13
ΔpobA
ubiC/EC
bsdBCD/BS
—


PHE14

ubiC/EC
bsdBCD/BS
aroG/CG


PHE15
Wild strain
ubiC/PP
bsdBCD/BS
—


PHE16

ubiC/PP
bsdBCD/BS
aroG/CG


PHE17
ΔpobA
ubiC/PP
bsdBCD/BS
—


PHE18

ubiC/PP
bsdBCD/BS
aroG/CG


PHE19-1
ΔpobAΔpoxF
ubiC/PP
bsdBCD/BS
aroG/CG


PHE19-2

ubiC/PP
dca/BAE
aroG/CG


PHE19-3

ubiC/PP
dca/BSS
aroG/CG


PHE19-4

ubiC/PP
dca/CKO
aroG/CG


PHE19-5

ubiC/PP
dca/EAE
aroG/CG


PHE19-6

ubiC/PP
dca/ECL
aroG/CG


PHE19-7

ubiC/PP
dca/EHO
aroG/CG


PHE19-8

ubiC/PP
dca/ESA
aroG/CG


PHE19-9

ubiC/PP
dca/ECK
aroG/CG


PHE19-10

ubiC/PP
dca/EFE
aroG/CG


PHE19-11

ubiC/PP
dca/PPY
aroG/CG


PHE19-12

ubiC/PP
dca/PAM
aroG/CG


PHE20-1
ΔpobAΔpoxF
ubiC/EC
dca/ECL
aroG/CG


PHE20-2

ubiC/ACB
dca/ECL
aroG/CG


PHE20-3

ubiC/AVN
dca/ECL
aroG/CG


PHE20-4

ubiC/CSA
dca/ECL
aroG/CG


PHE20-5

ubiC/CKO
dca/ECL
aroG/CG


PHE20-6

ubiC/CIT
dca/ECL
aroG/CG


PHE20-7

ubiC/ECL
dca/ECL
aroG/CG


PHE20-8

ubiC/MAQ
dca/ECL
aroG/CG


PHE20-9

ubiC/MME
dca/ECL
aroG/CG


PHE20-10

ubiC/PAM
dca/ECL
aroG/CG


PHE20-11

ubiC/PHA
dca/ECL
aroG/CG


PHE20-12

ubiC/REH
dca/ECL
aroG/CG


PHE20-13

ubiC/SPC
dca/ECL
aroG/CG


PHE20-14

ubiC/TBD
dca/ECL
aroG/CG





<Abbreviation for gene origin>


BS; Bacillus subtilis


EC; Escherichia coli


PP; Pseudomonas putida


CG; Corynebacterium glutamicum


BAE; Bacillus atrophaeus


BSS; Bacillus subtilis subsp. spizizenii


CKO; Citrobacter koseri


EAE; Enterobacter aerogenes


ECL; Enterobacter cloacae


EHO; Enterobacter hormaechei


ESA; Enterobacter sakazakii


ECK; Escherichia coli W


EFE; Escherichia fergusonii


PPY; Paenibacillus polymyxa


PAM; Pantoea ananatis


ACB; Acinetobacter baumannii


AVN; Azotobacter vinelandii


CSA; Chromohalobacter salexigens


CIT; Citrobacter youngae


MAQ; Marinobacter aquaeolei


MME; Marinomonas mediterranea


PHA; Pseudoalteromonas haloplanktis


REH; Ralstonia eutropha


SPC; Shewanella putrefaciens


TBD; Thiobacillus denitrificans


<Gene name>


ubiC; chorismate-pyruvate lyase gene


bsdBCD; 4-hydroxybenzoate decarboxylase gene


aroG; DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) gene


pobA; 4-hydroxybenzoate hydroxylase gene


dca; 4-hydroxybenzoate decarboxylase gene


poxF; phenol 2-monooxygenase gene






Example 2
Experiment of Phenol Production Using Corynebacterium glutamicum Phenol-Producing Gene Transgenic Strains and Corynebacterium Glutamicum by-Product Formation Pathway Disruptants
Phenol production was compared among the Corynebacterium glutamicum phenol-producing gene transgenic strains PHE11 to PHE18 prepared in Example 1.
Each of the Corynebacterium glutamicum phenol-producing gene transgenic strains (PHE11 to PHE18) was applied to A agar medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 L of distilled water) containing antibiotics shown in Table 2, and left stand in the dark at 28° C. for 20 hours.
An inoculation loop of each of the Corynebacterium glutamicum phenol-producing gene transgenic strains grown on a plate as above was inoculated into a test tube containing 10 mL of A liquid medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, and 40 g of glucose were dissolved in 1 L of distilled water) containing antibiotics shown in Table 2, and aerobically cultured with shaking at 28° C. for 15 hours.
Each of the Corynebacterium glutamicum phenol-producing gene transgenic strains grown in the above conditions was inoculated to 10 mL of A liquid medium containing the antibiotics, and aerobically cultured with shaking at 33° C. for 24 hours.
For quantitative determination of phenol, the reaction mixture sampled 24 hours later was centrifuged (15,000×g at 4° C. for 10 minutes), and the obtained supernatant was analyzed by liquid chromatography.
Also, using Corynebacterium glutamicum wild strain, phenol production was attempted in the same manner. However, no phenol production was detected.
The amounts of phenol produced by the strains are shown in Table 2 below.







TABLE 2







Experiment of phenol production using phenol -producing gene transgenic strains












Host strain






Corynebacterium
Transfected gene
Antibiotics
Amount of phenol


Strain
glutamicum
(gene name/origin)
added to medium
production (mM)
















PHE11
Wild strain
ubiC/EC
bsdBCD/BS
—
A, B
0.4


PHE12

ubiC/EC
bsdBCD/BS
aroG/C
A, B, C
0.9


PHE13
ΔpobA
ubiC/EC
bsdBCD/BS
—
A, B
0.8


PHE14

ubiC/EC
bsdBCD/BS
aroG/C
A, B, C
1.8


PHE15
Wild strain
ubiC/PP
bsdBCD/BS
—
A, B
1.1


PHE16

ubiC/PP
bsdBCD/BS
aroG/C
A, B, C
3.5


PHE17
ΔpobA
ubiC/PP
bsdBCD/BS
—
A, B
1.6


PHE18

ubiC/PP
bsdBCD/BS
aroG/C
A, B, C
6.3





<Abbreviation for gene origin>


BS; Bacillus subtilis


EC; Escherichia coli


PP; Pseudomonas putida


CG; Corynebacterium glutamicum


<Antibiotics added to the medium>


A; Chloramphenicol 5 μg/mL


B; Kanamycin 50 μg/mL


C; Zeocin 25 μg/mL


<Gene name>


ubiC; chorismate-pyruvate lyase gene


bsdBCD; 4-hydroxybenzoate decarboxylase gene


aroG; DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) gene


pobA; 4-hydroxybenzoate hydroxylase gene






As shown in Table 2, Corynebacterium glutamicum PHE11 produced 0.4 mM of phenol, PHE12 produced 0.9 mM of phenol, PHE13 produced 0.8 mM of phenol, PHE14 produced 1.8 mM of phenol, PHE15 produced 1.1 mM of phenol, PHE16 produced 3.5 mM of phenol, PHE17 produced 1.6 mM of phenol, and PHE18 produced 6.3 mM of phenol in the respective culture media.
These results show the following.
(1) Practical phenol production from glucose was first achieved by a Corynebacterium glutamicum transfected with both a ubiC gene and a bsdBCD gene.
(2) In the case where the ubiC gene derived from Pseudomonas putida was used, higher phenol productivity can be obtained than in the case where the ubiC gene derived from Escherichia coli was used.
(3) Phenol production was augmented by the use of an aroG gene in addition to a ubiC gene and a bsdBCD gene.
(4) Phenol production was further improved by pobA gene disruption.
Example 3
Experiment of Phenol Production Using Corynebacterium glutamicum Phenol-Producing Gene Transgenic Strains and Corynebacterium Glutamicum by-Product Formation Pathway Disruptants
Phenol production was compared among the Corynebacterium glutamicum phenol-producing gene transgenic strains PHE19-1 to PHE19-12 prepared in Example 1.
Each of the Corynebacterium glutamicum phenol-producing gene transgenic strains (PHE19-1 to PHE19-12) was applied to A agar medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 L of distilled water) containing antibiotics, namely, 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and left stand in the dark at 28° C. for 20 hours.
An inoculation loop of each of the Corynebacterium glutamicum phenol-producing gene transgenic strains grown on a plate as above was inoculated into a test tube containing 10 mL of A liquid medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, and 40 g of glucose were dissolved in 1 L of distilled water) containing antibiotics, namely, 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and aerobically cultured with shaking at 28° C. for 15 hours.
Each of the Corynebacterium glutamicum phenol-producing gene transgenic strains grown in the above conditions was inoculated to 10 mL of A liquid medium containing antibiotics, namely, 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and aerobically cultured with shaking at 33° C. for 24 hours.
For quantitative determination of phenol, the reaction mixture sampled 24 hours later was centrifuged (15,000×g at 4° C. for 10 minutes), and the obtained supernatant was analyzed by liquid chromatography.
The amounts of phenol produced by the strains are shown in Table 3 below.







TABLE 3







Experiment of phenol production using phenol-producing gene transgenic


strains













Amount





of





phenol



Host strain

produc-



Corynebacterium
Transfected gene
tion


Strain
glutamicum
(gene name/origin)
(mM)















PHE19-1
ΔpobAΔpoxF
ubiC/PP
bsdBCD/
aroG/CG
7.6





BS


PHE19-2

ubiC/PP
dca/BAE
aroG/CG
7.3


PHE19-3

ubiC/PP
dca/BSS
aroG/CG
7.3


PHE19-4

ubiC/PP
dca/CKO
aroG/CG
7.2


PHE19-5

ubiC/PP
dca/EAE
aroG/CG
8.5


PHE19-6

ubiC/PP
dca/ECL
aroG/CG
8.5


PHE19-7

ubiC/PP
dca/EHO
aroG/CG
8.3


PHE19-8

ubiC/PP
dca/ESA
aroG/CG
8.4


PHE19-9

ubiC/PP
dca/ECK
aroG/CG
8.5


PHE19-10

ubiC/PP
dca/EFE
aroG/CG
8.3


PHE19-11

ubiC/PP
dca/PPY
aroG/CG
7.0


PHE19-12

ubiC/PP
dca/PAM
aroG/CG
7.1





<Abbreviation for gene origin>


BS; Bacillus subtilis


EC; Escherichia coli


PP; Pseudomonas putida


CG; Corynebacterium glutamicum


BAE; Bacillus atrophaeus


BSS; Bacillus subtilis subsp. spizizenii


CKO; Citrobacter koseri


EAE; Enterobacter aerogenes


ECL; Enterobacter cloacae


EHO; Enterobacter hormaechei


ESA; Enterobacter sakazakii


ECK; Escherichia coli W


EFE; Escherichia fergusonii


PPY; Paenibacillus polymyxa


PAM; Pantoea ananatis


<Gene name>


ubiC; chorismate-pyruvate lyase gene


bsdBCD; 4-hydroxybenzoate decarboxylase gene


aroG; DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) gene


pobA; 4-hydroxybenzoate hydroxylase gene


dca; 4-hydroxybenzoate decarboxylase gene


poxF; phenol 2-monooxygenase gene






Comparison of the results in Table 3 with the result of PHE18 in Table 2 shows that poxF-gene disruption in addition to pobA-gene disruption further improved the phenol productivity.
As compared with the Corynebacterium glutamicum ΔpobAΔpoxF transfected with a bsdBCD gene derived from Bacillus subtilis in combination with a ubiC gene and an aroG gene, the counterpart transfected with a dca gene derived from Bacillus atrophaeus, a dca gene derived from Bacillus subtilis subsp. spizizenii, a dca gene derived from Citrobacter koseri, a dca gene derived from Enterobacter aerogenes, a dca gene derived from Enterobacter cloacae, a dca gene derived from Enterobacter hormaechei, a dca gene derived from Enterobacter sakazakii, a dca gene derived from Escherichia coli, a dca gene derived from Escherichia fergusonii, a dca gene derived from Paenibacillus polymyxa, or a dca gene derived from Pantoea ananatis instead of the bsdBCD gene derived from Bacillus subtilis showed an equal or a higher phenol productivity.
Example 4
Experiment of Phenol Production Using Corynebacterium glutamicum Phenol-Producing Gene Transgenic Strains and Corynebacterium Glutamicum by-Product Formation Pathway Disruptants
Phenol production was compared among the Corynebacterium glutamicum phenol-producing gene transgenic strains PHE19-6 and PHE20-1 to PHE20-14 prepared in Example 1.
Each of the Corynebacterium glutamicum phenol-producing gene transgenic strains (PHE19-6 and PHE20-1 to PHE20-14) was applied to A agar medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 L of distilled water) containing antibiotics, namely, 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and left stand in the dark at 28° C. for 20 hours.
An inoculation loop of each of the Corynebacterium glutamicum phenol-producing gene transgenic strains grown on a plate as above was inoculated into a test tube containing 10 mL of A liquid medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, and 40 g of glucose were dissolved in 1 L of distilled water) containing antibiotics, namely, 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and aerobically cultured with shaking at 28° C. for 15 hours.
Each of the Corynebacterium glutamicum phenol-producing gene transgenic strains grown in the above conditions was inoculated to 10 mL of A liquid medium containing antibiotics, namely, 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and aerobically cultured with shaking at 33° C. for 24 hours.
For quantitative determination of phenol, the reaction mixture sampled 24 hours later was centrifuged (15,000×g at 4° C. for 10 minutes), and the obtained supernatant was analyzed by liquid chromatography.
The amounts of phenol produced by the strains are shown in Table 4 below.







TABLE 4







Experiment of phenol production using phenol-producing gene transgenic


strains













Amount





of



Host strain

phenol



Coryne-

produc-



bacterium
Transfected gene
tion


Strain
glutamicum
(gene name/origin)
(mM)















PHE19-6
ΔpobAΔpoxF
ubiC/PP
dca/ECL
aroG/CG
8.5


PHE20-1

ubiC/EC
dca/ECL
aroG/CG
8.3


PHE20-2

ubiC/ACB
dca/ECL
aroG/CG
3.5


PHE20-3

ubiC/AVN
dca/ECL
aroG/CG
7.5


PHE20-4

ubiC/CSA
dca/ECL
aroG/CG
4.4


PHE20-5

ubiC/CKO
dca/ECL
aroG/CG
7.7


PHE20-6

ubiC/CIT
dca/ECL
aroG/CG
9.7


PHE20-7

ubiC/ECL
dca/ECL
aroG/CG
9.3


PHE20-8

ubiC/
dca/ECL
aroG/CG
4.1




MAQ


PHE20-9

ubiC/
dca/ECL
aroG/CG
3.9




MME


PHE20-10

ubiC/PAM
dca/ECL
aroG/CG
11.8


PHE20-11

ubiC/PHA
dca/ECL
aroG/CG
8.4


PHE20-12

ubiC/REH
dca/ECL
aroG/CG
4.5


PHE20-13

ubiC/SPC
dca/ECL
aroG/CG
2.2


PHE20-14

ubiC/TBD
dca/ECL
aroG/CG
1.9





<Abbreviation for gene origin>


PP; Pseudomonas putida


ECL; Enterobacter cloacae


CG; Corynebacterium glutamicum


EC; Escherichia coli


ACB; Acinetobacter baumannii


AVN; Azotobacter vinelandii


CSA; Chromohalobacter salexigens


CKO; Citrobacter koseri


CIT; Citrobacter youngae


MAQ; Marinobacter aquaeolei


MME; Marinomonas mediterranea


PAM; Pantoea ananatis


PHA; Pseudoalteromonas haloplanktis


REH; Ralstonia eutropha


SPC; Shewanella putrefaciens


TBD; Thiobacillus denitrificans


<Gene name>


ubiC; chorismate-pyruvate lyase gene


aroG; DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) gene


pobA; 4-hydroxybenzoate hydroxylase gene


dca; 4-hydroxybenzoate decarboxylase gene


poxF; phenol 2-monooxygenase gene






The results in Table 4 show that as is the case in the Corynebacterium glutamicum ΔpobAΔpoxF transfected with a ubiC gene derived from Pseudomonas putida in combination with a ubiC gene and an aroG gene, the counterpart transfected with a ubiC gene derived from Escherichia coli, a ubiC gene derived from Acinetobacter baumannii, a ubiC gene derived from Azotobacter vinelandii, a ubiC gene derived from Chromohalobacter salexigens, a ubiC gene derived from Citrobacter koseris, a ubiC gene derived from Citrobacter youngae, a ubiC gene derived from Marinobacter aquaeolei, a ubiC gene derived from Marinomonas mediterranea, a ubiC gene derived from Pantoea ananatis, a ubiC gene derived from Pseudoalteromonas haloplanktis, a ubiC gene derived from Ralstonia eutropha, a ubiC gene derived from Shewanella putrefaciens, or a ubiC gene derived from Thiobacillus denitrificans instead of the ubiC gene derived from Pseudomonas putida showed a significant phenol productivity.
Example 5
Experiment of Phenol Production Using Corynebacterium glutamicum PHE18 Under Reducing Conditions
The Corynebacterium glutamicum phenol-producing strain PHE18 created in Example 1 was applied to A agar medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and left stand in the dark at 28° C. for 20 hours.
An inoculation loop of the Corynebacterium glutamicum phenol-producing strain PHE18 grown on a plate as above was inoculated into a test tube containing 10 mL of A liquid medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and aerobically cultured with shaking at 28° C. for 10 hours.
The Corynebacterium glutamicum phenol-producing strain PHE18 grown in the above conditions was inoculated into a 2 L-conical flask containing 500 mL of A liquid medium containing 5 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 25 μg/mL of zeocin, and aerobically cultured with shaking at 33° C. for 15 hours.
The bacterial cells of the strain cultured and proliferated as above were collected by centrifugation (5,000×g at 4° C. for 15 minutes). The obtained bacterial cells were suspended in 50 mL of BT (−urea) liquid medium (0.7% ammonium sulfate, 0.05% potassium dihydrogen phosphate, 0.05% dipotassium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.0006% iron sulfate heptahydrate, 0.00042% manganese sulfate hydrate, 0.00002% biotin and 0.00002% thiamine hydrochloride) so that the final concentration of the bacterial cell was 10%. To a 100-mL medium bottle, 60 mL of the cell suspension was transferred, glucose was added, and the reaction was allowed to proceed under reducing conditions (the ORP of the reaction mixture: −450 mV) at 33° C. with stirring. As for the glucose addition, glucose was added so as to be 5% in concentration at first, and another 5% was added 12 hours later. During the reaction, the pH of the reaction mixture was kept at or above 7.0 through addition of 2.5 N aqueous ammonia controlled by a pH controller (Type: DT-1023 made by Able).
A sample of the reaction mixture was centrifuged (15,000×g at 4° C. for 10 minutes), and the obtained supernatant was used for quantitative determination of phenol.
As a result, in the reaction under reducing conditions, the Corynebacterium glutamicum phenol-producing strain PHE18 produced 9.2 mM of phenol 24 hours after the start of the reaction.
Thus, it was revealed that the transformant of the present invention further efficiently produced phenol under reducing conditions.
Example 6
Test for Suitability as a Host for Phenol Production
(1) Influence of Phenol on Aerobic Proliferation
A growth inhibition test in aerobic culture was performed to examine the influence of phenol on Corynebacterium glutamicum, Escherichia coli, and Pseudomonas putida. Pseudomonas putida S12, which was used for the test, is reported to be a solvent-resistant strain. In the report, disclosed is an unparalleled technology using the strain as a host in phenol production.
Corynebacterium glutamicum R was applied to A agar medium (2 g of (NH2)2C, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 L of distilled water) and was left stand in the dark at 33° C. for 15 hours.
An inoculation loop of the Corynebacterium glutamicum R grown on a plate as above was inoculated into a test tube containing 10 mL of A liquid medium (2 g of (NH2)2CO, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4.7H2O, 1 mL of 0.06% (w/v) Fe2SO4.7H2O+0.042% (w/v) MnSO4.2H2O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casamino acid, and 40 g of glucose were suspended in 1 L of distilled water) and was aerobically cultured with shaking at 33° C. for 13 hours.
The Corynebacterium glutamicum R grown in the above conditions was inoculated into 100 mL of A liquid medium in such a way that the initial bacterial cell concentration would be OD610=0.05, phenol was added at the same time in such a way that the final concentration would be 0, 0.16, 0.2, 0.24, or 0.32 mM, and aerobic culture was performed with shaking at 33° C. The growth of bacterial cells was determined by absorbance measurement at OD610.
Escherichia coli JM109 was applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl and 1.5% agar) and was left stand in the dark at 37° C. for 15 hours.
An inoculation loop of the Escherichia coli JM109 grown on a plate as above was inoculated into a test tube containing 10 mL of LB liquid medium (1% polypeptone, 0.5% yeast extract, and 0.5% NaCl), and aerobic culture was performed with shaking at 37° C. for 13 hours.
The Escherichia coli JM109 grown in the above conditions was inoculated into 100 mL of LB liquid medium in such a way that the initial bacterial cell concentration would be OD610=0.05, phenol was added at the same time in such a way that the final concentration would be 0, 0.16, or 0.20 mM, and aerobic culture was performed with shaking at 37° C. The growth of bacterial cells was determined by absorbance measurement at OD610.
Pseudomonas putida F1 and S12 were applied to LB agar medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl and 1.5% agar) and were left stand in the dark at 30° C. for 15 hours.
An inoculation loop of each of the Pseudomonas putida F1 and S12 grown on a plate as above was inoculated into a test tube containing 10 mL of LB (+glucose) liquid medium (1% polypeptone, 0.5% yeast extract, 0.5% NaCl and 0.4% glucose), and aerobic culture was performed with shaking at 30° C. for 13 hours.
The Pseudomonas putida F1 and S12 grown in the above conditions were each inoculated into 100 mL of LB (+glucose) liquid medium in such a way that the initial bacterial cell concentration would be OD610=0.05, phenol was added at the same time in such a way that the final concentration would be 0, 0.10, or 0.20 mM, and aerobic culture was performed with shaking at 30° C. The growth of bacterial cells was determined by absorbance measurement at OD610. FIG. 4 shows analysis results of the influence of phenol addition on aerobic proliferation.
The proliferation of Escherichia coli was significantly affected by 0.16% phenol and completely inhibited by 0.20% phenol.
Pseudomonas putida F1, and Pseudomonas putida S12, which was reported as a solvent-resistant strain, showed a similar tendency, and the proliferation thereof was significantly affected by 0.10% phenol and completely inhibited by 0.20% phenol.
In contrast, the proliferation of Corynebacterium glutamicum was hardly affected by 0.16% phenol, which significantly affected the proliferation of Escherichia coli. Even in the presence of 0.20% phenol, which completely inhibited the proliferation of Escherichia coli and Pseudomonas putida, Corynebacterium glutamicum showed favorable growth. Further, Corynebacterium glutamicum was able to proliferate in the presence of 0.24% phenol.
Thus, it was shown that Corynebacterium glutamicum has a higher resistance to phenol as compared with Escherichia coli and Pseudomonas putida, and is highly suitable as a host in phenol production.
(2) Influence of Phenol on Saccharide Metabolism Under Reducing Conditions
Corynebacterium glutamicum R was applied to A agar medium and was left stand in the dark at 33° C. for 20 hours.
An inoculation loop of the Corynebacterium glutamicum R grown on a plate as above was inoculated into a test tube containing 10 mL of A liquid medium and was aerobically cultured with shaking at 33° C. for 15 hours.
The Corynebacterium glutamicum R grown in the above conditions was inoculated into a 2 L-conical flask containing 500 mL of A liquid medium and was aerobically cultured with shaking at 33° C. for 15 hours.
The bacterial cells cultured and proliferated as above were collected by centrifugation (5,000×g at 4° C. for 15 minutes). The obtained bacterial cells were suspended in BT (−urea) liquid medium (0.7% ammonium sulfate, 0.05% potassium dihydrogen phosphate, 0.05% dipotassium hydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 0.0006% iron sulfate heptahydrate, 0.00042% manganese sulfate hydrate, 0.00002% biotin and 0.00002% thiamine hydrochloride) so that the concentration of the bacterial cell was 10% (w/v). To a 100-mL medium bottle, 60 mL of the cell suspension was transferred, glucose and phenol were added so as to be 8% and 0, 0.24, 0.38, or 0.46 mM in concentration, respectively, and the reaction was allowed to proceed under reducing conditions (the ORP of the reaction mixture: −450 mV) in a water bath kept at 33° C. with stirring. During the reaction, the pH of the reaction mixture was kept at or above 7.0 through addition of 2.5N aqueous ammonia controlled by a pH controller (Type: DT-1023 made by Able).
FIG. 5 shows the influence of phenol on the saccharide metabolism in Corynebacterium glutamicum R under reducing conditions.
Under reducing conditions, even in the presence of 0.24% phenol, which caused proliferation inhibition in aerobic culture, no influence of phenol was observed, and the saccharide consumption was comparable to that in the case free from phenol.
Further, saccharide consumption was observed even in the presence of 0.38% phenol, and was slightly observed even in the presence of 0.46% phenol.
Thus, it was shown that Corynebacterium glutamicum has a higher resistance to phenol under reducing conditions as compared with in aerobic culture, and that phenol production using Corynebacterium glutamicum as a host under reducing conditions is advantageous as compared with the production under aerobic conditions.
INDUSTRIAL APPLICABILITY
According to the process of the present invention, phenol can be produced with a practical efficiency using microorganisms.

## Claims

1. A phenol-producing transformant, wherein the phenol-producing transformant is a Corynebacterium transformed with a Pantoea ananatis chorismate-pyruvate lyase gene and an Enterobacter cloacae 4-hydroxybenzoate decarboxylase gene.

2. The phenol-producing transformant of claim 1, wherein the Pantoea ananatis chorismate-pyruvate lyase gene consists of the nucleotide sequence of SEQ ID NO: 89.

3. The phenol-producing transformant of claim 1, wherein the Enterobacter cloacae 4-hydroxybenzoate decarboxylase gene consists of the nucleotide sequence of SEQ ID NO: 56.

4. The phenol-producing transformant of claim 1, wherein a 4-hydroxybenzoate hydroxylase gene on the chromosome of the Corynebacterium is disrupted or deleted.

5. The phenol-producing transformant of claim 1, wherein a phenol 2-monooxygenase gene on the chromosome of the Corynebacterium is disrupted or deleted.

6. The phenol-producing transformant of claim 1, wherein the Corynebacterium is a Corynebacterium glutamicum.

7. The phenol-producing transformant of claim 1, wherein the Corynebacterium is Corynebacterium glutamicum R having accession number FERM BP-18976, ATCC13032, or ATCC13869.

8. The phenol-producing transformant of claim 7, wherein the Corynebacterium is Corynebacterium glutamicum R having accession number FERM BP-18976, or ATCC13032 or ATCC13869, and wherein a 4-hydroxybenzoate hydroxylase gene on the chromosome of the Corynebacterium glutamicum is disrupted or deleted.

9. The phenol-producing transformant of claim 7, wherein the Corynebacterium is Corynebacterium glutamicum R having accession number FERM BP-18976, or ATCC13032 or ATCC13869, and wherein a phenol 2-monooxygenase gene on the chromosome of the Corynebacterium glutamicum is disrupted or deleted.

10. A process for producing phenol, which comprises reacting the phenol-producing transformant of claim 1 with a saccharide in a reaction mixture under reducing conditions to produce phenol, and collecting phenol from the reaction mixture.

11. The process of claim 10, wherein the phenol-producing transformant does not proliferate in the reacting step.

12. The process of claim 10, wherein the oxidation-reduction potential of the reaction mixture under reducing conditions is −200 mV to −500 mV.

13. The process of claim 10, wherein the saccharide is selected from the group consisting of glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol.

14. The phenol-producing transformant of claim 1, wherein expression of a Corynebacterium aroG gene which encodes an enzyme having 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase activity is increased in the phenol-producing transformant as compared with a corresponding wild-type Corynebacterium.