JP7370587B2 - Method for producing organic compounds and coryneform bacteria - Google Patents

Description

The present invention relates to a method for producing an organic compound and a coryneform bacterium.

Currently, there is a strong desire to produce chemical products using bioresources, which are renewable resources, as raw materials instead of petroleum resources, which are considered to be the cause of global warming. Therefore, with the aim of producing chemical products from biological raw materials such as sugars, research is being conducted to produce chemical products for use as industrial raw materials, fuel, feed, food additives, etc. using microorganisms and their transformants. is being actively carried out. For example, it has been reported that yeast or E. coli transformants are used to produce organic compounds such as alcohols and amino acids from sugars (see US Pat. No. 9,926,577 and US Pat. No. 8,647,847).

An object of the present invention is to provide a new method for producing organic compounds and coryneform bacteria that can be used in this production method.
The production of chemical products using microorganisms is generally limited to microbial metabolites. Moreover, even among microbial metabolites, there are many microbial metabolites that are difficult to produce due to energy shortage or redox imbalance in the microbial metabolic system. One effective approach to solving such problems is to create transformants. However, the present inventors believe that if an attempt is made to solve the problem using only this method, it may take a lot of time to develop microbial cells that can produce the target compound. On the other hand, we believe that in the future there will be a need to produce a variety of compounds other than microbial metabolites from biological raw materials. Therefore, the present inventors investigated the development of a new process that does not rely solely on the creation of microbial transformants for the production of useful substances.

As a result of intensive studies, the present inventors discovered a production process in which intermediates, which are key to the synthesis of useful substances such as amino acids, can be obtained by culturing microorganisms having specific genes in the presence of carbonyl compounds. I was able to complete it.

According to the first aspect of the invention,
A microorganism containing a gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and a pyruvate-supplying compound selected from pyruvate and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(where R ₁₀₁ and R ₁₀₂ are independently of each other hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
Provided is a method for producing an α-keto acid, the method comprising the step of producing an α-keto acid represented by

According to the second aspect of the invention,
The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(b) a second gene encoding an enzyme that catalyzes the decarboxylation reaction of α-keto acids; and (c) a third gene encoding an enzyme that catalyzes the reduction reaction of aldehydes. a selected pyruvate-providing compound;
General formula (3) below:
R ₁₀₃ C(O)R ₁₀₄
(Here, R ₁₀₃ and R ₁₀₄ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms. However, when R ₁₀₃ is hydrogen, R ₁₀₄ is not hydrogen.)
by culturing in the presence of a carbonyl compound represented by
General formula (4) below:
R ₁₀₃ C(R ₁₀₄ )(OH)CH ₂ CH ₂ OH
(R ₁₀₃ and R ₁₀₄ in the above general formula (4) are the same groups as R ₁₀₃ and R ₁₀₄ in the above general formula (3), respectively)
Provided is a method for producing 1,3-diol, which includes the step of producing 1,3-diol represented by

According to a third aspect of the invention,
The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvate and a carbonyl compound;
(b) a second gene encoding an enzyme that catalyzes the decarboxylation reaction of α-keto acids; and (c) a third gene encoding an enzyme that catalyzes the reduction reaction of aldehydes. A method for producing 1,3-butanediol is provided, comprising culturing in the presence of a selected pyruvate-providing compound to produce 1,3-butanediol.

According to the fourth aspect of the invention,
The following genes:
A microorganism containing (a) a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (d) a fourth gene encoding an enzyme that catalyzes a reduction reaction of an α-keto acid was used to a pyruvate-providing compound selected from acids and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(where R ₁₀₁ and R ₁₀₂ are independently of each other hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (5) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(OH)COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (5) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
Provided is a method for producing 2,4-dihydroxycarboxylic acid, which includes the step of producing 2,4-dihydroxycarboxylic acid represented by

According to the fifth aspect of the invention,
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
α-keto acid, nitrogen source and reducing agent represented by
React in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase,
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (2), respectively)
Provided is a method for producing 2-amino-4-hydroxycarboxylic acid, which includes the step of producing 2-amino-4-hydroxycarboxylic acid represented by

According to the sixth aspect of the invention,
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
2-amino-4-hydroxycarboxylic acid represented by and an oxidizing agent,
React in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase,
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (6), respectively)
Provided is a method for producing an α-keto acid, the method comprising the step of producing an α-keto acid represented by

According to the seventh aspect of the invention,
The following genes:
(a) A microorganism comprising a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (e) a fifth gene encoding an enzyme that catalyzes a reductive amination reaction of an α-keto acid. a pyruvate-providing compound selected from pyruvate and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
Provided is a method for producing 2-amino-4-hydroxycarboxylic acid, which includes the step of producing 2-amino-4-hydroxycarboxylic acid represented by

According to the eighth aspect of the invention,
The following genes:
(a) A microorganism comprising a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (e) a fifth gene encoding an enzyme that catalyzes a reductive amination reaction of an α-keto acid. in the presence of formaldehyde and a pyruvate-providing compound selected from pyruvic acid and sugars to produce at least one of homoserine, threonine and 2-aminobutyric acid. A method for producing at least one aminobutyric acid is provided.

According to the ninth aspect of the invention,
The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(e) the fifth gene encoding an enzyme that catalyzes the reductive amination reaction of α-keto acids, and (f) the methyl mercaptan that has the phosphate group that 2-amino-4-phosphonooxycarboxylic acid has. A microorganism containing the sixth gene encoding an enzyme that catalyzes the reaction of substitution with a mercapto group is treated with a pyruvate-supplying compound selected from pyruvate and sugars, and methyl mercaptan.
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (7) below:
R ₁₀₁ C(R ₁₀₂ )(SCH ₃ )CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (7) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
Provided is a method for producing 2-amino-4-methylthiocarboxylic acid, which includes the step of producing 2-amino-4-methylthiocarboxylic acid represented by

According to the tenth aspect of the invention,
The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
A coryneform bacterium is provided.

According to the present invention, a method for producing an organic compound and a coryneform bacterium that can be used in this production method are provided.

Schematic diagram showing the process by which microorganisms produce 4-hydroxy-2-oxobutyric acid. A schematic diagram showing a process in which microorganisms produce 1,3-butanediol. Schematic diagram showing the process by which microorganisms produce 2,4-dihydroxybutyric acid. Schematic diagram showing the process by which microorganisms produce homoserine. Schematic diagram showing the process by which microorganisms produce methionine. A diagram showing the results of gas chromatograph mass spectrometry. A diagram showing the results of gas chromatograph mass spectrometry. A diagram showing the results of gas chromatograph mass spectrometry. A diagram showing the results of gas chromatograph mass spectrometry.

Hereinafter, the present invention will be explained in detail, but the following explanation is for the purpose of explaining the present invention in detail and is not intended to limit the present invention.
(1.) Method for producing α-keto acid The method for producing α-keto acid is as follows:
A microorganism containing a gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and a pyruvate-supplying compound selected from pyruvate and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(where R ₁₀₁ and R ₁₀₂ are independently of each other hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
The method includes a step of producing an α-keto acid represented by

In the following description, the above microorganism containing a gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound (hereinafter referred to as the first gene) is also referred to as an "α-keto acid producing microorganism". The α-keto acid-producing microorganism may be a microorganism that naturally has the first gene, or may be a microorganism obtained by transforming a host with the first gene. The case where the α-keto acid producing microorganism is a transformant will be explained below as an example.

(1.1) Transformants (1.1.1) Host Any microorganism can be used as the host. As the host, for example, yeast, bacteria, more specifically aerobic bacteria, more specifically coryneform bacteria, Escherichia coli, or Vibrio natriegens can be used. Preferably, the host is a coryneform bacterium.

Coryneform bacteria are a group of microorganisms defined in Bargeys Manual of Determinative Bacteriology, Vol. 8, 599 (1974), and are There are no particular limitations as long as it proliferates. Specific examples include bacteria of the genus Corynebacterium, bacteria of the genus Brevibacterium, bacteria of the genus Arthrobacter, bacteria of the genus Mycobacterium, and bacteria of the genus Micrococcus. Among the coryneform bacteria, Corynebacterium genus bacteria are preferred.

Corynebacterium genus bacteria include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkano. Examples include Corynebacterium alkanolyticum. Among them, Corynebacterium glutamicum is preferred because of its high productivity of α-keto acids. Suitable strains include ATCC13032 strain, ATCC13869 strain, ATCC13058 strain, ATCC13059 strain, ATCC13060 strain, ATCC13232 strain, ATCC13286 strain, ATCC13287 strain, ATCC13655 strain, ATCC13745 strain, ATCC13746 strain, ATCC13761 strain, ATCC1402. 0 shares, ATCC31831 shares, MJ-233 (FERM BP-1497), MJ-233AB-41 (FERM BP-1498), etc. Among these, ATCC13032 strain and ATCC13869 strain are preferred.

In addition, according to molecular biological classification, coryneforms such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, and Corynebacterium lilium The bacterial name is also unified 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), Kazuo Komagata et al., Classification of Coryneform bacteria, Fermentation and Industry, 45:944-963 (1987) ].

Older classifications of Brevibacterium lactofermentum ATCC13869 and Brevibacterium flavum MJ-233 (FERM BP-1497) and MJ-233AB-41 (FERM BP-1498) are also suitable Corynebacterium glutamicum. .

Examples of the Brevibacterium genus include Brevibacterium ammoniagenes (for example, ATCC6872 strain).

Examples of the Arthrobacter genus include Arthrobacter globiformis (eg, ATCC8010 strain, ATCC4336 strain, ATCC21056 strain, ATCC31250 strain, ATCC31738 strain, ATCC35698 strain), and the like.

Examples of the Mycobacterium genus include Mycobacterium bovis (eg, ATCC19210 strain, ATCC27289 strain), and the like.

Examples of Micrococcus include Micrococcus freudenreichii (e.g., strain No. 239 (FERM P-13221)), Micrococcus leuteus (e.g., strain No. 240 (FERM P-13222)), Examples include Micrococcus ureae (for example, IAM1010 strain), Micrococcus roseus (for example, IFO3764 strain), and the like.

Next, the first gene introduced into the host will be explained.

(1.1.2) First gene The first gene encodes "an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound.""An enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound" means a purified protein, pyruvate (initial substrate concentration 1mM), and carbonyl compound (initial substrate concentration 1mM) as an enzyme reaction solution according to the measurement method described below. An enzyme having an activity of 10 mU/mg protein or more when measured using an enzyme reaction solution containing The carbonyl compound in the "enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound" is, for example, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, isobutyraldehyde, valeraldehyde, or acetone. Butyraldehyde is specifically normal butyraldehyde or isobutyraldehyde. Valeraldehyde is specifically normal valeraldehyde. The "enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound" is, for example, aldolase.

The first gene may be a gene encoding an aldolase as exemplified below.

(a1) 2-dehydro-3-deoxy-phosphogluconate aldolase (2-dehydro-3-deoxy-phosphogluconate aldolase, EC number: 4.1.2.14),
(a2) 2-dehydro-3-deoxy-6-phosphogluconate aldolase (2-dehydro-3-deoxy-6-phosphogluconate aldolase, EC number 4.1.2.55),
(a3) 4-hydroxy-2-oxoglutarate aldolase (4-hydroxy-2-oxoglutarate aldolase, EC number: 4.1.3.16),
(a4) (4S)-4-hydroxyl-2-oxoglutarate aldolase ((4S)-4-hydroxyl-2-oxoglutarate aldolase, EC number 4.1.3.42),
(a5) 2-dehydro-3-deoxy-D-pentonate aldolase (2-dehydro-3-deoxy-D-pentonate aldolase, EC number 4.1.2.28),
(a6) 2-dehydro-3-deoxy-D-gluconate aldolase (2-dehydro-3-deoxy-D-gluconate aldolase, EC number 4.1.2.51),
(a7) 3-deoxy-D-manno-octulosonate aldolase (3-deoxy-D-manno-octulosonate aldolase, EC number 4.1.2.23),
(a8) 4-hydroxyl-2-oxovalerate aldolase (4-hydroxyl-2-oxovalerate aldolase, EC number 4.1.3.39),
(a9) 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase, EC number 4.1.2.34),
(a10) 4-hydroxy-2-oxoheptanedioate aldolase (4-hydroxy-2-oxoheptanedioate aldolase, EC number 4.1.2.52),
(a11) 2-dehydro-3-deoxyglucarate aldolase (2-dehydro-3-deoxyglucarate aldolase, EC number 4.1.2.20),
(a12) 2-keto-3-deoxy-L-rhamnonate aldolase (2-keto-3-deoxy-L-rhamnonate aldolase, EC number 4.1.2.53),
(a13) 4-hydroxyl-4-methyl-2-oxoglutarate aldolase (EC number 4.1.3.17), and (a14) 4-hydroxy- 2-oxohexanonate aldolase (4-hydroxy-2-oxohexanonate aldolase, EC number 4.1.3.43).

Examples of aldolases included in (a1), (a2), (a3), and (a4) include eda protein. Preferably, the eda protein is derived from E. coli.
Examples of the aldolase included in (a2) include dgoA protein. Preferably, the dgoA protein is derived from E. coli.
Examples of the aldolase included in (a5) include yjhH protein. Preferably, the yjhH protein is derived from E. coli.
Examples of the aldolase contained in (a6) include yagE protein. Preferably, the yagE protein is derived from E. coli.
Examples of the aldolase included in (a7) include nanA protein. Preferably, the nanA protein is derived from E. coli.
An example of the aldolase contained in (a8) is mhpE protein. Preferably, the mhpE protein is derived from E. coli. In addition, when the first gene is the mhpE gene, the mhpE gene may be introduced into the host in a state linked to the mhpF gene.
Examples of aldolases included in (a8) and (a10) include hpaI protein. Preferably, the hpaI protein is derived from E. coli.
Examples of the aldolase included in (a8) and (a14) include bphI protein. Preferably, the bphI protein is derived from Burkholderia xenovorans. In addition, when the first gene is the bphI gene, the bphI gene may be introduced into the host in a state linked to the bphJ gene.
Examples of the aldolase included in (a9) include phdJ protein. Preferably, the phdJ protein is derived from Nocardioides sp.
Examples of the aldolase contained in (a11) include garL protein. Preferably, the garL protein is derived from E. coli.
Examples of the aldolase included in (a12) include rhmA protein. Preferably, the rhmA protein is derived from E. coli.
Examples of the aldolase included in (a13) include galC protein. Preferably, the galC protein is derived from Pseudomonas putida.

The first gene may encode a class I aldolase or a class II aldolase.

Class I aldolases include, for example, eda protein, yjhH protein, yagE protein, dgoA protein, nanA protein, mphE protein, and phdJ protein.

Class II aldolases include, for example, hpaI protein, garL protein, rhmA protein, galC protein, and bphI protein.

Preferably, the first gene is selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 (i.e., hpaI protein derived from Escherichia coli) that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 (i.e. rhmA protein derived from Escherichia coli), a gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 (i.e., Escherichia coli-derived nanA protein), a gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-7) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8 (i.e., eda protein derived from Escherichia coli) and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10 (i.e., yjhH protein derived from Escherichia coli), a gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12 (i.e., dgoA protein derived from Escherichia coli), a gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14 (i.e., mhpE protein derived from Escherichia coli),
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16 (i.e., garL protein derived from Escherichia coli),
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18 (i.e., galC protein derived from Pseudomonas putida), a gene encoding a protein that catalyzes the aldol reaction between pyruvate and a carbonyl compound,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20 (i.e., phdJ protein derived from Nocardioides), which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound ,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22 (i.e. bphI protein derived from Burkholderia xenovorans), a gene encoding a protein that catalyzes the aldol reaction between pyruvate and a carbonyl compound; and (1-22) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 22, and has an activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. The gene that codes for.

In this specification, "one or several amino acids" in the expression "one or several amino acids have been deleted, substituted, or added" may vary depending on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. Although the number varies , it is more preferably 1 to 30 pieces, still more preferably 1 to 15 pieces, even more preferably 1 to 10 pieces, and even more preferably 1 to 5 pieces.

The above deletion, substitution, or addition of one or several amino acids is, for example, a conservative mutation that maintains normal protein function. A typical conservative variation is a conservative substitution. Conservative substitutions are between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid, and between polar amino acids. In the case of an amino acid with a hydroxyl group, between Gln and Asn; in the case of a basic amino acid, between Lys, Arg, and His; in the case of an acidic amino acid, between Asp and Glu; in the case of an amino acid with a hydroxyl group is a mutation in which Ser and Thr are substituted for each other. Specifically, substitutions that are considered conservative substitutions include Ala to Ser or Thr, Arg to Gln, His, or Lys, Asn to Glu, Gln, Lys, His, or Asp, Substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Gly, Asn, Gln, Lys or Asp. Substitution, Gly to Pro, His to Asn, Lys, Gln, Arg or Tyr, Ile to Leu, Met, Val or Phe, Leu to Ile, Met, Val or Phe, Substitution of Lys with Asn, Glu, Gln, His or Arg, Substitution of Met with Ile, Leu, Val or Phe, Substitution of Phe with Trp, Tyr, Met, Ile or Leu, Substitution of Ser with Thr or Ala substitutions, Thr to Ser or Ala, Trp to Phe or Tyr, Tyr to His, Phe or Trp, and Val to Met, He or Leu. Furthermore, deletions, substitutions, or additions of amino acids include those caused by naturally occurring mutations (mutants or variants) such as those based on individual differences in bacteria from which genes are derived or differences in species.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound" is " preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more with respect to the amino acid sequence set forth in SEQ ID NO: 2. A gene encoding a protein having an activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound may be used.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 4, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." is preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, with respect to the amino acid sequence set forth in SEQ ID NO: 4. It may also be a gene that encodes a protein consisting of a homologous amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 6, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.""An amino acid sequence having preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more homology to the amino acid sequence set forth in SEQ ID NO: 6. It may also be a gene that encodes a protein that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 8, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 8. It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 10, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 10. It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 12, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 12. It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 14" It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 16, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, still more preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 16" It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 18, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more with the amino acid sequence set forth in SEQ ID NO: 18. It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 20, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 20. It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 22, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound." "has a homology of 80% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 22. It may also be a gene encoding a protein consisting of an amino acid sequence and having the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

The UniprotKB accession number of the hpaI protein derived from E. coli is, for example, Q47098, the UniprotKB accession number of the rhmA protein derived from E. coli is, for example, P76469, and the UniprotKB accession number of the nanA protein derived from E. coli is, for example, Q47098. The ession number is, for example, P0A6L4, and is derived from E. coli. The UniprotKB accession number of the eda protein is, for example, P0A955, the UniprotKB accession number of the yjhH protein derived from E. coli is, for example, P39359, and the UniprotKB accession number of the dgoA protein derived from E. coli is, for example, P0A955. The sion number is, for example, Q6BF16, and mhpE derived from Escherichia coli The UniprotKB accession number of the protein is, for example, P51020, the UniprotKB accession number of the garL protein derived from E. coli is, for example, P23522, and the UniprotKB accession number of the galC protein derived from Pseudomonas putida. The ion number is, for example, Q88JX9, and Nocardioides spp. The UniprotKB accession number of the derived phdJ protein is, for example, Q79EM8, and the UniprotKB accession number of the bphI protein derived from Burkholderia xenovorans is, for example, P51015.

More preferably, the first gene is
hpaI gene derived from Escherichia coli, consisting of the base sequence set forth in SEQ ID NO: 1;
rhmA gene derived from E. coli, consisting of the base sequence set forth in SEQ ID NO: 3;
an E. coli-derived nanA gene consisting of the base sequence set forth in SEQ ID NO: 5;
an E. coli-derived eda gene consisting of the base sequence set forth in SEQ ID NO: 7;
yjhH gene derived from E. coli, consisting of the base sequence set forth in SEQ ID NO: 9;
dgoA gene derived from E. coli, consisting of the base sequence set forth in SEQ ID NO: 11;
mhpE gene derived from Escherichia coli, consisting of the base sequence set forth in SEQ ID NO: 13;
garL gene derived from E. coli, consisting of the base sequence set forth in SEQ ID NO: 15;
galC gene derived from Pseudomonas putida, consisting of the base sequence set forth in SEQ ID NO: 17;
The phdJ gene is derived from Nocardioides and consists of the base sequence set forth in SEQ ID NO: 19, or the bphI gene is derived from Burkholderia xenovorans and consists of the base sequence set forth in SEQ ID NO: 21.

Base sequences set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 The amino acid sequences described in and 22 are as follows.

Amino acid sequence homology is determined using the algorithms BLAST by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)]. be able to. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. Furthermore, when an amino acid sequence is analyzed by BLASTX based on BLAST, the parameters are, for example, score=50 and wordlength=3. To obtain gapped alignments, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform repeated searches that detect positional relationships (Id.) between molecules and relationships between molecules that share a common pattern. When using the BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters for each program can be used. Please see http://www.ncbi.nlm.nih.gov.

The activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound can be determined, for example, by performing the enzymatic reaction at 25°C in a 100mM HEPES-NaOH buffer (pH 8.0) containing 2mM magnesium chloride. It can be calculated by measuring the initial production rate of the aldol compound produced from the carbonyl compound and the carbonyl compound. The production rate of the aldol compound can be calculated, for example, from the time change in the concentration of the aldol compound itself. The concentration of the aldol compound can be measured, for example, by mixing the enzyme reaction solution and a 130 mM O-benzylhydroxyamine hydrochloride solution (pyridine:methanol:water=33:15:2) at a ratio of 1:5. This can be done by detecting the O-benzyloxime derivative produced from the aldol compound using high performance liquid chromatography and comparing it with a standard product whose concentration is known. Here, the activity in which 1 μmol of aldol compound is formed per minute at 25° C. is defined as 1 unit.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( hpaI gene variant) can be selected from DNA libraries of other species by, for example, PCR or hybridization using primers or probes designed according to the information based on the base sequence of SEQ ID NO: 2. . The genes selected in this way encode proteins that have a high probability of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 4, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( rhmA gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 6, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( nanA gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( eda gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 10, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( yjhH gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 12, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( dgoA gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( mhpE gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 16, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( garL gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( galC gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 20, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( phdJ gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 22, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound ( bphI gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

(1.1.3) Construction of recombinant vector for transformation The first gene can be integrated into a suitable plasmid vector for transformation.

As the plasmid vector, known plasmid vectors can be used depending on the host. The plasmid vector may contain a gene that controls an autonomous replication function within the host, or may contain a base sequence necessary for integration into the chromosome of the host.
Transformants can be produced using the obtained recombinant vector.

(1.1.4) Transformation As a transformation method, any known method can be used without restriction. Such known methods include, for example, the calcium chloride/rubidium chloride method, the calcium phosphate method, the DEAE-dextran mediated transfection, the electrical pulse method, and the like.

The obtained transformant may be cultured immediately after transformation using a medium commonly used for culturing transformants.
The culture temperature may be any temperature as long as it is suitable for growth of the transformant. The pH of the medium may be any pH suitable for growth of the transformant. Adjustment of the pH of the medium can be performed by known methods. The culture time may be any time as long as it is sufficient for the transformants to proliferate.

In addition, when a transformant is produced using a recombinant vector as described above, the first gene may be integrated into the chromosome of the host, or it may be inserted into the cytoplasm of the host in the form of a recombinant vector. May exist.

(1.1.5) Disruption or deletion of host chromosomal genes The host may be a wild strain, a mutant strain thereof, or an artificially genetically modified strain. When the host is a coryneform bacterium, the host may be a gene-disrupted strain or a gene-deleted strain obtained by disrupting or deleting at least one of the following genes originally possessed by a wild-type coryneform bacterium. Good: lactate dehydrogenase (ldhA) gene, phosphoenolpyrvate carboxylase (ppc) gene, pyruvate carboxylase (pyc) gene, pyruvate dehydrogenase, poxB) gene, glutamate-pyruvate aminotransferase (eg alaA) gene, acetolactate synthase (eg ilvB) gene, N-succinyldiaminopimelate aminotransferase (eg cg0931) genes, the S-(hydroxymethyl)mycothiol dehydrogenase (eg, adhE) gene, and the acetaldehyde dehydrogenase (eg, ald) gene. By using such a gene-disrupted strain or gene-deleted strain as a host, it is possible to improve the productivity of α-keto acids and suppress the production of by-products.

Construction of gene-disrupted strains or gene-deficient strains can be performed by known methods.

(1.1.6) Specific examples of α-keto acid producing microorganisms Specifically, α-keto acid producing microorganisms include:
The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
Corynebacteria containing coryneform bacteria can be used.

(1.2) Cultivation process α-keto acid can be produced by culturing an α-keto acid producing microorganism (for example, the transformant described above) in the presence of a pyruvate supplying compound and a carbonyl compound. "Cultivating in the presence of a pyruvate-supplying compound and a carbonyl compound" can be performed by culturing in a culture medium containing a pyruvate-supplying compound and a carbonyl compound. Preferably, "culturing in the presence of a pyruvate-supplying compound and a carbonyl compound" can be carried out by culturing in a culture medium to which the pyruvate-supplying compound and carbonyl compound are added.

Prior to culturing in the presence of a pyruvate-supplying compound and a carbonyl compound, it is preferable to culture and propagate the transformant described above under aerobic conditions. Culture under this aerobic condition is hereinafter referred to as growth culture, and culture in the presence of a pyruvate-supplying compound and a carbonyl compound is hereinafter referred to as production culture. Specifically, for example, culturing under aerobic conditions means culturing under conditions in which oxygen is supplied to the culture solution in an amount sufficient for the growth of the transformant during the culturing period. Cultivation under aerobic conditions can be performed by a known method. Cultivation under aerobic conditions can be achieved by supplying oxygen to the culture solution, for example, by aeration, stirring, shaking, or a combination thereof. More specifically, culture under aerobic conditions can be achieved by shaking culture or deep aeration agitation culture.

When propagation culture is performed prior to production culture, the propagation culture and subsequent production culture can be performed, for example, as follows. After propagation culture, first, a container containing the culture solution used in the propagation culture (hereinafter referred to as the propagation culture solution) and the transformant suspended in the propagation culture solution is centrifuged to perform transformation. body can be precipitated. Thereafter, the growth culture solution is removed from the container, and the isolated transformants are suspended in the culture solution used for production culture (hereinafter referred to as production culture solution) to perform production culture.

Alternatively, the propagation culture and subsequent production culture can be carried out by adding a pyruvate-providing compound and a carbonyl compound to the propagation medium containing the transformants after the propagation culture, changing the aerobic conditions to anaerobic conditions or microaerophilic conditions. By changing the conditions to the same conditions, production culture can be performed without replacing the culture medium. In this way, proliferation culture and production culture can also be performed continuously.

As used herein, the term "culture" means maintaining a transformant under specific controlled conditions suitable for growth of the transformant. During the culture period, the transformant may or may not proliferate. Therefore, the term "culture" can also be translated as "incubation."

(1.2.1) Culture medium for growth As the culture medium for growth, a medium commonly used for culturing transformants, such as a known medium containing a carbon source, a nitrogen source, inorganic salts, etc., may be used. can.
The culture temperature in propagation culture may be any temperature as long as it is suitable for propagation of the transformant. The pH of the growth culture solution may be any pH suitable for growth of the transformant. The pH of the growth culture solution can be adjusted by a known method. The culture time in proliferation culture may be any time as long as it is sufficient for the transformant to proliferate.

(1.2.2) Culture solution for production As the culture solution for production, a culture solution containing a pyruvic acid supplying compound, a carbonyl compound, and other necessary components can be used. As other necessary components, for example, inorganic salts, carbon sources other than sugars, or nitrogen sources can be used. The concentration of each component in the culture solution described herein refers to the final concentration in the culture solution at the time each component is added.

As the pyruvate-supplying compound, a compound selected from pyruvate and saccharides can be used. The pyruvate-supplying compound may be pyruvate itself or may be a saccharide that is converted to pyruvate in vivo by a microorganism. The pyruvate-providing compound can be pyruvate, a sugar, or a combination thereof.

As the saccharide, any saccharide can be used as long as it can be taken into the body by the transformant and can be converted into pyruvate. Specifically, sugars 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. etc. Among these, monosaccharides are preferred, and fructose and glucose are more preferred. Also preferred are saccharides containing glucose as a constituent monosaccharide, specifically disaccharides such as sucrose, oligosaccharides containing glucose as a constituent monosaccharide, and polysaccharides containing glucose as a constituent monosaccharide. One type of saccharide may be used or a mixture of two or more types may be used. In addition, sugars can be obtained from saccharified liquid obtained by saccharifying inedible agricultural waste such as rice straw, bagasse, and corn stover, and energy crops such as switch grass, napier grass, and miscanthus using saccharifying enzymes. can also be added in the form of sugars (including glucose, xylose, etc.).

The concentration of the pyruvate-supplying compound in the production culture solution can be made as high as possible without inhibiting the production of α-keto acid. The concentration of the pyruvate-supplying compound in the production culture solution is preferably within the range of 0.1 to 40 (w/v)%, more preferably within the range of 1 to 20 (w/v)%. preferable. Further, in accordance with the decrease in the pyruvate-supplying compound due to the production of α-keto acid, additional addition of the pyruvate-supplying compound can be carried out.

As the carbonyl compound, the following general formula (1):
R ₁₀₁ C(O)R ₁₀₂
(where R ₁₀₁ and R ₁₀₂ are independently of each other hydrogen or an alkyl group having 1 to 5 carbon atoms)
Carbonyl compounds represented by can be used.
According to one example, the carbonyl compound used is a carbonyl compound in which, in the above general formula (1), R ₁₀₁ is hydrogen or an alkyl group having 1 to 5 carbon atoms, and R ₁₀₂ is hydrogen. can do.
According to another example, the carbonyl compound is a carbonyl compound in which R ₁₀₁ is a methyl group and R ₁₀₂ is an alkyl group having 1 to 5 carbon atoms in the above general formula (1). Can be done.

Specifically, as the carbonyl compound, in the above general formula (1),
A carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen (i.e., formaldehyde);
A carbonyl compound in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen (i.e., acetaldehyde);
A carbonyl compound in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen (i.e., propionaldehyde);
A carbonyl compound in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen (i.e., butyraldehyde), specifically a carbonyl compound in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen ( or a carbonyl compound in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen (i.e., isobutyraldehyde); or a carbonyl compound in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen; A certain carbonyl compound (i.e., valeraldehyde), specifically a carbonyl compound in which R ₁₀₁ is a normal pentyl group and R ₁₀₂ is hydrogen (i.e., normal valeraldehyde);
can be used.
Further, as the carbonyl compound, a carbonyl compound (ie, acetone) in which R ₁₀₁ and R ₁₀₂ in the above general formula (1) are both methyl groups can also be used.
One type of carbonyl compound may be used or a mixture of two or more types may be used.

The concentration of carbonyl compound in the production culture solution is preferably within the range of 0.001 to 10 (w/v)%, more preferably within the range of 0.01 to 1 (w/v)%. preferable. Furthermore, additional carbonyl compounds can be added in accordance with the decrease in carbonyl compounds accompanying the production of α-keto acids.

In addition, when one type of carbonyl compound is used in the production culture solution, it is possible to easily perform the separation and purification described in the section "(1.2.4) Recovery of α-keto acid" described below. be.

Furthermore, inorganic salts can be added as necessary. As the inorganic salts, metal salts such as phosphates, sulfates, magnesium, potassium, manganese, iron, and zinc can be used. Specifically, potassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, iron (II) sulfate, manganese sulfate, zinc sulfate, cobalt sulfate, calcium carbonate, etc. can be used. can. One kind of inorganic salt may be used or a mixture of two or more kinds may be used. The concentration of inorganic salts in the production culture solution varies depending on the inorganic salt used, but can usually be about 0.01 to 1 (w/v)%.

Furthermore, if necessary, carbon sources other than sugars can also be added. Carbon sources other than sugars include sugar alcohols such as mannitol, sorbitol, xylitol, glycerol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; and carbonization such as normal paraffin. Hydrogen etc. can be used.

Furthermore, a nitrogen source can be added if necessary. As the nitrogen source, inorganic or organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate; urea; aqueous ammonia; nitrates such as sodium nitrate, potassium nitrate, etc. can be used. Also usable are corn steep liquor; meat extract; yeast extract; soybean hydrolyzate; protein hydrolyzate such as peptone, NZ-amine or casein decomposition product; nitrogen-containing organic compounds such as amino acids. One type of nitrogen source may be used or a mixture of two or more types may be used. The concentration of the nitrogen source in the production culture solution varies depending on the nitrogen compound used, but can usually be about 0.1 to 10 (w/v)%.

Furthermore, vitamins can be added if necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

A specific production culture solution contains glucose, formaldehyde, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, iron (II) sulfate, and manganese (II) sulfate, and has a pH of 6.0. A culture medium can be used.

Specific production culture fluids include pyruvic acid, formaldehyde, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, iron (II) sulfate, manganese (II) sulfate, and 4-morpholinopropanesulfonic acid. A culture medium can also be used.

Furthermore, as the production culture solution, a culture solution obtained by adding a pyruvic acid supplying compound and a carbonyl compound to a base culture solution having the same composition as the proliferation culture solution used in the proliferation culture can also be used. Note that if the pyruvic acid-supplying compound is present in a sufficient amount in the above-mentioned base culture solution, it is not necessary to add the pyruvic acid-supplying compound.

(1.2.3) Conditions for production culture The culture temperature for production culture may be any temperature as long as it is suitable for producing α-keto acids. The culture temperature in production culture is preferably about 20 to 50°C, more preferably about 25 to 47°C. Within the above temperature range, α-keto acids can be produced efficiently. The pH of the production culture solution may be any pH suitable for producing α-keto acids. The pH of the production culture solution is preferably maintained within the range of about 6-8. The pH of the production culture solution should be adjusted using a pH buffer solution containing an inorganic or organic acid; an alkaline solution such as an aqueous potassium hydroxide solution; urea; calcium carbonate; ammonia; 4-morpholinopropanesulfonic acid, etc. Can be done. Even during production culture, the pH of the production culture solution can be adjusted as necessary. The culture time in production culture may be any time as long as it is sufficient for the transformant to produce α-keto acid. The culture time in production culture is preferably about 3 hours to 7 days, more preferably about 1 to 3 days.
Production culture may be carried out under aerobic conditions, anaerobic conditions or microaerobic conditions. This is because the transformant described above has a pathway for producing α-keto acids that works under both aerobic, anaerobic, or microaerobic conditions. The pathway for producing α-keto acids will be described in the section “(1.3) Actions and Effects” below.

<Anaerobic conditions or microaerobic conditions>
Production culture is preferably carried out under anaerobic or microaerobic conditions.

When the transformant is cultured under anaerobic or microaerobic conditions, the transformant does not substantially proliferate and can produce α-keto acids more efficiently.

"Anaerobic conditions or microaerobic conditions" refer to, for example, conditions in which the concentration of dissolved oxygen in the production culture solution is within the range of 0 to 2 ppm. "Anaerobic conditions or microaerobic conditions" preferably refer to conditions in which the concentration of dissolved oxygen in the production culture solution is within the range of 0 to 1 ppm, and more preferably, dissolved oxygen in the production culture solution. Refers to conditions where the concentration is within the range of 0 to 0.5 ppm. The dissolved oxygen concentration in the culture solution can be measured using, for example, a dissolved oxygen meter.

Strictly speaking, "anaerobic conditions" is a technical term that refers to conditions in the absence of dissolved oxygen and electron acceptors such as oxides such as nitric acid. On the other hand, the preferable conditions for production culture are such that there are not enough electron acceptors or the transformants do not grow due to the nature of the electron acceptors, and the supplied carbon source is insufficient for the production of α-keto acids. The above strict conditions do not need to be adopted as long as the production efficiency can be increased. In other words, the preferable conditions for production culture are to suppress the growth of transformants and increase the production efficiency of α-keto acids. Electron acceptors may also be present. Accordingly, the expression "anaerobic or microaerobic conditions" as used herein also includes such conditions.

Anaerobic and microaerobic conditions can be achieved, for example, by not aerating oxygen-containing gas, so that the oxygen supplied from the gas-liquid interface is insufficient for the growth of the transformants. Alternatively, this can be achieved by aerating an inert gas, specifically nitrogen gas, into the production culture solution. Alternatively, this can be achieved by sealing the container used for production culture.

<High density conditions>
Production culture is preferably carried out with the transformant suspended in a production culture solution at high density. By culturing the transformant under such conditions, α-keto acids can be produced more efficiently.

"A state in which the transformant is suspended in a production culture solution at high density" means, for example, that the transformant is suspended in a production culture solution so that the wet bacterial weight% of the transformant is 1 to 50 (w). /v)%. "The state in which the transformant is suspended in a production culture solution at high density" is preferably a state in which the transformant is suspended in a production culture solution so that the wet bacterial weight% of the transformant is 3 to 30 ( w/v)% range.

Production culture is more preferably carried out under anaerobic or microaerobic conditions with the transformant suspended in a production culture solution at high density.

(1.2.4) Recovery of α-keto acid As described above, when a transformant containing the first gene is cultured in a production culture solution, α-keto acid is produced in the production culture solution.

Specifically, the following general formula (1) is added to the production culture solution:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
When using a carbonyl compound represented by the following general formula (2):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
α-keto acids represented by can be produced.

More specifically, when using a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (1), when R ₁₀₁ is hydrogen in the above general formula (2), , and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxobutyric acid).
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen, in the above general formula (2), R ₁₀₁ is a methyl group, and R An α-keto acid in which ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxovaleric acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen, in the above general formula (2), R ₁₀₁ is an ethyl group, and R An α-keto acid (ie, 4-hydroxy-2-oxocaproic acid) in which ₁₀₂ is hydrogen can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen, in the above general formula (2), R ₁₀₁ is a propyl group, and R α-keto acids (ie, 4-hydroxy-2-oxoenanto acids) in which ₁₀₂ is hydrogen can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen in the above general formula (1), in the above general formula (2), R ₁₀₁ is a normal propyl group. and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxonormalheptanoic acid) can be produced. Further, specifically, when using a carbonyl compound in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen in the above general formula (1), in the above general formula (2), R ₁₀₁ is isopropyl and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxoisoenanto acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen, in the above general formula (2), R ₁₀₁ is a pentyl group, and R An α-keto acid in which ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxocaprylic acid) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal pentyl group in the above general formula (1) and R ₁₀₂ is hydrogen, in the above general formula (2), R ₁₀₁ is a normal pentyl group. and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxonormal caprylic acid) can be produced.
When a carbonyl compound in which R ₁₀₁ and R ₁₀₂ in the above general formula (1) are both methyl groups is used, an α-keto compound in which R ₁₀₁ and R ₁₀₂ in the above general formula (2) are both methyl groups is used. acid (ie, 4-methyl-4-hydroxy-2-oxovaleric acid).

The above α-keto acid can be recovered by recovering the production culture solution, but it is also possible to separate and purify the α-keto acid from the production culture solution using a known method. Such known methods include a crystallization method, a chromatography method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, and the like.

Furthermore, when multiple types of carbonyl compounds are used, multiple types of α-keto acids are produced in the culture solution. Multiple types of α-keto acids can be separated from each other by crystallization or chromatography.

(1.3) Actions and Effects The process by which α-keto acid-producing microorganisms produce α-keto acids is schematically shown in Figure 1. In the process shown in FIG. 1, an example will be described in which the pyruvate-supplying compound is glucose, the carbonyl compound is formaldehyde, and the α-keto acid is 4-hydroxy-2-oxobutyric acid.

As shown in FIG. 1, first, the α-keto acid producing microorganism 1 takes in glucose 2 and formaldehyde 3. Glucose 2 is then converted to pyruvate 4 through glycolysis. Next, pyruvate 4 and formaldehyde 3 are converted to 4-hydroxy-2-oxobutyric acid 5 by the "enzyme that catalyzes the aldol reaction between pyruvate and aldehyde" encoded by the first gene. In the manner described above, 4-hydroxy-2-oxobutyric acid is produced.

In Figure 1, we explained the case where an aldehyde is used as a carbonyl compound, but even when a ketone is used as a carbonyl compound, the ketone and pyruvate are The reaction of converting the and into an α-keto acid proceeds. This is because aldehydes and ketones both belong to the same type of compound (i.e., carbonyl compound), so both aldehydes and ketones can serve as substrates for the same reaction in organic chemical reactions such as enzymatic reactions. .

The manufacturing method explained above was developed by cultivating microorganisms with specific genes in the presence of carbonyl compounds, and developed a production process in which intermediates, which are key to the synthesis of useful substances such as amino acids, can be obtained. This is what I did. Since carbonyl compounds are generally harmful to the growth of microorganisms, it was surprising that useful compounds were obtained in their presence. In addition, because the carbonyl compound is taken into the microbial metabolic system, an intermediate that is not produced in the normal microbial metabolic system is obtained, which will support future needs for producing a variety of compounds from biological raw materials. This can be expected as a method that satisfies the above requirements. In fact, as described later in this specification, the fact that we were able to develop the production of various useful substances through this intermediate shows some of the possibilities of producing a variety of compounds using the technology of the present invention.

(2.) Method for producing 1,3-diol The method for producing 1,3-diol is as follows:
The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(b) a second gene encoding an enzyme that catalyzes the decarboxylation reaction of α-keto acids; and (c) a third gene encoding an enzyme that catalyzes the reduction reaction of aldehydes. a selected pyruvate-providing compound;
General formula (3) below:
R ₁₀₃ C(O)R ₁₀₄
(Here, R ₁₀₃ and R ₁₀₄ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms. However, when R ₁₀₃ is hydrogen, R ₁₀₄ is not hydrogen.)
by culturing in the presence of a carbonyl compound represented by
General formula (4) below:
R ₁₀₃ C(R ₁₀₄ )(OH)CH ₂ CH ₂ OH
(R ₁₀₃ and R ₁₀₄ in the above general formula (4) are the same groups as R ₁₀₃ and R ₁₀₄ in the above general formula (3), respectively)
The method includes a step of producing a 1,3-diol represented by

In the following description, the above-mentioned microorganism containing the first, second, and third genes is also referred to as a "1,3-diol-producing microorganism." The 1,3-diol-producing microorganism may be a microorganism that naturally has the first, second, and third genes, or it may be obtained by transforming a host with the first, second, and third genes. It may also be a microorganism. Hereinafter, the case where the 1,3-diol-producing microorganism is a transformant will be explained as an example.

(2.1) Transformants (2.1.1) Host As the host, the same hosts as those described in the column "(1.1.1) Host" can be used.
Next, the first, second and third genes introduced into the host will be explained.

(2.1.2) First gene As the first gene, a gene similar to the first gene described in the column "(1.1.2) First gene" can be used. .

(2.1.3) Second gene The second gene encodes "an enzyme that catalyzes the decarboxylation reaction of α-keto acids.""An enzyme that catalyzes the decarboxylation reaction of α-keto acids" is an enzyme that catalyzes the decarboxylation reaction of α-keto acids using an enzyme reaction solution containing purified protein and α-keto acids (initial substrate concentration 1mM) according to the measurement method described below. An enzyme that has an activity of 1 mU/mg protein or more when measured. The second gene encodes, for example, an enzyme that catalyzes the decarboxylation reaction of α-keto acids obtained by the aldol reaction catalyzed by the protein encoded by the first gene. The α-keto acid in the “enzyme that catalyzes the decarboxylation reaction of α-keto acid” is, for example, represented by the general formula (2) described in the column “(1.) Method for producing α-keto acid”. It is an α-keto acid. The "enzyme that catalyzes the decarboxylation reaction of α-keto acids" is, for example, decarboxylase.

The second gene may be a gene encoding a decarboxylase as exemplified below.

(b1) pyruvate decarboxylase (EC number 4.1.1.1),
(b2) benzoylformate decarboxylase (EC number 4.1.1.7), and (b3) indolepyruvate decarboxylase (EC number 4.1.1.74).

Examples of the decarboxylase included in (b1) include pdc protein. Preferably, the pdc protein is derived from Zymomonas mobilis.
Examples of the decarboxylase included in (b2) include mdlC protein. Preferably, the mdlC protein is derived from Pseudomonas putida.
Examples of the decarboxylase included in (b3) include kivD protein. Preferably, the kivD protein is derived from Lactococcus lactis.

Preferably, the second gene is selected from the group consisting of:
(2-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 24 (i.e., mdlC protein derived from Pseudomonas putida) that catalyzes the decarboxylation reaction of α-keto acids, and (2 -2) A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 24, and which has an activity of catalyzing the decarboxylation reaction of α-keto acids. .

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 24, and that has the activity of catalyzing the decarboxylation reaction of α-keto acids" , " Preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more of the amino acid sequence set forth in SEQ ID NO: 24. It may also be a gene encoding a protein consisting of a homologous amino acid sequence and having the activity of catalyzing the decarboxylation reaction of α-keto acids.

The UniprotKB accession number of the mdlC protein derived from Pseudomonas putida is, for example, P20906.

More preferably, the second gene is the mdlC gene derived from Pseudomonas putida and has the base sequence set forth in SEQ ID NO: 23.

The base sequence set forth in SEQ ID NO: 23 and the amino acid sequence set forth in SEQ ID NO: 24 are as follows.

The activity of catalyzing the decarboxylation reaction of α-keto acids can be determined, for example, by catalyzing the enzymatic reaction in a 100 mM potassium phosphate buffer (pH 6.0) containing 0.5 mM thiamine pyrophosphate, 1 mM magnesium chloride, 10 mM NADH, and an excess amount of alcohol dehydrogenase. It can be calculated by measuring the initial production rate of the alcohol compound produced by successive reduction of the aldehyde compound produced from the raw material α-keto acid. The production rate of the alcohol compound can be calculated, for example, from the time change in the concentration of the alcohol compound itself. The concentration of an alcohol compound can be measured, for example, by detecting the alcohol compound using high performance liquid chromatography and comparing it with a standard sample whose concentration is known. Here, the activity of decarboxylating 1 μmol of α-keto acid per minute at 30° C. is defined as 1 unit.

"A gene (mdlC Gene mutants)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

(2.1.4) Third gene The third gene encodes "an enzyme that catalyzes the reduction reaction of aldehyde.""Enzyme that catalyzes the reduction reaction of aldehyde" means 10 mU/ An enzyme with an activity greater than mg protein. The third gene encodes, for example, an enzyme that catalyzes a reduction reaction of an aldehyde obtained by a decarboxylation reaction catalyzed by a protein encoded by the second gene. The aldehyde in the "enzyme that catalyzes the reduction reaction of aldehyde" is, for example, 3-hydroxybutyraldehyde, 3-hydroxyvaleraldehyde, 3-hydroxyhexylaldehyde, 3-hydroxyisohexylaldehyde, 3-hydroxyheptyraldehyde, or 3-methyl -3-hydroxybutyraldehyde. The "enzyme that catalyzes the reduction reaction of aldehyde" is, for example, alcohol dehydrogenase.

The third gene may be, for example, a gene encoding 1,3-propanediol dehydrogenase (EC number 1.1.1.202).

Examples of 1,3-propanediol dehydrogenase include dhaT protein or lpo protein. Preferably, the dhaT protein is derived from Klebsiella pneumoniae. Also preferably, the lpo protein is derived from Lactobacillus reuteri.

Preferably, the third gene is selected from the group consisting of:
(3-1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 26 (i.e., dhaT protein derived from Klebsiella pneumoniae), a gene encoding a protein that catalyzes the reduction reaction of aldehyde, and (3-2) the sequence A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in number 26, and which has an activity of catalyzing an aldehyde reduction reaction.

"A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 26, and which has the activity of catalyzing an aldehyde reduction reaction" Preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, even more preferably 98% or more, still more preferably 99% or more homology to the amino acid sequence described in 26. It may also be a gene encoding a protein consisting of an amino acid sequence and having an activity of catalyzing an aldehyde reduction reaction.

The UniprotKB accession number of the dhaT protein derived from Klebsiella pneumoniae is, for example, Q59477.

More preferably, the third gene is a dhaT gene derived from Klebsiella pneumoniae and consisting of the base sequence set forth in SEQ ID NO: 25.

The base sequence set forth in SEQ ID NO: 25 and the amino acid sequence set forth in SEQ ID NO: 26 are as follows.

The activity of catalyzing the reduction reaction of aldehyde can be determined by, for example, performing the enzymatic reaction at 30°C in a 50mM MES-NaOH buffer (pH 6.5) containing 0.2mM NADH, and reducing the raw material aldehyde to an alcohol compound. It can be calculated by measuring the initial consumption rate of NADH consumed when The initial consumption rate of NADH can be calculated, for example, by measuring the rate of decrease in absorbance at 340 nm derived from NADH in the enzyme reaction solution. Here, the activity of reducing 1 μmol of aldehyde per minute at 30° C. is defined as 1 unit.

"A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 26, and which has the activity of catalyzing an aldehyde reduction reaction (dhaT gene variant) can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

(2.1.5) Construction of recombinant vectors for transformation The first, second, and third genes can be incorporated into a suitable plasmid vector for transformation.

As the plasmid vector, a plasmid vector similar to the plasmid vector described in "(1.1.3) Construction of recombinant vector for transformation" can be used.

The first, second and third genes may be incorporated into separate plasmid vectors, or may be integrated into the same plasmid vector. Transformants can be produced using the obtained recombinant vector.

(2.1.6) Transformation Any known transformation method can be used without restriction. As such a known method, the same method as described in the column "(1.1.4) Transformation" can be used.

In addition, when a transformant is produced using a recombinant vector as described above, each of the first, second, and third genes may be integrated into the chromosome of the host, or the recombinant vector It may exist in the cytoplasm of the host in the form of

(2.1.7) Disruption or deletion of host chromosomal genes The host may be a wild strain, a mutant strain thereof, or an artificially genetically modified strain. When the host is a coryneform bacterium, the host must be a gene-disrupted strain or a gene-deleted strain similar to the gene-disrupted strain or gene-deleted strain described in “(1.1.5) Disruption or deletion of host chromosomal genes.” Deletion strains can be used. In addition, as a method for producing gene-disrupted strains or gene-deleted strains, methods similar to those described in the column "(1.1.5) Disruption or deletion of host chromosomal genes" can be used. . By using such a gene-disrupted strain or gene-deleted strain as a host, it is possible to improve the productivity of 1,3-diol and to suppress the production of by-products.

(2.1.8) Specific examples of 1,3-diol-producing microorganisms Specifically, 1,3-diol-producing microorganisms include:
The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that encodes a protein that is a protein consisting of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
A second gene selected from the group consisting of:
(2-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 24, which catalyzes the decarboxylation reaction of α-keto acids; and (2-2) 1 or several genes in SEQ ID NO: 24. A gene that encodes a protein consisting of an amino acid sequence in which five amino acids are deleted, substituted, or added, and that has the activity of catalyzing the decarboxylation reaction of α-keto acids; and a gene selected from the group consisting of: Third gene:
(3-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 26, which catalyzes the reduction reaction of aldehyde, and (3-2) A gene in which one or several amino acids in SEQ ID NO: 26 A coryneform bacterium containing a gene encoding a protein consisting of a deleted, substituted, or added amino acid sequence and having an activity of catalyzing an aldehyde reduction reaction can be used.

(2.2) Cultivation process 1,3-diol can be produced by culturing a 1,3-diol-producing microorganism (for example, the transformant described above) in the presence of a pyruvate-supplying compound and a carbonyl compound. can. "Cultivating in the presence of a pyruvate-supplying compound and a carbonyl compound" can be performed by culturing in a culture medium containing a pyruvate-supplying compound and a carbonyl compound. Preferably, "culturing in the presence of a pyruvate-supplying compound and a carbonyl compound" can be carried out by culturing in a culture medium to which the pyruvate-supplying compound and carbonyl compound are added.

Prior to culturing in the presence of a pyruvate-supplying compound and a carbonyl compound, it is preferable to carry out the proliferation culture described in the column "(1.2) Cultivation step".

When propagation culture is performed prior to production culture, the propagation culture and subsequent production culture can be performed, for example, as described in the column "(1.2) Cultivation step".

(2.2.1) Culture solution for proliferation As the culture solution for proliferation, the same culture solution as the culture solution for proliferation described in the column "(1.2.1) Culture solution for proliferation" can be used. can.

(2.2.2) Culture solution for production As the culture solution for production, a culture solution containing a pyruvic acid supplying compound, a carbonyl compound, and other necessary components can be used. As other necessary components, for example, inorganic salts, carbon sources other than sugars, or nitrogen sources can be used.

As the pyruvic acid supplying compound, the pyruvic acid supplying compound described in the column of "(1.2.2) Production culture solution" can be used.

As the carbonyl compound, the following general formula (3):
R ₁₀₃ C(O)R ₁₀₄
(Here, R ₁₀₃ and R ₁₀₄ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms. However, when R ₁₀₃ is hydrogen, R ₁₀₄ is not hydrogen.)
Carbonyl compounds represented by can be used.
According to one example, the carbonyl compound used is a carbonyl compound in which, in the above general formula (3), R ₁₀₃ is hydrogen or an alkyl group having 1 to 5 carbon atoms, and R ₁₀₄ is hydrogen. can do.
According to another example, the carbonyl compound is a carbonyl compound in the general formula (3) above, in which R ₁₀₃ is a methyl group and R ₁₀₄ is an alkyl group having 1 to 5 carbon atoms. I can do it.

Specifically, as the carbonyl compound, in the above general formula (1),
A carbonyl compound in which R ₁₀₃ is a methyl group and R ₁₀₄ is hydrogen (i.e., acetaldehyde);
A carbonyl compound in which R ₁₀₃ is an ethyl group and R ₁₀₄ is hydrogen (i.e., propionaldehyde);
A carbonyl compound in which R ₁₀₃ is a propyl group and R ₁₀₄ is hydrogen (i.e., butyraldehyde), specifically a carbonyl compound in which R ₁₀₃ is a normal propyl group and R ₁₀₄ is hydrogen ( or a carbonyl compound in which R ₁₀₃ is an isopropyl group and R ₁₀₄ is hydrogen (i.e., isobutyraldehyde); or a carbonyl compound in which R ₁₀₃ is a pentyl group and R ₁₀₄ is hydrogen; A certain carbonyl compound (i.e., valeraldehyde), specifically a carbonyl compound in which R ₁₀₃ is a normal pentyl group and R ₁₀₄ is hydrogen (i.e., normal valeraldehyde);
can be used.
Further, as the carbonyl compound, a ketone (ie, acetone) in which R ₁₀₃ and R ₁₀₄ in the above general formula (3) are both methyl groups can be used.
One type of carbonyl compound may be used or a mixture of two or more types may be used.

The concentration of carbonyl compound in the production culture solution is preferably within the range of 0.001 to 10 (w/v)%, more preferably within the range of 0.01 to 1 (w/v)%. preferable. Furthermore, additional carbonyl compounds can be added in accordance with the decrease in carbonyl compounds accompanying the production of 1,3-diol.

Note that when one type of carbonyl compound is used in the production culture solution, it is possible to easily perform the separation and purification described in "(2.2.4) Recovery of 1,3-diol" below.

As other necessary components, the same components as those described in the column of "(1.2.2) Production culture solution" can be used.

A specific production culture solution contains glucose, acetaldehyde, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, iron (II) sulfate, and manganese (II) sulfate, and has a pH of 6.0. A culture medium can be used.
Furthermore, as the production culture solution, a culture solution obtained by adding a pyruvic acid supplying compound and a carbonyl compound to a base culture solution having the same composition as the proliferation culture solution used in the proliferation culture can also be used. Note that if the pyruvic acid-supplying compound is present in a sufficient amount in the above-mentioned base culture solution, it is not necessary to add the pyruvic acid-supplying compound.

(2.2.3) Conditions for production culture The culture temperature for production culture may be any temperature as long as it is suitable for producing 1,3-diol. The culture temperature in production culture is preferably about 20 to 50°C, more preferably about 25 to 47°C. Within the above temperature range, 1,3-diol can be produced efficiently. The pH of the production culture solution may be any pH as long as it is suitable for producing 1,3-diol. The pH of the production culture solution is preferably maintained within the range of about 6-8. The pH of the production culture solution should be adjusted using a pH buffer solution containing an inorganic or organic acid; an alkaline solution such as an aqueous potassium hydroxide solution; urea; calcium carbonate; ammonia; 4-morpholinopropanesulfonic acid, etc. Can be done. Even during production culture, the pH of the production culture solution can be adjusted as necessary. The culture time in production culture may be any time as long as it is sufficient for the transformant to produce 1,3-diol. The culture time in production culture is preferably about 3 hours to 7 days, more preferably about 1 to 3 days.
Production culture may be carried out under aerobic conditions, anaerobic conditions or microaerobic conditions. This is because the transformant described above has a pathway for producing 1,3-diol that works under both aerobic, anaerobic, or microaerobic conditions. The route for producing 1,3-diol will be described in the "(2.3) Actions and Effects" section below.

<Anaerobic conditions or microaerobic conditions>
Production culture is preferably carried out under anaerobic or microaerobic conditions.

When the transformant is cultured under anaerobic or microaerobic conditions, the transformant does not substantially proliferate and can produce 1,3-diol more efficiently.

“Anaerobic conditions or microaerobic conditions” can be the same conditions as “anaerobic conditions or microaerobic conditions” described in “(1.2.3) Production culture conditions”. can.

<High density conditions>
Production culture is preferably carried out with the transformant suspended in a production culture solution at high density. By culturing the transformant under such conditions, 1,3-diol can be produced more efficiently.

"A state in which the transformant is suspended in a production culture solution at high density" means "a state in which the transformant is suspended in a production culture solution" as described in the column of "(1.2.3) Production culture conditions". It can be in a state similar to "a state in which it is suspended at a high density in".

Production culture is more preferably carried out under anaerobic or microaerobic conditions with the transformant suspended in a production culture solution at high density.

(2.2.4) Recovery of 1,3-diol As mentioned above, when the transformant containing the first, second, and third genes is cultured in the production culture solution, 1,3-diol is added to the production culture solution. 3-diol is produced.

Specifically, the following general formula (3) is contained in the production culture solution:
General formula (3) below:
R ₁₀₃ C(O)R ₁₀₄
(Here, R ₁₀₃ and R ₁₀₄ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms. However, neither R ₁₀₃ nor R ₁₀₄ is hydrogen.)
When using a carbonyl compound represented by the following general formula (4):
R ₁₀₃ C(R ₁₀₄ )(OH) CH ₂ CH ₂ OH
(R ₁₀₃ and R ₁₀₄ in the above general formula (4) are the same groups as R ₁₀₃ and R ₁₀₄ in the above general formula (3), respectively)
A 1,3-diol represented by can be produced.

More specifically, when using a carbonyl compound in which R ₁₀₃ in the above general formula (3) is a methyl group and R ₁₀₄ is hydrogen, in the above general formula (4), R ₁₀₃ is a methyl group. and R ₁₀₄ is hydrogen (ie, 1,3-butanediol) can be produced.
In the above general formula (3), when using a carbonyl compound in which R ₁₀₃ is an ethyl group and R ₁₀₄ is hydrogen, in the above general formula (4), R ₁₀₃ is an ethyl group, and R A 1,3-diol in which ₁₀₄ is hydrogen (ie, 1,3-pentanediol) can be produced.
In the above general formula (3), when using a carbonyl compound in which R ₁₀₃ is a propyl group and R ₁₀₄ is hydrogen, in the above general formula (4), R ₁₀₃ is a propyl group, and R A 1,3-diol in which ₁₀₄ is hydrogen (ie, 1,3-hexanediol) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₃ is a normal propyl group and R ₁₀₄ is hydrogen in the above general formula (3), in the above general formula (4), R ₁₀₃ is a normal propyl group. and R ₁₀₄ is hydrogen (ie, 1,3-n-hexanediol) can be produced. Further, specifically, when using a carbonyl compound in which R ₁₀₃ is an isopropyl group in the above general formula (3) and R ₁₀₃ is hydrogen, in the above general formula (4), R ₁₀₃ is an isopropyl group. and R ₁₀₄ is hydrogen (ie, 4-methyl-1,3-pentanediol).
In the above general formula (3), when using a carbonyl compound in which R ₁₀₃ is a pentyl group and R ₁₀₄ is hydrogen, in the above general formula (4), R ₁₀₃ is a pentyl group, and R A 1,3-diol in which ₁₀₄ is hydrogen (ie, 1,3-heptanediol) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₃ in the above general formula (3) is a normal pentyl group and R ₁₀₄ is hydrogen, in the above general formula (4), R ₁₀₃ is a normal pentyl group. and R ₁₀₄ is hydrogen (ie, 1,3-n-heptanediol) can be produced.
When using a carbonyl compound in which R ₁₀₃ and R ₁₀₄ in the above general formula (3) are both methyl groups, 1,3 in which R ₁₀₃ and R ₁₀₄ in the above general formula (4) are both methyl groups is used. -diol (ie, 3-methyl-1,3-butanediol).

Although the above-mentioned 1,3-diol can be recovered by collecting the production culture solution, 1,3-diol can also be separated and purified from the production culture solution using a known method. Such known methods include a distillation method, a concentration method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, a crystallization method, and the like.
Note that substances such as α-keto acids produced by the aldol reaction catalyzed by the enzyme encoded by the first gene can also be recovered from the production culture solution.

Furthermore, when multiple types of carbonyl compounds are used, multiple types of 1,3-diols are produced in the culture solution. Multiple types of 1,3-diols can be separated from each other by distillation or chromatography.

(2.3) Actions and Effects The process by which 1,3-diol-producing microorganisms produce 1,3-diol is schematically shown in FIG. 2. In the process shown in FIG. 2, an example will be described in which the pyruvic acid supplying compound is glucose, the carbonyl compound is acetaldehyde, and the 1,3-diol is 1,3-butanediol.

As shown in FIG. 2, first, the 1,3-diol-producing microorganism 6 takes in glucose 2 and acetaldehyde 7. Glucose 2 is then converted to pyruvate 4 through glycolysis. Next, pyruvic acid 4 and acetaldehyde 7 are converted to 4-hydroxy-2-oxovaleric acid 8 by the "enzyme that catalyzes the aldol reaction between pyruvic acid and aldehyde" encoded by the first gene. Next, 4-hydroxy-2-oxovaleric acid 8 is converted to 3-hydroxybutyraldehyde 9 by the "enzyme that catalyzes the decarboxylation reaction of α-keto acid" encoded by the second gene. Next, 3-hydroxybutyraldehyde 9 is converted to 1,3-butanediol 10 by the "enzyme that catalyzes the reduction reaction of aldehyde" encoded by the third gene. In the above manner, 1,3-butanediol is produced.

Alternatively, as another embodiment, in the method for producing 1,3-diol described in the section "(2.) Method for producing 1,3-diol," only when producing 1,3-butanediol, The production culture solution does not need to contain acetaldehyde.
That is, the method for producing 1,3-butanediol according to this embodiment includes a step of culturing a 1,3-diol-producing microorganism in the presence of a pyruvate-supplying compound to produce 1,3-butanediol. include.
This embodiment can be carried out according to the same procedure as the above-mentioned "method for producing 1,3-diol" except that acetaldehyde is not added to the production culture medium.

The present inventors believe that the reason why 1,3-butanediol can be produced even if the production culture solution does not contain acetaldehyde is as follows. First, a 1,3-diol-producing microorganism takes into the body a pyruvate-supplying compound in a production culture solution. Next, when the pyruvate-supplying compound is pyruvate, part of the pyruvate is converted to acetaldehyde by the enzyme encoded by the second gene. When the pyruvate-supplying compound is a saccharide, the saccharide is converted to pyruvate, and then a portion of the pyruvate is converted to acetaldehyde by an enzyme encoded by the second gene. Next, acetaldehyde and pyruvate are converted to 4-hydroxy-2-oxovaleric acid by the enzyme encoded by the first gene. 4-hydroxy-2-oxovaleric acid is then converted to 3-hydroxybutyraldehyde by an enzyme encoded by the second gene. 3-hydroxybutyraldehyde is then converted to 1,3-butanediol. In this way, 1,3-diol-producing microorganisms can produce acetaldehyde in vivo. Therefore, 1,3-butanediol is produced even if the production culture solution does not contain acetaldehyde. Note that acetaldehyde produced by the 1,3-diol-producing microorganism may be secreted into the culture solution from the 1,3-diol-producing microorganism.

(3.) Method for producing 2,4-dihydroxycarboxylic acid The method for producing 2,4-dihydroxycarboxylic acid is as follows:
The following genes:
(a) A microorganism containing a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (d) a fourth gene encoding an enzyme that catalyzes a reduction reaction of an α-keto acid. a pyruvate-providing compound selected from acids and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (5) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(OH)COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (5) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
The method includes a step of producing a 2,4-dihydroxycarboxylic acid represented by

In the following description, the microorganism containing the first and fourth genes is also referred to as a "2,4-dihydroxycarboxylic acid producing microorganism." The 2,4-dihydroxycarboxylic acid-producing microorganism may be a microorganism that naturally has the first and fourth genes, or a microorganism obtained by transforming a host with the first and fourth genes. Good too. The case where the 2,4-dihydroxycarboxylic acid-producing microorganism is a transformant will be explained below as an example.

(3.1) Transformants (3.1.1) Host As the host, the same hosts as those described in the column "(1.1.1) Host" can be used.
Next, the first and fourth genes introduced into the host will be explained.

(3.1.2) First gene As the first gene, a gene similar to the first gene described in the column "(1.1.2) First gene" can be used.

(3.1.3) Fourth gene The fourth gene encodes "an enzyme that catalyzes the reduction reaction of α-keto acid.""Enzyme that catalyzes the reduction reaction of α-keto acid" is measured using an enzyme reaction solution containing purified protein and α-keto acid (initial substrate concentration 1mM) according to the measurement method described below. An enzyme with an activity of 10 mU/mg protein or more. The fourth gene encodes, for example, an enzyme that catalyzes the reduction reaction of α-keto acid obtained by the aldol reaction catalyzed by the protein encoded by the first gene. The α-keto acid in the “enzyme that catalyzes the reduction reaction of α-keto acid” is, for example, represented by the general formula (2) described in the column “(1.) Method for producing α-keto acid” It is an α-keto acid. The "enzyme that catalyzes the reduction reaction of α-keto acid" is, for example, dehydrogenase.

The fourth gene may be, for example, a gene encoding lactate dehydrogenase (EC number 1.1.1.27).

Examples of lactate dehydrogenase include ldhA protein. Preferably, the ldhA protein is a protein derived from Lactococcus lactis. In addition, "protein derived from Lactococcus lactis" may be a protein isolated from Lactococcus lactis, or may be a variant of the isolated protein as long as the activity of the protein is maintained. It's okay. More preferably, the ldhA protein is a protein derived from Lactococcus lactis NBRC100676 strain. More preferably, it is ldhA protein derived from Lactococcus lactis NBRC100676 strain, in which the 85th glutamine residue from the N-terminus is replaced with a cysteine residue.

Preferably, the fourth gene is selected from the group consisting of:
(4-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 28 (i.e., ldhA protein derived from Lactococcus lactis NBRC100676 strain, in which the 85th glutamine residue from the N-terminus is substituted with a cysteine residue) (4-2) A gene encoding a protein that catalyzes the reduction reaction of α-keto acids, and (4-2) an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 28. A gene encoding a protein that has the activity of catalyzing the reduction reaction of α-keto acids.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 28, and that has the activity of catalyzing the reduction reaction of α-keto acid" "80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more of the amino acid sequence set forth in SEQ ID NO: 28. It may also be a gene that encodes a protein consisting of an amino acid sequence having homology to , and having the activity of catalyzing the reduction reaction of α-keto acids.

"A gene that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 28, and that has the activity of catalyzing the reduction reaction of α-keto acid" Preferably, the protein encodes a protein that maintains a cysteine residue at the 85th residue from the N-terminus.

More preferably, the fourth gene is the ldhA gene derived from Lactococcus lactis NBRC100676 strain, which has the base sequence set forth in SEQ ID NO: 27. The base sequence set forth in SEQ ID NO: 27 encodes a protein having a cysteine residue at the 85th residue from the N-terminus.

The base sequence set forth in SEQ ID NO: 27 and the amino acid sequence set forth in SEQ ID NO: 28 are as follows.

The activity of catalyzing the reduction reaction of α-keto acids can be determined, for example, by catalyzing the enzyme reaction with 60mM containing 0.25mM NADH, 50mM potassium chloride, 5mM magnesium chloride, 5mM magnesium chloride, 5mM D-fructose and 1,6-bisphosphate. It can be calculated by measuring the initial consumption rate of NADH, which is consumed when the raw material α-keto acid is reduced, in a HEPES-NaOH buffer (pH 7.0) at 30°C. The initial consumption rate of NADH can be calculated, for example, by measuring the rate of decrease in absorbance at 340 nm derived from NADH in the enzyme reaction solution. Here, the activity of reducing 1 μmol of α-keto acid per minute at 30° C. is defined as 1 unit.

``A gene (ldhA gene) that encodes a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 28, and has the activity of catalyzing the reduction reaction of α-keto acids. mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

(3.1.4) Construction of recombinant vector for transformation The first and fourth genes can be integrated into a suitable plasmid vector for transformation.

As the plasmid vector, a plasmid vector similar to the plasmid vector described in "(1.1.3) Construction of recombinant vector for transformation" can be used.

The first and fourth genes may be integrated into separate plasmid vectors, or may be integrated into the same plasmid vector. Transformants can be produced using the obtained recombinant vector.

(3.1.5) Transformation Any known transformation method can be used without restriction. As such a known method, the same method as described in the column "(1.1.4) Transformation" can be used.

In addition, when a transformant is produced using a recombinant vector as described above, each of the first and fourth genes may be integrated into the chromosome of the host, or alternatively, the first and fourth genes may be integrated into the host chromosome in the form of a recombinant vector. may be present in the cytoplasm of

(3.1.6) Disruption or deletion of host chromosomal genes The host may be a wild strain, a mutant strain thereof, or an artificially genetically modified strain. When the host is a coryneform bacterium, the host must be a gene-disrupted strain or a gene-deleted strain similar to the gene-disrupted strain or gene-deleted strain described in “(1.1.5) Disruption or deletion of host chromosomal genes.” Deletion strains can be used. In addition, as a method for producing gene-disrupted strains or gene-deleted strains, methods similar to those described in the column "(1.1.5) Disruption or deletion of host chromosomal genes" can be used. . By using such a gene-disrupted strain or gene-deleted strain as a host, it is possible to improve the productivity of 2,4-dihydroxycarboxylic acid and to suppress the production of by-products.

(3.1.7) Specific examples of 2,4-dihydroxycarboxylic acid-producing microorganisms Specifically, 2,4-dihydroxycarboxylic acid-producing microorganisms include:
The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that is a protein consisting of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that encodes a protein that has the activity of catalyzing the aldol reaction between pyruvate and a carbonyl compound; and a gene selected from the group consisting of: The fourth gene to be:
(4-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 28, which catalyzes the reduction reaction of α-keto acid; and (4-2) one or several genes in SEQ ID NO: 28. A coryneform bacterium can be used that contains a gene encoding a protein having an amino acid sequence in which the following amino acids have been deleted, substituted, or added, and which has an activity of catalyzing the reduction reaction of α-keto acids.

(3.2) Cultivation step A 2,4-dihydroxycarboxylic acid-producing microorganism (for example, the transformant described above) is cultured in the presence of a pyruvate-supplying compound and a carbonyl compound to produce 2,4-dihydroxycarboxylic acid. can be produced. "Cultivating in the presence of a pyruvate-supplying compound and a carbonyl compound" can be performed by culturing in a culture medium containing a pyruvate-supplying compound and a carbonyl compound. Preferably, "culturing in the presence of a pyruvate-supplying compound and a carbonyl compound" can be carried out by culturing in a culture medium to which the pyruvate-supplying compound and carbonyl compound are added.

Prior to culturing in the presence of a pyruvate-supplying compound and a carbonyl compound, it is preferable to carry out the proliferation culture described in the column "(1.2) Cultivation step".

When propagation culture is performed prior to production culture, the propagation culture and subsequent production culture can be performed, for example, as described in the column "(1.2) Cultivation step".

(3.2.1) Culture solution for proliferation As the culture solution for proliferation, the same culture solution as the culture solution for proliferation described in the column "(1.2.1) Culture solution for proliferation" can be used. can.

(3.2.2) Culture solution for production As the culture solution for production, the same culture solution as the production culture solution described in the column "(1.2.2) Culture solution for production" can be used. can.

A specific production culture solution contains glucose, formaldehyde, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, iron (II) sulfate and manganese (II) sulfate, and has a pH of 7.0. A culture medium can be used.

In addition, when one type of carbonyl compound is used in the production culture solution, the separation and purification described in the section "(3.2.4) Recovery of 2,4-dihydroxycarboxylic acid" described below must be easily performed. is possible.

(3.2.3) Conditions for production culture The culture temperature for production culture may be any temperature as long as it is suitable for producing 2,4-dihydroxycarboxylic acid. The culture temperature in production culture is preferably about 20 to 50°C, more preferably about 25 to 47°C. Within the above temperature range, 2,4-dihydroxycarboxylic acid can be efficiently produced. The pH of the production culture solution may be any pH suitable for producing 2,4-dihydroxycarboxylic acid. The pH of the production culture solution is preferably maintained within the range of about 6-8. The pH of the production culture solution should be adjusted using a pH buffer solution containing an inorganic or organic acid; an alkaline solution such as an aqueous potassium hydroxide solution; urea; calcium carbonate; ammonia; 4-morpholinopropanesulfonic acid, etc. I can do it. Even during production culture, the pH of the production culture solution can be adjusted as necessary. The culture time in production culture may be any time as long as it is sufficient for the transformant to produce 2,4-dihydroxycarboxylic acid. The culture time in production culture is preferably about 3 hours to 7 days, more preferably about 1 to 3 days.
Production culture may be carried out under aerobic conditions, anaerobic conditions or microaerobic conditions. This is because the above-mentioned transformant has a pathway for producing 2,4-dihydroxycarboxylic acid that works under both aerobic, anaerobic, or microaerobic conditions. . The route for producing 2,4-dihydroxycarboxylic acid will be described in the "(3.3) Actions and Effects" section below.

<Anaerobic conditions or microaerobic conditions>
Production culture is preferably carried out under anaerobic or microaerobic conditions.

When the transformant is cultured under anaerobic or microaerobic conditions, the transformant does not substantially proliferate and can produce 2,4-dihydroxycarboxylic acid more efficiently.

“Anaerobic conditions or microaerobic conditions” can be the same conditions as “anaerobic conditions or microaerobic conditions” described in “(1.2.3) Production culture conditions”. can.

<High density conditions>
Production culture is preferably carried out with the transformant suspended in a production culture solution at high density. By culturing the transformant under such conditions, 2,4-dihydroxycarboxylic acid can be produced more efficiently.

"A state in which the transformant is suspended in a production culture solution at high density" means "a state in which the transformant is suspended in a production culture solution" as described in the column of "(1.2.3) Production culture conditions". It can be in a state similar to "a state in which it is suspended at a high density in".

Production culture is more preferably carried out under anaerobic or microaerobic conditions with the transformant suspended in a production culture solution at high density.

(3.2.4) Recovery of 2,4-dihydroxycarboxylic acid As mentioned above, when the transformant containing the first and fourth genes is cultured in the production culture solution, 2,4-dihydroxycarboxylic acid is added to the production culture solution. -Dihydroxycarboxylic acids are produced.

Specifically, the following general formula (1) is added to the production culture solution:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
When using a carbonyl compound represented by the following general formula (5):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(OH)COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (5) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
A 2,4-dihydroxycarboxylic acid represented by can be produced.

More specifically, when using a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (1), when R ₁₀₁ is hydrogen in the above general formula (5), , and R ₁₀₂ is hydrogen (ie, 2,4-dihydroxybutyric acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen, in the above general formula (5), R ₁₀₁ is a methyl group, and R A 2,4-dihydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2,4-dihydroxyvaleric acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen, in the above general formula (5), R ₁₀₁ is an ethyl group, and R A 2,4-dihydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2,4-dihydroxycaproic acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen, in the above general formula (5), R ₁₀₁ is a propyl group, and R A 2,4-dihydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2,4-dihydroxyenantoic acid) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen in the above general formula (1), in the above general formula (5), R ₁₀₁ is a normal propyl group. and R ₁₀₂ is hydrogen (ie, 2,4-dihydroxynormalheptanoic acid) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen in the general formula (1), R ₁₀₁ in the general formula (5) is hydrogen. A 2,4-dihydroxycarboxylic acid that is an isopropyl group and R ₁₀₂ is hydrogen (ie, 2,4-dihydroxyisoenanto acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen, in the above general formula (5), R ₁₀₁ is a pentyl group, and R A 2,4-dihydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2,4-dihydroxycaprylic acid) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal pentyl group in the above general formula (1) and R ₁₀₂ is hydrogen, in the above general formula (5), R ₁₀₁ is a normal pentyl group. and R ₁₀₂ is hydrogen (ie, 2,4-dihydroxyn-caprylic acid) can be produced.
More specifically, when using a carbonyl compound in which R ₁₀₁ and R ₁₀₂ in the above general formula (1) are both methyl groups, in the above general formula (5), both R ₁₀₁ and R ₁₀₂ are methyl groups. The radical 2,4-dihydroxycarboxylic acid (ie, 4-methyl-2,4-dihydroxyvaleric acid) can be produced.

The above-mentioned 2,4-dihydroxycarboxylic acid can be recovered by recovering the production culture solution, but 2,4-dihydroxycarboxylic acid can also be separated and purified from the production culture solution by a known method. Such known methods include a crystallization method, a chromatography method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, and the like.

Note that substances such as α-keto acids produced by the aldol reaction catalyzed by the enzyme encoded by the first gene can also be recovered from the production culture solution.

Furthermore, when multiple types of carbonyl compounds are used, multiple types of 2,4-dihydroxycarboxylic acids are produced in the culture solution. Multiple types of 2,4-dihydroxycarboxylic acids can be separated from each other by crystallization or chromatography.

(3.3) Actions and Effects The process by which 2,4-dihydroxycarboxylic acid-producing microorganisms produce 2,4-dihydroxycarboxylic acid is schematically shown in FIG. In the process shown in FIG. 3, an example will be explained in which the pyruvate supplying compound is glucose, the carbonyl compound is formaldehyde, and the 2,4-dihydroxycarboxylic acid is 2,4-dihydroxybutyric acid.

As shown in FIG. 3, first, the 2,4-dihydroxycarboxylic acid producing microorganism 11 takes in glucose 2 and formaldehyde 3. Glucose 2 is then converted to pyruvate 4 through glycolysis. Next, pyruvate 4 and formaldehyde 3 are converted to 4-hydroxy-2-oxobutyric acid 5 by the "enzyme that catalyzes the aldol reaction between pyruvate and aldehyde" encoded by the first gene. Next, 4-hydroxy-2-oxobutyric acid 5 is converted to 2,4-dihydroxybutyric acid 12 by the "enzyme that catalyzes the reduction reaction of α-keto acid" encoded by the fourth gene. In the manner described above, 2,4-dihydroxybutyric acid is produced.

(4.) Method for producing 2-amino-4-hydroxycarboxylic acid (biological method)
The method for producing 2-amino-4-hydroxycarboxylic acid is as follows:
The following genes:
(a) A microorganism comprising a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (e) a fifth gene encoding an enzyme that catalyzes a reductive amination reaction of an α-keto acid. a pyruvate-providing compound selected from pyruvate and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
The method includes a step of producing 2-amino-4-hydroxycarboxylic acid represented by

In the following description, the microorganism containing the first and fifth genes is also referred to as a "2-amino-4-hydroxycarboxylic acid producing microorganism." The 2-amino-4-hydroxycarboxylic acid-producing microorganism may be a microorganism that naturally has the first and fifth genes, or a microorganism obtained by transforming a host with the first and fifth genes. There may be. Hereinafter, the case where the 2-amino-4-hydroxycarboxylic acid-producing microorganism is a transformant will be explained as an example.

(4.1) Transformants (4.1.1) Host As the host, the same hosts as those described in the column "(1.1.1) Host" can be used.
Next, the first and fifth genes introduced into the host will be explained.

(4.1.2) First gene As the first gene, a gene similar to the first gene described in the column "(1.1.2) First gene" can be used.

(4.1.3) Fifth gene The fifth gene encodes "an enzyme that catalyzes the reductive amination reaction of α-keto acids.""Enzyme that catalyzes the reductive amination reaction of α-keto acid" means using an enzyme reaction solution containing purified protein and α-keto acid (initial substrate concentration 1mM) according to the measurement method described below. An enzyme that has an activity of 10 mU/mg protein or more when measured as follows. The fifth gene encodes, for example, an enzyme that catalyzes the reductive amination reaction of α-keto acids obtained by the aldol reaction catalyzed by the protein encoded by the first gene. The α-keto acid in the “enzyme that catalyzes the reductive amination reaction of α-keto acid” is, for example, expressed by the general formula (2) described in the column “(1.) Method for producing α-keto acid”. It is an α-keto acid represented by The "enzyme that catalyzes the reductive amination reaction of α-keto acids" is, for example, glutamate/phenylalanine/leucine/valine dehydrogenase (IPR006095). "Glutamate/phenylalanine/leucine/valine dehydrogenase" is a term used in the art to refer to a family of dehydrogenases that includes glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase, and valine dehydrogenase. According to one example, "glutamate/phenylalanine/leucine/valine dehydrogenase" is at least one dehydrogenase selected from the group consisting of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase and valine dehydrogenase.

The fifth gene may be a gene encoding glutamic acid/phenylalanine/leucine/valine dehydrogenase as exemplified below.

(d1) leucine dehydrogenase (EC number 1.4.1.9),
(d2) phenylalanine dehydrogenase (EC number 1.4.1.20), and (d3) valine dehydrogenase (EC number 1.4.1.8 and 1.4.1.23).

Examples of the dehydrogenase included in (d1) include leuDH protein. Preferably, the leuDH protein is derived from Lysinibacillus sphaericus, Bacillus cereus or Geobacillus stearothermophillus.
Examples of the dehydrogenase included in (d2) include phedh protein. Preferably, the phedh protein is derived from Bacillus badius.
Examples of the dehydrogenase included in (d3) include valdh protein. Preferably, the valdh protein is from Streptomyces fradiae.

Preferably, the fifth gene is selected from the group consisting of:
(5-1) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30 (i.e., leuDH protein derived from Ricinibacillus sphaericus), a gene encoding a protein that catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32 (i.e., phedh protein derived from Bacillus badius), which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34 (i.e., valdh protein derived from Streptomyces Fraziae), a gene encoding a protein that catalyzes the reductive amination reaction of α-keto acids; and (5-6) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 34, and has an activity of catalyzing the reductive amination reaction of α-keto acids. Genes that code for proteins.

``A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 30, and which has the activity of catalyzing the reductive amination reaction of α-keto acids. ” means “at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 98%, with respect to the amino acid sequence set forth in SEQ ID NO: 30.” It may also be a gene that encodes a protein consisting of an amino acid sequence having 99% or more homology and having the activity of catalyzing the reductive amination reaction of α-keto acids.

``A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 32, and which has the activity of catalyzing the reductive amination reaction of α-keto acids. ” means “at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 98%, with respect to the amino acid sequence set forth in SEQ ID NO: 32.” It may also be a gene that encodes a protein consisting of an amino acid sequence having 99% or more homology and having the activity of catalyzing the reductive amination reaction of α-keto acids.

``A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 34, and which has the activity of catalyzing the reductive amination reaction of α-keto acids. ” means “at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 98%, with respect to the amino acid sequence set forth in SEQ ID NO: 34.” It may also be a gene that encodes a protein consisting of an amino acid sequence having 99% or more homology and having the activity of catalyzing the reductive amination reaction of α-keto acids.

More preferably, the fifth gene is
leuDH gene derived from Ricinibacillus sphaericus, consisting of the nucleotide sequence set forth in SEQ ID NO: 30,
The phedh gene derived from Bacillus badius consists of the base sequence set forth in SEQ ID NO: 32, or the valdh gene derived from Streptomyces frasiae consists of the base sequence set forth in SEQ ID NO: 34.

The base sequence set forth in SEQ ID NO: 29 and the amino acid sequence set forth in SEQ ID NO: 30 are as follows.

The base sequence set forth in SEQ ID NO: 31 and the amino acid sequence set forth in SEQ ID NO: 32 are as follows.

The base sequence set forth in SEQ ID NO: 33 and the amino acid sequence set forth in SEQ ID NO: 34 are as follows.

α－ケト酸の還元的アミノ化反応を触媒する活性は、例えば、当該酵素反応を０．２ｍＭ ＮＡＤＨ、２００ｍＭ 塩化アンモニウムを含有する１００ｍＭ ＨＥＰＥＳ－ＫＯＨバッファー（ｐＨ７．５）中で２５℃にて行い、原料のα－ケト酸がα－アミノ酸に還元される際に消費されるＮＡＤＨの初期消費速度を測定することで算出できる。ＮＡＤＨの初期消費速度は、例えば、酵素反応液中のＮＡＤＨに由来する３４０ｎｍにおける吸光度の減少速度を測定することで算出できる。ここでは、２５℃で１分間に１μｍｏｌのα－ケト酸がα－アミノ酸に還元される活性を１ユニットとする。

The activity of catalyzing the reductive amination reaction of α-keto acids can be determined, for example, by performing the enzyme reaction at 25°C in 100mM HEPES-KOH buffer (pH 7.5) containing 0.2mM NADH and 200mM ammonium chloride. can be calculated by measuring the initial consumption rate of NADH consumed when the raw material α-keto acid is reduced to α-amino acid. The initial consumption rate of NADH can be calculated, for example, by measuring the rate of decrease in absorbance at 340 nm derived from NADH in the enzyme reaction solution. Here, 1 unit is defined as the activity of reducing 1 μmol of α-keto acid to α-amino acid in 1 minute at 25°C.

``A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 30, and which has the activity of catalyzing the reductive amination reaction of α-keto acids. (leuDH gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

``A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 32, and which has the activity of catalyzing the reductive amination reaction of α-keto acids. (phedh gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

``A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 34, and which has the activity of catalyzing the reductive amination reaction of α-keto acids. (valdh gene mutant)" can be selected from DNA libraries of other species by a method similar to the above method for selecting hpaI gene mutants.

(4.1.4) Construction of recombinant vector for transformation The first and fifth genes can be integrated into a suitable plasmid vector for transformation.

As the plasmid vector, a plasmid vector similar to the plasmid vector described in "(1.1.3) Construction of recombinant vector for transformation" can be used.

The first and fifth genes may be integrated into separate plasmid vectors, or may be integrated into the same plasmid vector. Transformants can be produced using the obtained recombinant vector.

(4.1.5) Transformation Any known transformation method can be used without restriction. As such a known method, the same method as described in the column "(1.1.4) Transformation" can be used.

In addition, when a transformant is produced using a recombinant vector as described above, each of the first and fifth genes may be integrated into the chromosome of the host, or may be inserted into the host in the form of a recombinant vector. may be present in the cytoplasm of

(4.1.6) Disruption or deletion of host chromosomal genes The host may be a wild strain, a mutant strain thereof, or an artificially genetically modified strain. When the host is a coryneform bacterium, the host must be a gene-disrupted strain or a gene-deleted strain similar to the gene-disrupted strain or gene-deleted strain described in “(1.1.5) Disruption or deletion of host chromosomal genes.” Deletion strains can be used. In addition, as a method for producing gene-disrupted strains or gene-deleted strains, methods similar to those described in the column "(1.1.5) Disruption or deletion of host chromosomal genes" can be used. . By using such a gene-disrupted strain or gene-deleted strain as a host, it is possible to improve the productivity of 2-amino-4-hydroxycarboxylic acid and to suppress the production of by-products.

(4.1.7) Specific examples of 2-amino-4-hydroxycarboxylic acid-producing microorganisms Specifically, 2-amino-4-hydroxycarboxylic acid-producing microorganisms include:
The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that is a protein consisting of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that encodes a protein that has the activity of catalyzing the aldol reaction between pyruvate and a carbonyl compound; and a gene selected from the group consisting of: The fifth gene to be:
(5-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, and (5-6) 1 in SEQ ID NO: 34. or a coryneform bacterium that contains a gene encoding a protein that is a protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted, or added, and that has the activity of catalyzing the reductive amination reaction of α-keto acids. can be used.

(4.2) Cultivation step A 2-amino-4-hydroxycarboxylic acid-producing microorganism (for example, the above-mentioned transformant) is cultured in the presence of a pyruvate-supplying compound and a carbonyl compound to Hydroxycarboxylic acids can be produced. "Cultivating in the presence of a pyruvate-supplying compound and a carbonyl compound" can be performed by culturing in a culture medium containing a pyruvate-supplying compound and a carbonyl compound. Preferably, "culturing in the presence of a pyruvate-supplying compound and a carbonyl compound" can be carried out by culturing in a culture medium to which the pyruvate-supplying compound and carbonyl compound are added.

Prior to culturing in the presence of a pyruvate-supplying compound and a carbonyl compound, it is preferable to carry out the proliferation culture described in the column "(1.2) Cultivation step".

When propagation culture is performed prior to production culture, the propagation culture and subsequent production culture can be performed, for example, as described in the column "(1.2) Cultivation step".

(4.2.1) Culture solution for proliferation As the culture solution for proliferation, the same culture solution as the culture solution for proliferation described in the column "(1.2.1) Culture solution for proliferation" can be used. can.

(4.2.2) Culture solution for production As the culture solution for production, the same culture solution as the production culture solution described in the column "(1.2.2) Culture solution for production" can be used. can.

The specific production culture medium contains glucose, formaldehyde, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, iron (II) sulfate, and manganese (II) sulfate, and has a pH of 7.0. A medium having the following can be used.

In addition, when one type of carbonyl compound is used in the production culture solution, the separation and purification described in "(4.2.4) Recovery of 2-amino-4-hydroxycarboxylic acid" described below can be easily carried out. It is possible to do so.

(4.2.3) Conditions for production culture The culture temperature for production culture may be any temperature as long as it is suitable for producing 2-amino-4-hydroxycarboxylic acid. The culture temperature in production culture is preferably about 20 to 50°C, more preferably about 25 to 47°C. Within the above temperature range, 2-amino-4-hydroxycarboxylic acid can be produced efficiently. The pH of the production culture solution may be any pH suitable for producing 2-amino-4-hydroxycarboxylic acid. The pH of the production culture solution is preferably maintained within the range of about 6-8. The pH of the production culture solution should be adjusted using a pH buffer solution containing an inorganic or organic acid; an alkaline solution such as an aqueous potassium hydroxide solution; urea; calcium carbonate; ammonia; 4-morpholinopropanesulfonic acid, etc. Can be done. Even during production culture, the pH of the production culture solution can be adjusted as necessary. The culture time in production culture may be any time as long as it is sufficient for the transformant to produce 2-amino-4-hydroxycarboxylic acid. The culture time in production culture is preferably about 3 hours to 7 days, more preferably about 1 to 3 days.
Production culture may be carried out under aerobic conditions, anaerobic conditions or microaerobic conditions. This is because the above-mentioned transformants have a pathway for producing 2-amino-4-hydroxycarboxylic acid that works under both aerobic, anaerobic, or microaerobic conditions. It is. The route for producing 2-amino-4-hydroxycarboxylic acid will be described in the "(4.3) Actions and Effects" section below.

<Anaerobic conditions or microaerobic conditions>
Production culture is preferably carried out under anaerobic or microaerobic conditions.

When the transformant is cultured under anaerobic or microaerobic conditions, the transformant does not substantially proliferate and can produce 2-amino-4-hydroxycarboxylic acid more efficiently.

“Anaerobic conditions or microaerobic conditions” can be the same conditions as “anaerobic conditions or microaerobic conditions” described in “(1.2.3) Production culture conditions”. can.

<High density conditions>
Production culture is preferably carried out with the transformant suspended in a production culture solution at high density. By culturing the transformant under such conditions, 2-amino-4-hydroxycarboxylic acid can be produced more efficiently.

"A state in which the transformant is suspended in a production culture solution at high density" means "a state in which the transformant is suspended in a production culture solution" as described in the column of "(1.2.3) Production culture conditions". It can be in a state similar to "a state in which it is suspended at a high density in".

Production culture is more preferably carried out under anaerobic or microaerobic conditions with the transformant suspended in a production culture solution at high density.

(4.2.4) Recovery of 2-amino-4-hydroxycarboxylic acid As mentioned above, when a transformant containing the first and fifth genes is cultured in a production culture solution, 2-amino-4-hydroxycarboxylic acid is -amino-4-hydroxycarboxylic acid is produced.

Specifically, the following general formula (1) is contained in the production culture solution:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
When using a carbonyl compound represented by the following general formula (6):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
A 2-amino-4-hydroxycarboxylic acid represented by can be produced.

More specifically, when using a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (1), when R ₁₀₁ is hydrogen in the above general formula (6), , and R ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxybutyric acid).
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is a methyl group, and R A 2-amino-4-hydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxyvaleric acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is an ethyl group, and R A 2-amino-4-hydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxycaproic acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is a propyl group, and R A 2-amino-4-hydroxycarboxylic acid (ie, 4-hydroxy-2-oxoenantoic acid) in which ₁₀₂ is hydrogen can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen in the above general formula (1), in the above general formula (6), R ₁₀₁ is a normal propyl group. and R ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxynormalheptanoic acid) can be produced. Further, specifically, when using a carbonyl compound in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen in the above general formula (1), in the above general formula (6), R ₁₀₁ is an isopropyl group. and R ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxyisoenanto acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is a pentyl group, and R A 2-amino-4-hydroxycarboxylic acid in which ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxycaprylic acid) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal pentyl group in the above general formula (1) and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is a normal pentyl group. and R ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxyn-caprylic acid) can be produced.
More specifically, when using a carbonyl compound in which R ₁₀₁ and R ₁₀₂ in the above general formula (1) are both methyl groups, in the above general formula (6), R ₁₀₁ and R ₁₀₂ are both methyl groups. The group 2-amino-4-hydroxycarboxylic acid (ie, 4-methyl-2-amino-4-hydroxyvaleric acid) can be produced.

In the above general formula (1), 2-amino-4-hydroxybutyric acid obtained when a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen is also called homoserine. Homoserine is, for example, L-homoserine. When a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (1) is used, a homoserine derivative is produced in addition to homoserine in the production culture solution. Homoserine derivatives include threonine and 2-aminobutyric acid.

Threonine is produced from homoserine, for example, by enzymes (eg, homoserine kinase and threonine synthase) that convert homoserine to threonine, which are carried by 2-amino-4-hydroxycarboxylic acid-producing microorganisms. Threonine is, for example, L-threonine.

2-Aminobutyric acid is produced by, for example, an enzyme that converts threonine into 2-aminobutyric acid (for example, threonine deaminase, amino acid dehydrogenase, or aminotransferase), which is held by a 2-amino-4-hydroxycarboxylic acid-producing microorganism. is produced from threonine by a combination of Amino acid dehydrogenase is, for example, glutamate/phenylalanine/leucine/valine dehydrogenase. Glutamic acid/phenylalanine/leucine/valine dehydrogenase is specifically leucine dehydrogenase.

The above-mentioned 2-amino-4-hydroxycarboxylic acid and homoserine derivatives can be recovered by collecting the production culture solution, but 2-amino-4-hydroxycarboxylic acid can also be separated from the production culture solution using a known method. It can also be purified. Such known methods include a crystallization method, a chromatography method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, and the like.

Note that substances such as α-keto acids produced by the aldol reaction catalyzed by the enzyme encoded by the first gene can also be recovered from the production culture solution.

As mentioned above, when using a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the general formula (1), homoserine, threonine and 2-aminobutyric acid are produced in the culture solution. . Homoserine, threonine and 2-aminobutyric acid can be separated from each other by crystallization or chromatography.
Furthermore, when multiple types of carbonyl compounds are used, multiple types of 2-amino-4-hydroxycarboxylic acids are produced in the culture solution. Multiple types of 2-amino-4-hydroxycarboxylic acids can be separated from each other by crystallization or chromatography.

(4.3) Action and Effect The process by which 2-amino-4-hydroxycarboxylic acid-producing microorganisms produce 2-amino-4-hydroxycarboxylic acid is schematically shown in FIG. The process shown in FIG. 4 will be explained using an example in which the pyruvic acid supplying compound is glucose, the carbonyl compound is formaldehyde, and the 2-amino-4-hydroxycarboxylic acid is homoserine.

As shown in FIG. 4, first, the 2-amino-4-hydroxycarboxylic acid producing microorganism 13 takes in glucose 2 and formaldehyde 3. Glucose 2 is then converted to pyruvate 4 through glycolysis. Next, pyruvate 4 and formaldehyde 3 are converted to 4-hydroxy-2-oxobutyric acid 5 by the "enzyme that catalyzes the aldol reaction between pyruvate and aldehyde" encoded by the first gene. Next, 4-hydroxy-2-oxobutyric acid 5 is converted to homoserine 14 by the "enzyme that catalyzes the reductive amination reaction of α-keto acids" encoded by the fifth gene. Homoserine is produced in the above manner.

(5.) Method for producing 2-amino-4-hydroxycarboxylic acid (chemical method)
In the column "(4.) Method for producing 2-amino-4-hydroxycarboxylic acid," we explained a method for producing 2-amino-4-hydroxycarboxylic acid by a biological method using microorganisms. The method can also be carried out by chemical methods without the use of microorganisms.
That is, the method for producing 2-amino-4-hydroxycarboxylic acid is as follows:
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
α-keto acid, nitrogen source and reducing agent represented by
React in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase,
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (2), respectively)
The method includes a step of producing 2-amino-4-hydroxycarboxylic acid represented by

Reacting an α-keto acid, a nitrogen source, and a reducing agent in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase can be performed, for example, by reacting an α-keto acid, a nitrogen source, a reducing agent, and glutamic acid/phenylalanine/leucine/valine dehydrogenase. and a buffer. Hereinafter, a case where the reaction of an α-keto acid, a nitrogen source, and a reducing agent in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase is performed in a reaction solution will be described as an example.

(5.1) Reaction liquid Each component contained in the reaction liquid will be explained below. The concentration of each component in the reaction solution described herein refers to the final concentration in the reaction solution at the time each component is added.
(5.1.1) α-keto acid α-keto acid has the following general formula (2):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
Represented by
According to one example, an α-keto acid is an α-keto acid in which R ₁₀₁ is hydrogen or an alkyl group having 1 to 5 carbon atoms, and R ₁₀₂ is hydrogen in the above general formula (2). Acids can be used.
According to another example, an α-keto acid is an α-keto acid in the general formula (2) above, in which R ₁₀₁ is a methyl group and R ₁₀₂ is an alkyl group having 1 to 5 carbon atoms. can be used.

Specifically, as the α-keto acid represented by the above general formula (2), in the above general formula (2),
an α-keto acid in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen (i.e., 4-hydroxy-2-oxobutyric acid);
an α-keto acid in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen (i.e., 4-hydroxy-2-oxovaleric acid);
an α-keto acid in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen (i.e., 4-hydroxy-2-oxocaproic acid);
α-keto acid (i.e., 4-hydroxy-2-oxoenanto acid) in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen, specifically, R ₁₀₁ is a normal propyl group, and R an α-keto acid in which ₁₀₂ is hydrogen (i.e., 4-hydroxy-2-oxonormalheptanoic acid), or an α-keto acid in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen (i.e., 4-hydroxy-2-oxonalheptanoic acid); or α-keto acids (i.e., 4-hydroxy-2-oxocaprylic acid) in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen, specifically , an α-keto acid in which R ₁₀₁ is a normal pentyl group and R ₁₀₂ is hydrogen (i.e., 4-hydroxy-2-oxonormal caprylic acid);
can be used.
Further, as the α-keto acid represented by the above general formula (2), in the above general formula (2), R ₁₀₁ and R ₁₀₂ are both methyl groups (i.e., 4-methyl- 4-hydroxy-2-oxovaleric acid) can also be used.
One type of α-keto acid may be used, or two or more types may be used in combination.

The concentration of α-keto acid in the reaction solution can be made as high as possible without inhibiting the production of 2-amino-4-hydroxycarboxylic acid. The concentration of α-keto acid in the reaction solution is preferably within the range of 0.1 mM to 1M, more preferably within the range of 1 mM to 300 mM. Additionally, α-keto acid can be additionally added in accordance with the decrease in α-keto acid accompanying the production of 2-amino-4-hydroxycarboxylic acid.

In addition, when one type of α-keto acid is used in the reaction solution, the separation and purification described in the section "(5.3) Recovery of 2-amino-4-hydroxycarboxylic acid" described below should be easily performed. is possible.

(5.1.2) Nitrogen Source As the nitrogen source, any compound that provides an amino group in the reductive amination reaction catalyzed by glutamic acid/phenylalanine/leucine/valine dehydrogenase can be used. As the nitrogen source, specifically, ammonium salts that dissociate in the reaction solution to produce ammonium ions, such as ammonium chloride, ammonium sulfate, and ammonium carbonate, can be used.

The concentration of the nitrogen source in the reaction solution can be made as high as possible without inhibiting the production of 2-amino-4-hydroxycarboxylic acid. The concentration of the nitrogen source in the reaction solution is preferably within the range of 0.1mM to 1M, more preferably within the range of 1mM to 300mM. Additionally, a nitrogen source can be added in response to a decrease in the nitrogen source accompanying the production of 2-amino-4-hydroxycarboxylic acid.

(5.1.3) Reducing Agent As the reducing agent, any electron donor that can be used by glutamic acid/phenylalanine/leucine/valine dehydrogenase can be used. For example, NADH, NADPH can be used.

The concentration of the reducing agent in the reaction solution is preferably within the range of 0.01mM to 100mM, more preferably within the range of 0.1mM to 10mM. Furthermore, the reducing agent that decreases with the production of 2-amino-4-hydroxycarboxylic acid can be regenerated using an appropriate compound and enzyme. Suitable compounds and enzymes are, for example, ethanol and alcohol dehydrogenase.

(5.1.4) Glutamic acid/phenylalanine/leucine/valine dehydrogenase As the glutamic acid/phenylalanine/leucine/valine dehydrogenase, any enzyme included in glutamic acid/phenylalanine/leucine/valine dehydrogenase can be used. According to one example, "glutamate/phenylalanine/leucine/valine dehydrogenase" is at least one dehydrogenase selected from the group consisting of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase and valine dehydrogenase.

As the glutamic acid/phenylalanine/leucine/valine dehydrogenase, the following dehydrogenases can be used.

(d1) leucine dehydrogenase (EC number 1.4.1.9),
(d2) phenylalanine dehydrogenase (EC number 1.4.1.20), and (d3) valine dehydrogenase (EC number 1.4.1.8 and 1.4.1.23).

Examples of the dehydrogenase included in (d1) include leuDH protein. Preferably, the leuDH protein is derived from Lysinibacillus sphaericus, Bacillus cereus or Geobacillus stearothermophillus.
Examples of the dehydrogenase included in (d2) include phedh protein. Preferably, the phedh protein is derived from Bacillus badius.
Examples of the dehydrogenase included in (d3) include valdh protein. Preferably, the valdh protein is from Streptomyces fradiae.

Preferably, the glutamate/phenylalanine/leucine/valine dehydrogenase is selected from the group consisting of:
(5'-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5'-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added to SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. protein,
(5'-3) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5'-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. protein,
(5'-5) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes a reductive amination reaction of α-keto acid, and (5'-6) 1 or a number in SEQ ID NO: 34. A protein consisting of an amino acid sequence in which six amino acids have been deleted, substituted, or added, and has the activity of catalyzing the reductive amination reaction of α-keto acids.

"A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 30, and which has the activity of catalyzing the reductive amination reaction of α-keto acids" is defined as " 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more of the amino acid sequence set forth in SEQ ID NO: 30. It may also be a protein consisting of a homologous amino acid sequence and having an activity of catalyzing the reductive amination reaction of α-keto acids.

"A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 32, and which has the activity of catalyzing the reductive amination reaction of α-keto acids" is defined as " 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more of the amino acid sequence set forth in SEQ ID NO: 32. It may also be a protein consisting of a homologous amino acid sequence and having an activity of catalyzing the reductive amination reaction of α-keto acids.

"A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 34, and which has the activity of catalyzing the reductive amination reaction of α-keto acids" 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more, still more preferably 98% or more, even more preferably 99% or more of the amino acid sequence set forth in SEQ ID NO: 34. It may also be a protein consisting of a homologous amino acid sequence and having an activity of catalyzing the reductive amination reaction of α-keto acids.

The activity of catalyzing the reductive amination reaction of α-keto acids can be measured by a method similar to the method for measuring activity described in the column of “(4.1.3) Fifth gene”.

Glutamic acid/phenylalanine/leucine/valine dehydrogenase may be produced by, for example, using a commercially available enzyme, or by expressing a gene encoding glutamic acid/phenylalanine/leucine/valine dehydrogenase in a host such as Escherichia coli or coryneform bacteria. The obtained enzyme may be used. In the latter case, a cell lysate of a microorganism expressing the enzyme can be used as the enzyme, or an enzyme-containing liquid obtained by purifying the cell lysate can also be used as the enzyme.

The concentration of glutamic acid/phenylalanine/leucine/valine dehydrogenase in the reaction solution can be made as high as possible without inhibiting the production of 2-amino-4-hydroxycarboxylic acid. The concentration of glutamic acid/phenylalanine/leucine/valine dehydrogenase in the reaction solution is preferably within the range of 0.01 ug/mL to 1 mg/mL, more preferably within the range of 0.1 to 100 ug/mL.

(5.1.5) Buffer A buffer contains, for example, a buffer and other necessary components.

Any buffer can be used as the buffer. As a buffer, for example HEPES or glycine can be used. The concentration of the buffer in the reaction solution can be in the range of 1mM to 1M.

Furthermore, an inorganic salt or the like may be added as necessary.

(5.2) Reaction conditions The reaction temperature may be any temperature as long as it is suitable for producing 2-amino-4-hydroxycarboxylic acid. The reaction temperature is preferably about 4 to 80°C, more preferably about 20 to 60°C. Within the above temperature range, 2-amino-4-hydroxycarboxylic acid can be produced efficiently. The pH of the reaction solution may be any pH suitable for producing 2-amino-4-hydroxycarboxylic acid. The pH of the reaction solution is preferably maintained within the range of about 5-11. The pH of the reaction solution can be adjusted using hydrochloric acid, an aqueous potassium hydroxide solution, or the like. During the reaction, the pH of the reaction solution can be adjusted as necessary. The reaction time may be any time that is sufficient to produce 2-amino-4-hydroxycarboxylic acid. The reaction time is preferably about 1 minute to 10 days, more preferably about 1 hour to 3 days.

(5.3) Recovery of 2-amino-4-hydroxycarboxylic acid As mentioned above, when α-keto acid, nitrogen source, and reducing agent are reacted in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase, 2- Amino-4-hydroxycarboxylic acid is produced.

Specifically, the following general formula (2):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
When using an α-keto acid represented by the following general formula (6):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (2), respectively)
A 2-amino-4-hydroxycarboxylic acid represented by can be produced.

More specifically, when using an α-keto acid in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (2), in the above general formula (6), R ₁₀₁ is hydrogen. and R ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxybutyric acid) can be produced.
In the above general formula (2), when using an α-keto acid in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is a methyl group, and , 2-amino-4-hydroxycarboxylic acid (ie, 2-amino-4-hydroxyvaleric acid) in which R ₁₀₂ is hydrogen can be produced.
In the above general formula (2), when using an α-keto acid in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is an ethyl group, and , 2-amino-4-hydroxycarboxylic acid (ie, 2-amino-4-hydroxycaproic acid) in which R ₁₀₂ is hydrogen can be produced.
In the above general formula (2), when using an α-keto acid in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is a propyl group, and , 2-amino-4-hydroxycarboxylic acid (ie, 2-amino-4-hydroxyenantoic acid) in which R ₁₀₂ is hydrogen can be produced. Specifically, when using an α-keto acid in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen in the above general formula (2), in the above general formula (6), R ₁₀₁ is A 2-amino-4-hydroxycarboxylic acid (ie, 2-amino-4-hydroxynormalheptanoic acid) which is a normal propyl group and R ₁₀₂ is hydrogen can be produced. Further, specifically, when using an α-keto acid in which R ₁₀₁ in the above general formula (2) is an isopropyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen (ie, 2-amino-4-hydroxyisoenanto acid) can be produced.
In the above general formula (2), when using an α-keto acid in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen, in the above general formula (6), R ₁₀₁ is an isopentyl group, and , 2-amino-4-hydroxycarboxylic acid (ie, 2-amino-4-hydroxycaprylic acid) in which R ₁₀₂ is hydrogen can be produced. Specifically, when using an α-keto acid in which R ₁₀₁ is a normal pentyl group and R ₁₀₂ is hydrogen in the above general formula (2), in the above general formula (6), R ₁₀₁ is A 2-amino-4-hydroxycarboxylic acid (ie, 2-amino-4-hydroxynormal caprylic acid) which is a normal pentyl group and R ₁₀₂ is hydrogen can be produced.
When using an α-keto acid in which R ₁₀₁ and R ₁₀₂ in the above general formula (2) are both methyl groups, 2 in the above general formula (6) in which R ₁₀₁ and R ₁₀₂ are both methyl groups. -amino-4-hydroxycarboxylic acid (ie, 4-methyl-2-amino-4-hydroxyvaleric acid).

Although 2-amino-4-hydroxycarboxylic acid can be recovered by recovering the reaction solution, 2-amino-4-hydroxycarboxylic acid can also be separated and purified from the reaction solution using a known method. Such known methods include a crystallization method, a chromatography method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, and the like.

Furthermore, when multiple types of α-keto acids are used, multiple types of 2-amino-4-hydroxycarboxylic acids are produced in the reaction solution. Multiple types of 2-amino-4-hydroxycarboxylic acids can be separated from each other by crystallization or chromatography.

(5.4) Method for producing α-keto acid using oxidative deamination reaction of glutamic acid/phenylalanine/leucine/valine dehydrogenase The method for producing 2-amino-4-hydroxycarboxylic acid described above is based on glutamic acid/phenylalanine/leucine/valine dehydrogenase. Utilizes the reductive amination reaction of leucine/valine dehydrogenase.
Generally, amino acid dehydrogenases such as glutamic acid/phenylalanine/leucine/valine dehydrogenase catalyze both reductive amination reactions and their reverse reactions, that is, oxidative deamination reactions, and the activity of these dehydrogenases. There is a correlation between the strengths (for example, Biochim. Biophys. Acta 96, 248-262 (1965), J. Biol. Chem. 253, 5719-5725 (1978), Eur. J. Biochem. 100, 29-39 ( 1979)). Therefore, oxidative deamination of glutamic acid/phenylalanine/leucine/valine dehydrogenase can be used to produce α-keto acids from 2-amino-4-hydroxycarboxylic acids.
Therefore, according to another embodiment, a method for producing an α-keto acid using the oxidative deamination reaction of glutamic acid/phenylalanine/leucine/valine dehydrogenase is provided. That is, the method for producing an α-keto acid according to this embodiment,
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
2-amino-4-hydroxycarboxylic acid selected from 2-amino-4-hydroxycarboxylic acids represented by and an oxidizing agent,
React in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase,
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (6), respectively)
The method includes a step of producing an α-keto acid represented by

Reacting 2-amino-4-hydroxycarboxylic acid in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase can be performed, for example, by reacting 2-amino-4-hydroxycarboxylic acid with an oxidizing agent and glutamic acid/phenylalanine/leucine/valine dehydrogenase. and a buffer.

As the 2-amino-4-hydroxycarboxylic acid represented by the above general formula (6), the general formula (6) described in the column "(5.3) Recovery of 2-amino-4-hydroxycarboxylic acid" A 2-amino-4-hydroxycarboxylic acid represented by can be used.
One type of 2-amino-4-hydroxycarboxylic acid may be used, or two or more types may be used in combination.
Note that when one type of 2-amino-4-hydroxycarboxylic acid is used in the reaction solution, it is possible to easily separate and purify the α-keto acid, which will be described later.

As the glutamic acid/phenylalanine/leucine/valine dehydrogenase, the glutamic acid/phenylalanine/leucine/valine dehydrogenase described in the column "(5.1.4) Glutamic acid/phenylalanine/leucine/valine dehydrogenase" can be used.

As the oxidizing agent, any electron acceptor that can be used by glutamic acid/phenylalanine/leucine/valine dehydrogenase can be used. For example, NAD ⁺ , NADP ⁺ can be used.

The concentration of the oxidizing agent in the reaction solution is preferably within the range of 0.01mM to 100mM, more preferably within the range of 0.1mM to 10mM. In addition, the oxidizing agent that decreases with the production of α-keto acids can be regenerated using appropriate compounds and enzymes. Suitable compounds and enzymes are, for example, pyruvate and lactate dehydrogenase.

As the buffer, the buffer described in the column "(5.1.5) Buffer" can be used.

The reaction temperature may be any temperature suitable for producing an α-keto acid. The reaction temperature is preferably about 4 to 80°C, more preferably about 20 to 60°C. Within the above temperature range, α-keto acids can be produced efficiently. The pH of the reaction solution may be any pH suitable for producing an α-keto acid. The pH of the reaction solution is preferably maintained within the range of about 5-11. The pH of the reaction solution can be adjusted using hydrochloric acid, an aqueous potassium hydroxide solution, or the like. During the reaction, the pH of the reaction solution can be adjusted as necessary. The reaction time may be any time that is sufficient to produce the α-keto acid. The reaction time is preferably about 1 minute to 10 days, more preferably about 1 hour to 3 days.

As mentioned above, when 2-amino-4-hydroxycarboxylic acid and an oxidizing agent are reacted in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase, an α-keto acid is produced.

Specifically, the following general formula (6):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
When using 2-amino-4-hydroxycarboxylic acid represented by
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (6), respectively)
α-keto acids represented by can be produced.

More specifically, when using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (6), in the above general formula (2), , R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxobutyric acid).
When using a 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen in the above general formula (6), in the above general formula (2), R ₁₀₁ is a methyl group. and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxovaleric acid) can be produced.
When using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen in the above general formula (6), in the above general formula (2), R ₁₀₁ is an ethyl group. and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxocaproic acid) can be produced.
When using a 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen in the above general formula (6), in the above general formula (2), R ₁₀₁ is a propyl group. and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxoenanto acid) can be produced. Specifically, when using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen in the above general formula (6), the above general formula (2) Among them, an α-keto acid (ie, 4-hydroxy-2-oxonormalheptanoic acid) in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen can be produced. Specifically, when using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen in the general formula (6), ) in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxoisoenanto acid) can be produced.
When using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen in the above general formula (6), in the above general formula (2), R ₁₀₁ is a pentyl group. and R ₁₀₂ is hydrogen (ie, 4-hydroxy-2-oxocaprylic acid) can be produced. Specifically, when using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ is a normal pentyl group and R ₁₀₂ is hydrogen in the above general formula (6), the above general formula (2) Among them, an α-keto acid (ie, 4-hydroxy-2-oxonormal caprylic acid) in which R ₁₀₁ is a normal pentyl group and R ₁₀₂ is hydrogen can be produced.
When using 2-amino-4-hydroxycarboxylic acid in which R ₁₀₁ and R ₁₀₂ in the above general formula (6) are both methyl groups, in the above general formula (2), both R ₁₀₁ and R ₁₀₂ are An α-keto acid that is a methyl group (ie, 4-methyl-4-hydroxy-2-oxovaleric acid) can be produced.

The above α-keto acid can be recovered by recovering the reaction solution, but it is also possible to separate and purify the α-keto acid from the reaction solution using a known method. Such known methods include a crystallization method, a chromatography method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, and the like.

Furthermore, when multiple types of 2-amino-4-hydroxycarboxylic acids are used, multiple types of α-keto acids are produced in the reaction solution. Multiple types of α-keto acids can be separated from each other by crystallization or chromatography.

(6.) Method for producing 2-amino-4-methylthiocarboxylic acid The method for producing 2-amino-4-methylthiocarboxylic acid is as follows:
The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(e) the fifth gene encoding an enzyme that catalyzes the reductive amination reaction of α-keto acids, and (f) the methyl mercaptan that has the phosphate group that 2-amino-4-phosphonooxycarboxylic acid has. A microorganism containing the sixth gene encoding an enzyme that catalyzes the reaction of substitution with a mercapto group is treated with a pyruvate-supplying compound selected from pyruvate and sugars, and methyl mercaptan.
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(where R ₁₀₁ and R ₁₀₂ are independently of each other hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (7) below:
R ₁₀₁ C(R ₁₀₂ )(SCH ₃ )CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (7) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
The method includes a step of producing 2-amino-4-methylthiocarboxylic acid represented by

In the following description, the above-mentioned microorganism containing the first, fifth, and sixth genes is also referred to as a "2-amino-4-methylthiocarboxylic acid-producing microorganism." The 2-amino-4-methylthiocarboxylic acid-producing microorganism may be a microorganism that naturally has the first, fifth, and sixth genes, or the host may be transformed with the first, fifth, and sixth genes. Microorganisms obtained by Hereinafter, the case where the 2-amino-4-methylthiocarboxylic acid-producing microorganism is a transformant will be explained as an example.

(6.1) Transformants (6.1.1) Host As the host, the same hosts as those described in the column "(1.1.1) Host" can be used.
Next, the first, fifth and sixth genes introduced into the host will be explained.

(6.1.2) First gene As the first gene, a gene similar to the first gene described in the column "(1.1.2) First gene" can be used.

(6.1.3) Fifth gene As the fifth gene, a gene similar to the first gene described in the column "(4.1.3) Fifth gene" can be used.

(6.1.4) Sixth gene The sixth gene is an enzyme that catalyzes the reaction of substituting the phosphate group of 2-amino-4-phosphonooxycarboxylic acid with the methylmercapto group of methylmercaptan. Code. "An enzyme that catalyzes the reaction of substituting the phosphoric acid group of 2-amino-4-phosphonooxycarboxylic acid with the methyl mercapto group of methyl mercaptan" means that purified protein is , an activity of 0.1 mU/mg protein or more when measured using an enzyme reaction solution containing 2-amino-4-phosphonooxycarboxylic acid (initial substrate concentration 1mM) and methyl mercaptan (initial substrate concentration 5mM). An enzyme that has The 6th gene, for example, converts the phosphate group of 2-amino-4-phosphonooxycarboxylic acid obtained through a reductive amination reaction catalyzed by the protein encoded by the 5th gene into the methyl mercaptan. Encodes an enzyme that catalyzes the substitution reaction with mercapto groups. The "enzyme that catalyzes the reaction of substituting the phosphate group of 2-amino-4-phosphonooxycarboxylic acid with the methylmercapto group of methylmercaptan" is, for example, a transsulfuration enzyme.

Note that there is an additional reaction between the reaction catalyzed by the protein encoded by the fifth gene and the reaction catalyzed by the protein encoded by the sixth gene (see FIG. 5). Specifically, 2-amino-4-hydroxycarboxylic acid obtained by a reaction catalyzed by the protein encoded by the fifth gene is converted into 2-amino-4-hydroxycarboxylic acid held by a 2-amino-4-methylthiocarboxylic acid-producing microorganism. -There is a reaction in which hydroxycarboxylic acid is converted to 2-amino-4-phosphonooxycarboxylic acid by an enzyme (eg, homoserine kinase) that phosphorylates it. 2-Amino-4-phosphonooxycarboxylic acid is used as a substrate for the reaction catalyzed by the protein encoded by the sixth gene.

The sixth gene may be, for example, a gene encoding cystathionine γ-synthase (EC number 2.5.1.48).

Examples of cystathionine γ-synthase include CGS1 protein. Preferably, the CGS1 protein is derived from Arabidopsis thaliana. More preferably, the CGS1 protein is a CGS1 mature protein derived from Arabidopsis. The Arabidopsis-derived CGS1 mature protein is a protein in which the 2nd to 68th amino acid residues from the N-terminus are deleted from the Arabidopsis-derived wild-type CGS1 protein.

Preferably, the sixth gene is selected from the group consisting of:
(6-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 36 (i.e. CGS1 mature protein derived from Arabidopsis), in which the phosphate group of 2-amino-4-phosphonooxycarboxylic acid is replaced by methyl mercaptan. and (6-2) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 36. , a gene encoding a protein having the activity of catalyzing a reaction in which the phosphoric acid group of 2-amino-4-phosphonooxycarboxylic acid is replaced with the methyl mercapto group of methyl mercaptan.

"A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 36, in which methyl mercaptan has the phosphate group that 2-amino-4-phosphonooxycarboxylic acid has. A gene encoding a protein having the activity of catalyzing the reaction of substitution with a methyl mercapto group is defined as a gene encoding a protein having an activity of 80% or more, preferably 90% or more, more preferably 95% or more of the amino acid sequence set forth in SEQ ID NO: 36. , more preferably 97% or more, still more preferably 98% or more, and even more preferably 99% or more, a protein consisting of an amino acid sequence having a homology of 2-amino-4-phosphonooxycarboxylic acid. It may also be a gene encoding a protein having an activity of catalyzing the reaction of substituting a group with a methyl mercapto group of methyl mercaptan.

The UniprotKB accession number of CGS1 protein derived from Arabidopsis is P55217.

More preferably, the sixth gene is a gene synthesized so that the portion of the Arabidopsis-derived CGS1 gene consisting of the base sequence set forth in SEQ ID NO: 35 is optimally expressed in coryneform bacteria. It is.

The base sequence set forth in SEQ ID NO: 35 and the amino acid sequence set forth in SEQ ID NO: 36 are as follows.

The activity of catalyzing the reaction of substituting the phosphoric acid group of 2-amino-4-phosphonooxycarboxylic acid with the methyl mercapto group of methyl mercaptan can be determined by, for example, catalyzing the enzyme reaction with 100 mM HEPES containing 1 mM dithiothreitol. It can be calculated by performing the reaction in a KOH buffer (pH 7.5) at 25° C. and measuring the initial production rate of inorganic phosphate ions liberated during the reaction. If necessary, a small amount of pyridoxal phosphate may be added. The production rate of inorganic phosphate ions can be calculated, for example, from the time change in the concentration of inorganic phosphate ions. The concentration of inorganic phosphate ions can be measured, for example, by mixing the enzyme reaction solution and 20% (w/v) trichloroacetic acid at a ratio of 1:2, stopping the reaction, and then using the malachite green method. Here, the activity in which 1 μmol of a sulfide compound is formed per minute at 25° C. is defined as 1 unit.

"A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 36, in which methyl mercaptan has the phosphate group that 2-amino-4-phosphonooxycarboxylic acid has. A gene encoding a protein having the activity of catalyzing the reaction of substitution with a methyl mercapto group (CGS1 gene variant) was obtained from a DNA library of other species by a method similar to the above method for selecting the hpaI gene variant. You can choose.

(6.1.5) Construction of recombinant vector for transformation The first, fifth and sixth genes can be incorporated into a suitable plasmid vector for transformation.

As the plasmid vector, a plasmid vector similar to the plasmid vector described in "(1.1.3) Construction of recombinant vector for transformation" can be used.

The first, fifth and sixth genes may be incorporated into separate plasmid vectors, or may be integrated into the same plasmid vector. Transformants can be produced using the obtained recombinant vector.

(6.1.6) Transformation Any known transformation method can be used without restriction. As such a known method, the same method as described in the column "(1.1.4) Transformation" can be used.

In addition, when a transformant is produced using a recombinant vector as described above, each of the first, fifth, and sixth genes may be integrated into the chromosome of the host, or may be integrated into the host chromosome. It may exist in the cytoplasm of the host in the form of

(6.1.7) Disruption or deletion of host chromosomal genes The host may be a wild strain, a mutant strain thereof, or an artificially genetically modified strain. When the host is a coryneform bacterium, the host must be a gene-disrupted strain or a gene-deleted strain similar to the gene-disrupted strain or gene-deleted strain described in “(1.1.5) Disruption or deletion of host chromosomal genes.” Deletion strains can be used. In addition, as a method for producing gene-disrupted strains or gene-deleted strains, methods similar to those described in the column "(1.1.5) Disruption or deletion of host chromosomal genes" can be used. . By using such gene-disrupted or gene-deleted strains as hosts, it is possible to improve the productivity of 2-amino-4-methylthiocarboxylic acid and to suppress the production of by-products.

(6.1.8) Specific examples of 2-amino-4-methylthiocarboxylic acid producing microorganisms Specifically, 2-amino-4-methylthiocarboxylic acid producing microorganisms include:
The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
A fifth gene selected from the group consisting of:
(5-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, and (5-6) 1 in SEQ ID NO: 34. or a gene encoding a protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted, or added, and which has the activity of catalyzing the reductive amination reaction of α-keto acids; and a group consisting of the following: The 6th gene selected from:
(6-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 36, which catalyzes the reaction of replacing the phosphate group of 2-amino-4-phosphonooxycarboxylic acid with the methylmercapto group of methylmercaptan. (6-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 36, which is 2-amino-4-phosphonooxy A coryneform bacterium containing a gene encoding a protein having the activity of catalyzing the reaction of replacing the phosphate group of carboxylic acid with the methyl mercapto group of methyl mercaptan can be used.

(6.2) Cultivation step A 2-amino-4-methylthiocarboxylic acid-producing microorganism (for example, the above-mentioned transformant) is cultured in the presence of a pyruvic acid supplying compound, a carbonyl compound, and methyl mercaptan. Amino-4-methylthiocarboxylic acid can be produced. "Cultivating in the presence of a pyruvic acid supplying compound, a carbonyl compound, and methyl mercaptan" can be carried out by culturing in a culture solution containing a pyruvic acid supplying compound, a carbonyl compound, and methyl mercaptan. Preferably, "cultivating in the presence of a pyruvate-supplying compound, a carbonyl compound, and methyl mercaptan" can be carried out by culturing in a culture medium to which a pyruvate-supplying compound, a carbonyl compound, and methyl mercaptan are added. can.

Prior to culturing in the presence of the pyruvic acid-supplying compound, the carbonyl compound, and methyl mercaptan, it is preferable to perform the proliferation culture described in the column of "(1.2) Cultivation step".

When propagation culture is performed prior to production culture, the propagation culture and subsequent production culture can be performed, for example, as follows. After propagation culture, first, a container containing the culture solution used in the propagation culture (hereinafter referred to as the propagation culture solution) and the transformant suspended in the propagation culture solution is centrifuged to perform transformation. body can be precipitated. Thereafter, the growth culture solution is removed from the container, and the isolated transformants are suspended in the culture solution used for production culture (hereinafter referred to as production culture solution) to perform production culture.

Alternatively, the propagation culture and subsequent production culture can be carried out by adding pyruvate-providing compounds, carbonyl compounds, and methyl mercaptan to the propagation medium containing the transformants after propagation culture, changing the aerobic conditions to anaerobic conditions or By changing to microaerobic conditions, production culture can be performed without replacing the culture medium. In this way, proliferation culture and production culture can also be performed continuously.

(6.2.1) Culture solution for proliferation As the culture solution for proliferation, the same culture solution as the culture solution for proliferation described in the column "(1.2.1) Culture solution for proliferation" can be used. can.

(6.2.2) Culture solution for production As the culture solution for production, a culture solution containing a pyruvic acid supplying compound, a carbonyl compound, methyl mercaptan, and other necessary components can be used. As other necessary components, for example, inorganic salts, carbon sources other than sugars, or nitrogen sources can be used.

As the pyruvic acid supplying compound, the pyruvic acid supplying compound described in the column of "(1.2.2) Production culture solution" can be used.

As the carbonyl compound, the carbonyl compounds described in the column of "(1.2.2) Production culture solution" can be used.

In addition, when one type of carbonyl compound is used in the production culture solution, the separation and purification described in "(6.2.4) Recovery of 2-amino-4-methylthiocarboxylic acid" described later can be easily performed. It is possible.

The concentration of methyl mercaptan in the production culture solution is preferably in the range of 1mM to 1M, more preferably in the range of 10mM to 300mM. Additionally, methyl mercaptan can be additionally added in accordance with the decrease in methyl mercaptan accompanying the production of 2-amino-4-methylthiocarboxylic acid.

As other necessary components, the same components as those described in the column of "(1.2.2) Production culture solution" can be used.

Specific production culture fluids include glucose, formaldehyde, methyl mercaptan, ammonium sulfate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, iron (II) sulfate, and manganese (II) sulfate; 7. A medium with a pH of 0 can be used.

In addition, as the production culture solution, a culture solution obtained by adding a pyruvate-supplying compound, a carbonyl compound, and methyl mercaptan to a base culture solution with the same composition as the growth culture solution used in the proliferation culture can also be used. can. Note that if the pyruvic acid-supplying compound is present in a sufficient amount in the above-mentioned base culture solution, it is not necessary to add the pyruvic acid-supplying compound.

(6.2.3) Conditions for production culture The culture temperature for production culture may be any temperature as long as it is suitable for producing 2-amino-4-methylthiocarboxylic acid. The culture temperature in production culture is preferably about 20 to 50°C, more preferably about 25 to 47°C. Within the above temperature range, 2-amino-4-methylthiocarboxylic acid can be produced efficiently. The pH of the production culture solution may be any pH suitable for producing 2-amino-4-methylthiocarboxylic acid. The pH of the production culture solution is preferably maintained within the range of about 6-8. The pH of the production culture solution should be adjusted using a pH buffer solution containing an inorganic or organic acid; an alkaline solution such as an aqueous potassium hydroxide solution; urea; calcium carbonate; ammonia; 4-morpholinopropanesulfonic acid, etc. Can be done. Even during production culture, the pH of the production culture solution can be adjusted as necessary. The culture time in production culture may be any time as long as it is sufficient for the transformant to produce 2-amino-4-methylthiocarboxylic acid. The culture time in production culture is preferably about 3 hours to 7 days, more preferably about 1 to 3 days.
Production culture may be carried out under aerobic conditions, anaerobic conditions or microaerobic conditions. This is because the above-mentioned transformants have a pathway for producing 2-amino-4-methylthiocarboxylic acid that works under both aerobic, anaerobic, or microaerobic conditions. It is. The route for producing 2-amino-4-methylthiocarboxylic acid will be described in the "(6.3) Actions and Effects" section below.

<Anaerobic conditions or microaerobic conditions>
Production culture is preferably carried out under anaerobic or microaerobic conditions.

When the transformant is cultured under anaerobic or microaerobic conditions, the transformant does not substantially proliferate and can produce 2-amino-4-methylthiocarboxylic acid more efficiently.

"Anaerobic conditions or microaerobic conditions" are the same as "anaerobic conditions or microaerobic conditions" described in the "(1.2.3) Production culture conditions" column. It can be a condition.

<High density conditions>
Production culture is preferably carried out with the transformant suspended in a production culture solution at high density. By culturing the transformant under such conditions, 2-amino-4-methylthiocarboxylic acid can be produced more efficiently.

"A state in which the transformant is suspended in a production culture solution at high density" means "a state in which the transformant is suspended in a production culture solution" as described in the column of "(1.2.3) Production culture conditions". It can be in a state similar to "a state in which it is suspended at a high density in".

Production culture is more preferably carried out under anaerobic or microaerobic conditions with the transformant suspended in a production culture solution at high density.

(6.2.4) Recovery of 2-amino-4-methylthiocarboxylic acid As mentioned above, when the transformant containing the first, fifth, and sixth genes is cultured in the production culture medium, In the process, 2-amino-4-methylthiocarboxylic acid is produced.

Specifically, the following general formula (1) is contained in the production culture solution:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
When using a carbonyl compound represented by the following general formula (7):
R ₁₀₁ C(R ₁₀₂ )(SCH ₃ )CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (7) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
2-amino-4-methylthiocarboxylic acid represented by can be produced.

More specifically, when using a carbonyl compound in which R ₁₀₁ is hydrogen and R ₁₀₂ is hydrogen in the above general formula (1), when R ₁₀₁ is hydrogen in the above general formula (7), , and R ₁₀₂ is hydrogen (ie, 2-amino-4-methylthiobutyric acid).
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a methyl group and R ₁₀₂ is hydrogen, in the above general formula (7), R ₁₀₁ is a methyl group, and R 2-amino-4-methylthiocarboxylic acid in which ₁₀₂ is hydrogen (ie, 2-amino-4-methylthiovaleric acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is an ethyl group and R ₁₀₂ is hydrogen, in the above general formula (7), R ₁₀₁ is an ethyl group, and R 2-amino-4-methylthiocarboxylic acid (ie, 2-amino-4-methylthiocaproic acid) in which ₁₀₂ is hydrogen can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a propyl group and R ₁₀₂ is hydrogen, in the above general formula (7), R ₁₀₁ is a propyl group, and R 2-amino-4-methylthiocarboxylic acid (ie, 2-amino-4-methylthioenantoic acid) in which ₁₀₂ is hydrogen can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal propyl group and R ₁₀₂ is hydrogen in the above general formula (1), in the above general formula (7), R ₁₀₁ is a normal propyl group. and R ₁₀₂ is hydrogen (ie, 2-amino-4-methylthionormalheptanoic acid) can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is an isopropyl group and R ₁₀₂ is hydrogen in the general formula (7), R ₁₀₁ is isopropyl in the general formula (7). and R ₁₀₂ is hydrogen (ie, 2-amino-4-methylthioisoenantoic acid) can be produced.
In the above general formula (1), when using a carbonyl compound in which R ₁₀₁ is a pentyl group and R ₁₀₂ is hydrogen, in the above general formula (7), R ₁₀₁ is a pentyl group, and R 2-amino-4-methylthiocarboxylic acid (ie, 2-amino-4-methylthiocaprylic acid) in which ₁₀₂ is hydrogen can be produced. Specifically, when using a carbonyl compound in which R ₁₀₁ is a normal pentyl group in the above general formula (1) and R ₁₀₂ is hydrogen, in the above general formula (7), R ₁₀₁ is a normal pentyl group. and R ₁₀₂ is hydrogen (ie, 2-amino-4-methylthionormalcaprylic acid) can be produced.
When a carbonyl compound in which R ₁₀₁ and R ₁₀₂ in the above general formula (1) are both methyl groups is used, in the above general formula (7), a 2-amino compound in which R ₁₀₁ and R ₁₀₂ are both methyl groups is used. -4-methylthiocarboxylic acid (ie, 4-methyl-2-amino-4-methylthiovaleric acid) can be produced.
Note that 2-amino-4-methylthiobutyric acid is also called methionine. Methionine is, for example, L-methionine.

Although the above-mentioned 2-amino-4-methylthiocarboxylic acid can be recovered by collecting the production culture solution, 2-amino-4-methylthiocarboxylic acid can be further separated and purified from the production culture solution by a known method. You can also do it. Such known methods include a crystallization method, a chromatography method, an ion exchange resin method, an activated carbon adsorption/elution method, a solvent extraction method, and the like.

Note that substances such as α-keto acids produced by the aldol reaction catalyzed by the enzyme encoded by the first gene can also be recovered from the production culture solution.

Furthermore, when multiple types of carbonyl compounds are used, multiple types of 2-amino-4-methylthiocarboxylic acids are produced in the culture solution. Multiple types of 2-amino-4-methylthiocarboxylic acids can be separated from each other by crystallization or chromatography.

(6.3) Actions and Effects The process by which 2-amino-4-methylthiocarboxylic acid-producing microorganisms produce 2-amino-4-methylthiocarboxylic acid is schematically shown in FIG. In the process shown in FIG. 5, an example will be explained in which the pyruvic acid supplying compound is glucose, the carbonyl compound is formaldehyde, and the 2-amino-4-methylthiocarboxylic acid is methionine.

As shown in FIG. 5, first, the 2-amino-4-methylthiocarboxylic acid producing microorganism 15 takes in glucose 2, formaldehyde 3 and methyl mercaptan 16. Glucose 2 is then converted to pyruvate 4 through glycolysis. Next, pyruvate 4 and formaldehyde 3 are converted to 4-hydroxy-2-oxobutyric acid 5 by the "enzyme that catalyzes the aldol reaction between pyruvate and aldehyde" encoded by the first gene. Next, 4-hydroxy-2-oxobutyric acid 5 is converted to homoserine 14 by the "enzyme that catalyzes the reductive amination reaction of α-keto acids" encoded by the fifth gene. Next, homoserine 14 is converted to O-phosphohomoserine 17 by homoserine kinase, which is naturally present in the 2-amino-4-methylthiocarboxylic acid producing microorganism. Methyl mercaptan 16 and O-phosphohomoserine 17 are converted to methionine 18 by the "enzyme that converts O-phosphohomoserine to methionine" encoded by the sixth gene. Methionine is produced in the above manner.

(7.) Preferred Embodiments Preferred embodiments of the present invention are summarized below.
[A1] A microorganism containing a gene encoding an enzyme that catalyzes an aldol reaction between pyruvic acid and a carbonyl compound, and a pyruvic acid-supplying compound selected from pyruvic acid and saccharides;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
A method for producing an α-keto acid, the method comprising the step of producing an α-keto acid represented by:
[A2] The method according to [A1], wherein in the general formulas (1) and (2), R ₁₀₂ is hydrogen.
[A3] In the general formulas (1) and (2), R ₁₀₁ is hydrogen, methyl group, ethyl group, propyl group or pentyl group, and R ₁₀₂ is hydrogen [A1] or [A2] The method described in.
[A4] In the general formulas (1) and (2), R ₁₀₁ is hydrogen, a methyl group, an ethyl group, a normal propyl group, an isopropyl group, or a normal pentyl group, and R ₁₀₂ is hydrogen [A1 ] to [A3].
[A5] The method according to any one of [A1] to [A4], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[A6] The method according to [A5], wherein the culture solution is a culture solution to which the pyruvic acid supplying compound and carbonyl compound are added.
[A7] The pyruvate-supplying compound is added to the culture medium at a concentration of 0.1 to 40 (w/v)%, preferably 1 to 20 (w/v)% [A6 The method described in ].
[A8] The carbonyl compound is added to the culture solution at a concentration of 0.001 to 10 (w/v)%, preferably 0.01 to 1 (w/v)% [A6] Or the method described in [A7].
[A9] The method according to any one of [A1] to [A8], wherein the pyruvic acid supplying compound is a saccharide.
[A10] The saccharide may be a monosaccharide, such as fructose or glucose; a disaccharide, such as sucrose; a polysaccharide, such as a polysaccharide containing glucose as a constituent monosaccharide; The method according to any one of [A1] to [A9], which is a saccharified liquid obtained by processing.
[A11] The method according to any one of [A1] to [A10], wherein the saccharide is glucose.
[A12] The method according to any one of [A1] to [A8], wherein the pyruvic acid supplying compound is pyruvic acid.
[A13] The method according to any one of [A1] to [A12], wherein the culturing is performed under anaerobic conditions or microaerobic conditions.
[A14] The method according to [A13], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[A15] The anaerobic condition or the microaerobic condition is such that the dissolved oxygen concentration in the culture solution is within the range of 0 to 2 ppm, preferably within the range of 0 to 1 ppm, and more preferably 0 to 1 ppm. The method according to [A14], wherein the condition is within the range of 0.5 ppm.
[A16] The method according to any one of [A1] to [A15], wherein the culturing is performed with the microorganism suspended in a culture solution at high density.
[A17] The method according to [A16], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[A18] The state in which the microorganism is suspended in the culture solution at a high density is the state in which the microorganism is suspended in the culture solution in such a manner that the wet bacterial weight% of the microorganism is 1 to 50 (w/v)%. Preferably, the microorganism is suspended in the culture medium so that the wet weight percentage of the microorganism is within the range of 3 to 30 (w/v)%. The method according to [A17], which is in a cloudy state.
[A19] The method according to any one of [A1] to [A18], wherein the gene encodes aldolase.
[A20] The method according to any one of [A1] to [A19], wherein the gene encodes type I aldolase.
[A21] The method according to any one of [A1] to [A19], wherein the gene encodes type II aldolase.
[A22] The gene encodes an aldolase selected from the group consisting of:
(a1) 2-dehydro-3-deoxy-phosphogluconate aldolase (2-dehydro-3-deoxy-phosphogluconate aldolase),
(a2) 2-dehydro-3-deoxy-6-phosphogluconate aldolase (2-dehydro-3-deoxy-6-phosphogluconate aldolase),
(a3) 4-hydroxy-2-oxoglutarate aldolase,
(a4) (4S)-4-hydroxyl-2-oxoglutarate aldolase (4S)-4-hydroxyl-2-oxoglutarate aldolase,
(a5) 2-dehydro-3-deoxy-D-pentonate aldolase (2-dehydro-3-deoxy-D-pentonate aldolase),
(a6) 2-dehydro-3-deoxy-D-gluconate aldolase,
(a7) 3-deoxy-D-manno-octulosonate aldolase (3-deoxy-D-manno-octulosonate aldolase),
(a8) 4-hydroxyl-2-oxovalerate aldolase (4-hydroxyl-2-oxovalerate aldolase),
(a9) 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase),
(a10) 4-hydroxy-2-oxoheptanedioate aldolase (4-hydroxy-2-oxoheptanedioate aldolase),
(a11) 2-dehydro-3-deoxyglucarate aldolase (2-dehydro-3-deoxyglucarate aldolase),
(a12) 2-keto-3-deoxy-L-rhamnonate aldolase,
(a13) 4-hydroxyl-4-methyl-2-oxoglutarate aldolase, and (a14) 4-hydroxyl-2-oxohexanonate aldolase (4-hydroxyl-4-methyl-2-oxoglutarate aldolase). hydroxy-2-oxohexanonate aldolase);
The method according to any one of [A1] to [A21].
[A23] The gene includes eda protein, preferably E. coli-derived eda protein; dgoA protein, preferably E. coli-derived dgoA protein; yjhH protein, preferably E. coli-derived yjhH protein; yagE protein, preferably E. coli-derived dgoA protein, preferably E. coli-derived dgoA protein; yagE protein derived from E. coli; nanA protein, preferably nanA protein derived from E. coli; mhpE protein, preferably mhpE protein derived from E. coli; hpaI protein, preferably hpaI protein derived from E. coli; bphI protein, preferably Burkholderia bphI protein derived from Xenovorans; phdJ protein, preferably phdJ protein derived from Nocardioides; garL protein, preferably garL protein derived from Escherichia coli; rhmA protein, preferably rhmA protein derived from Escherichia coli; and galC protein, Preferably, the method according to any one of [A1] to [A22], which encodes an aldolase selected from the group consisting of galC protein derived from Pseudomonas putida.
[A24] The gene is selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6,
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-7) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10,
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12,
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-22) 1 or 1 in SEQ ID NO: 22. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
The method according to any one of [A1] to [A23].
[A25] The gene is selected from the group consisting of:
A gene consisting of the base sequence set forth in SEQ ID NO: 1,
A gene consisting of the base sequence set forth in SEQ ID NO: 3,
A gene consisting of the base sequence set forth in SEQ ID NO: 5,
A gene consisting of the base sequence set forth in SEQ ID NO: 7,
A gene consisting of the base sequence set forth in SEQ ID NO: 9,
A gene consisting of the base sequence set forth in SEQ ID NO: 11,
A gene consisting of the base sequence set forth in SEQ ID NO: 13,
A gene consisting of the base sequence set forth in SEQ ID NO: 15,
A gene consisting of the base sequence set forth in SEQ ID NO: 17,
A gene consisting of the base sequence set forth in SEQ ID NO: 19, and a gene consisting of the base sequence set forth in SEQ ID NO: 21;
The method according to any one of [A1] to [A24].
[A26] The microorganism is an aerobic bacterium, preferably a coryneform bacterium, more preferably a Corynebacterium genus, more preferably a Corynebacterium glutamicum [A1] to [ A25].
[A27] The method according to any one of [A1] to [A26], wherein the microorganism is a microorganism transformed by the gene.
[A28] The microorganism has a lactate dehydrogenase gene, a phosphoenolpyrvate carboxylase gene, a pyruvate carboxylase gene, a pyruvate dehydrogenase gene, and a glutamate dehydrogenase gene. pyruvate aminotransferase gene, acetolactate synthase gene, N-succinyldiaminopimelate aminotransferase gene, S-(hydroxymethyl)mycothiol dehydrogenase (S-(hydroxymethyl)mycothiol dehydrogenase ) mycothiol dehydrogenase gene, and the acetaldehyde dehydrogenase (acetaldehyde dehydrogenase) gene.
[A29] The method according to any one of [A1] to [A28], further comprising culturing the microorganism under aerobic conditions before the culturing.
[A30] The method according to any one of [A1] to [A29], further comprising recovering the produced α-keto acid.

[B1] The following genes:
The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(b) a second gene encoding an enzyme that catalyzes the decarboxylation reaction of α-keto acids; and (c) a third gene encoding an enzyme that catalyzes the reduction reaction of aldehydes. a selected pyruvate-providing compound;
General formula (3) below:
R ₁₀₃ C(O)R ₁₀₄
(Here, R ₁₀₃ and R ₁₀₄ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms. However, when R ₁₀₃ is hydrogen, R ₁₀₄ is not hydrogen.)
by culturing in the presence of a carbonyl compound represented by
General formula (4) below:
R ₁₀₃ C(R ₁₀₄ )(OH)CH ₂ CH ₂ OH
(R ₁₀₃ and R ₁₀₄ in the above general formula (4) are the same groups as R ₁₀₃ and R ₁₀₄ in the above general formula (3), respectively)
A method for producing 1,3-diol, comprising the step of producing 1,3-diol represented by
[B2] The method according to [B1], wherein R ₁₀₄ in the general formulas (3) and (4) is hydrogen.
[B3] In the general formulas (3) and (4), R ₁₀₃ is hydrogen, methyl group, ethyl group, propyl group or pentyl group, and R ₁₀₄ is hydrogen [B1] or [B2] The method described in.
[B4] In the general formulas (3) and (4), R ₁₀₃ is a methyl group, ethyl group, normal propyl group, isopropyl group, or normal pentyl group, and R ₁₀₄ is hydrogen [B1] ~ The method according to any one of [B3].
[B5] The method according to any one of [B1] to [B4], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[B6] The method according to [B5], wherein the culture solution is a culture solution to which the pyruvic acid supplying compound and carbonyl compound are added.
[B7] The pyruvate-supplying compound is added to the culture solution at a concentration of 0.1 to 40 (w/v)%, preferably 1 to 20 (w/v)% [B6 The method described in ].
[B8] The carbonyl compound is added to the culture solution at a concentration of 0.001 to 10 (w/v)%, preferably 0.01 to 1 (w/v)% [B6] Or the method described in [B7].
[B9] The method according to any one of [B1] to [B8], wherein the pyruvic acid supplying compound is a saccharide.
[B10] The saccharide may be a monosaccharide, such as fructose or glucose; a disaccharide, such as sucrose; a polysaccharide, such as a polysaccharide containing glucose as a constituent monosaccharide; The method according to any one of [B1] to [B9], which is a saccharified liquid obtained by processing.
[B11] The method according to any one of [B1] to [B10], wherein the saccharide is glucose.
[B12] The method according to any one of [B1] to [B8], wherein the pyruvic acid supplying compound is pyruvic acid.
[B13] The method according to any one of [B1] to [B12], wherein the culturing is performed under anaerobic conditions or microaerobic conditions.
[B14] The method according to [B13], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[B15] The anaerobic condition or the microaerobic condition is such that the dissolved oxygen concentration in the culture solution is within the range of 0 to 2 ppm, preferably within the range of 0 to 1 ppm, and more preferably 0 to 1 ppm. The method according to [B14], wherein the conditions are within the range of 0.5 ppm.
[B16] The method according to any one of [B1] to [B15], wherein the culturing is performed with the microorganism suspended in a culture solution at high density.
[B17] The method according to [B16], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[B18] The state in which the microorganism is suspended in the culture solution at a high density is the state in which the microorganism is suspended in the culture solution so that the wet weight percentage of the microorganism is 1 to 50 (w/v)%. Preferably, the microorganism is suspended in the culture medium so that the wet weight percentage of the microorganism is within the range of 3 to 30 (w/v)%. The method according to [B17], which is in a cloudy state.
[B19] The method according to any one of [B1] to [B18], further comprising recovering the produced 1,3-diol.
[B20] The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(b) a second gene encoding an enzyme that catalyzes the decarboxylation reaction of α-keto acids; and (c) a third gene encoding an enzyme that catalyzes the reduction reaction of aldehydes. A method for producing 1,3-butanediol, comprising the step of culturing in the presence of a selected pyruvate-supplying compound to produce 1,3-butanediol.
[B21] The method according to [B20], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound.
[B22] The method according to [B21], wherein the culture solution is a culture solution to which the pyruvate-supplying compound is added.
[B23] The pyruvate-supplying compound is added to the culture solution at a concentration of 0.1 to 40 (w/v)%, preferably 1 to 20 (w/v)% [B22 The method described in ].
[B24] The method according to any one of [B20] to [B23], wherein the pyruvic acid supplying compound is a saccharide.
[B25] The saccharide may be a monosaccharide, such as fructose or glucose; a disaccharide, such as sucrose; a polysaccharide, such as a polysaccharide containing glucose as a constituent monosaccharide; The method according to any one of [B20] to [B24], which is a saccharified liquid obtained by processing.
[B26] The method according to any one of [B20] to [B25], wherein the saccharide is glucose.
[B27] The method according to any one of [B20] to [B26], wherein the culturing is performed under anaerobic conditions or microaerobic conditions.
[B28] The method according to [B27], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound.
[B29] The anaerobic condition or the microaerobic condition is such that the dissolved oxygen concentration in the culture solution is within the range of 0 to 2 ppm, preferably 0 to 1 ppm, and more preferably 0 to 1 ppm. The method according to [B28], wherein the conditions are within the range of 0.5 ppm.
[B30] The method according to any one of [B20] to [B29], wherein the culturing is performed with the microorganism suspended in a culture solution at high density.
[B31] The method according to [B30], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound.
[B32] The state in which the microorganism is suspended in the culture solution at a high density is the state in which the microorganism is suspended in the culture solution so that the wet weight percentage of the microorganism is 1 to 50 (w/v)%. Preferably, the microorganism is suspended in the culture medium so that the wet weight percentage of the microorganism is within the range of 3 to 30 (w/v)%. The method according to [B31], which is in a cloudy state.
[B33] The method according to any one of [B20] to [B32], further comprising recovering the produced 1,3-butanediol.
[B34] The method according to any one of [B1] to [B33], wherein the first gene encodes aldolase.
[B35] The method according to any one of [B1] to [B34], wherein the first gene encodes type I aldolase.
[B36] The method according to any one of [B1] to [B34], wherein the first gene encodes type II aldolase.
[B37] The first gene encodes an aldolase selected from the group consisting of:
(a1) 2-dehydro-3-deoxy-phosphogluconate aldolase (2-dehydro-3-deoxy-phosphogluconate aldolase),
(a2) 2-dehydro-3-deoxy-6-phosphogluconate aldolase (2-dehydro-3-deoxy-6-phosphogluconate aldolase),
(a3) 4-hydroxy-2-oxoglutarate aldolase,
(a4) (4S)-4-hydroxyl-2-oxoglutarate aldolase (4S)-4-hydroxyl-2-oxoglutarate aldolase,
(a5) 2-dehydro-3-deoxy-D-pentonate aldolase (2-dehydro-3-deoxy-D-pentonate aldolase),
(a6) 2-dehydro-3-deoxy-D-gluconate aldolase,
(a7) 3-deoxy-D-manno-octulosonate aldolase (3-deoxy-D-manno-octulosonate aldolase),
(a8) 4-hydroxyl-2-oxovalerate aldolase (4-hydroxyl-2-oxovalerate aldolase),
(a9) 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase),
(a10) 4-hydroxy-2-oxoheptanedioate aldolase (4-hydroxy-2-oxoheptanedioate aldolase),
(a11) 2-dehydro-3-deoxyglucarate aldolase (2-dehydro-3-deoxyglucarate aldolase),
(a12) 2-keto-3-deoxy-L-rhamnonate aldolase,
(a13) 4-hydroxyl-4-methyl-2-oxoglutarate aldolase, and (a14) 4-hydroxyl-2-oxohexanonate aldolase (4-hydroxyl-4-methyl-2-oxoglutarate aldolase). hydroxy-2-oxohexanonate aldolase);
The method according to any one of [B1] to [B36].
[B38] The first gene is an eda protein, preferably an E. coli-derived eda protein; a dgoA protein, preferably an E. coli-derived dgoA protein; a yjhH protein, preferably an E. coli-derived yjhH protein; a yagE protein, preferably , yagE protein from E. coli; nanA protein, preferably nanA protein from E. coli; mhpE protein, preferably mhpE protein from E. coli; hpaI protein, preferably hpaI protein from E. coli; bphI protein, preferably Burke bphI protein from Holderia xenovorans; phdJ protein, preferably phdJ protein from Nocardioides; garL protein, preferably garL protein from E. coli; rhmA protein, preferably rhmA protein from E. coli; and galC The method according to any one of [B1] to [B37], which encodes an aldolase selected from the group consisting of a protein, preferably galC protein derived from Pseudomonas putida.
[B39] The first gene is selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6,
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-7) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10,
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12,
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-22) 1 or 1 in SEQ ID NO: 22. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
The method according to any one of [B1] to [B38].
[B40] The first gene is selected from the group consisting of:
A gene consisting of the base sequence set forth in SEQ ID NO: 1,
A gene consisting of the base sequence set forth in SEQ ID NO: 3,
A gene consisting of the base sequence set forth in SEQ ID NO: 5,
A gene consisting of the base sequence set forth in SEQ ID NO: 7,
A gene consisting of the base sequence set forth in SEQ ID NO: 9,
A gene consisting of the base sequence set forth in SEQ ID NO: 11,
A gene consisting of the base sequence set forth in SEQ ID NO: 13,
A gene consisting of the base sequence set forth in SEQ ID NO: 15,
A gene consisting of the base sequence set forth in SEQ ID NO: 17,
A gene consisting of the base sequence set forth in SEQ ID NO: 19, and a gene consisting of the base sequence set forth in SEQ ID NO: 21;
The method according to any one of [B1] to [B39].
[B41] The method according to any one of [B1] to [B40], wherein the second gene encodes the enzyme that catalyzes the decarboxylation reaction of the α-keto acid obtained by the aldol reaction.
[B42] The method according to any one of [B1] to [B41], wherein the second gene encodes a decarboxylase.
[B43] The second gene encodes a decarboxylase selected from the group consisting of:
(b1) pyruvate decarboxylase,
(b2) benzoylformate decarboxylase, and (b3) indolepyruvate decarboxylase;
The method according to any one of [B1] to [B42].
[B44] The second gene is a pdc protein, preferably a pdc protein derived from Zymomonas mobilis; an mdlC protein, preferably an mdlC protein derived from Pseudomonas putida; a kivD protein, preferably a kivD protein derived from Lactococcus lactis; The method according to any one of [B1] to [B43], which encodes a decarboxylase selected from the group consisting of:
[B45] The method according to any one of [B1] to [B44], wherein the second gene encodes the mdlC protein, preferably the mdlC protein derived from Pseudomonas putida.
[B46] The second gene is selected from the group consisting of:
(2-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 24, which catalyzes the decarboxylation reaction of α-keto acids; and (2-2) 1 or several genes in SEQ ID NO: 24. A gene encoding a protein consisting of an amino acid sequence with deletion, substitution, or addition of amino acids, which has the activity of catalyzing the decarboxylation reaction of α-keto acids;
The method according to any one of [B1] to [B45].
[B47] The method according to any one of [B1] to [B46], wherein the second gene is a gene consisting of the base sequence set forth in SEQ ID NO: 23.
[B48] The third gene encodes the enzyme that catalyzes the reduction reaction of an aldehyde that is different in type from the aldehyde that is the substrate of the aldol reaction catalyzed by the enzyme encoded by the first gene [B1] The method according to any one of [B47].
[B49] The method according to any one of [B1] to [B48], wherein the third gene encodes the enzyme that catalyzes the reduction reaction of the aldehyde obtained by the decarboxylation reaction.
[B50] The method according to any one of [B1] to [B49], wherein the third gene encodes alcohol dehydrogenase.
[B51] The method according to any one of [B1] to [B50], wherein the third gene encodes 1,3-propanediol dehydrogenase.
[B52] The third gene is a 1,3-propanediol selected from the group consisting of dhaT protein, preferably dhaT protein derived from Klebsiella pneumoniae; and lpo protein, preferably lpo protein derived from Lactobacillus reuteri. The method according to any one of [B1] to [B51], which encodes a dehydrogenase.
[B53] The third gene is selected from the group consisting of:
(3-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 26, which catalyzes the reduction reaction of aldehyde, and (3-2) A gene in which one or several amino acids in SEQ ID NO: 26 A gene encoding a protein consisting of a deleted, substituted, or added amino acid sequence and having an activity of catalyzing an aldehyde reduction reaction;
The method according to any one of [B1] to [B52].
[B54] The method according to any one of [B1] to [B53], wherein the third gene is a gene consisting of the base sequence set forth in SEQ ID NO: 25.
[B55] The microorganism is an aerobic bacterium, preferably a coryneform bacterium, more preferably a Corynebacterium genus, more preferably a Corynebacterium glutamicum [B1] to [ B54].
[B56] The method according to any one of [B1] to [B55], wherein the microorganism is a microorganism transformed by the first, second, and third genes.
[B57] The microorganism has a lactate dehydrogenase gene, a phosphoenolpyrvate carboxylase gene, a pyruvate carboxylase gene, a pyruvate dehydrogenase gene, and a glutamate dehydrogenase gene. pyruvate aminotransferase gene, acetolactate synthase gene, N-succinyldiaminopimelate aminotransferase gene, S-(hydroxymethyl)mycothiol dehydrogenase (S-(hydroxymethyl)mycothiol dehydrogenase ) mycothiol dehydrogenase) gene, and the acetaldehyde dehydrogenase (acetaldehyde dehydrogenase) gene.
[B58] The method according to any one of [B1] to [B57], further comprising culturing the microorganism under aerobic conditions before the culturing.

[C1] The following genes:
(a) A microorganism containing a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (d) a fourth gene encoding an enzyme that catalyzes a reduction reaction of an α-keto acid. a pyruvate-providing compound selected from acids and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (5) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(OH)COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (5) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
A method for producing 2,4-dihydroxycarboxylic acid, comprising the step of producing 2,4-dihydroxycarboxylic acid represented by
[C2] The method according to [C1], wherein in the general formulas (1) and (5), R ₁₀₂ is hydrogen.
[C3] In the general formulas (1) and (5), R ₁₀₁ is hydrogen, methyl group, ethyl group, propyl group or pentyl group, and R ₁₀₂ is hydrogen [C1] or [C2] The method described in any one of .
[C4] The method according to any one of [C1] to [C3], wherein R ₁₀₁ and R ₁₀₂ in the general formulas (1) and (5) are hydrogen.
[C5] The method according to any one of [C1] to [C4], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[C6] The method according to [C5], wherein the culture solution is a culture solution to which the pyruvic acid supplying compound and carbonyl compound are added.
[C7] The pyruvate-supplying compound is added to the culture solution at a concentration of 0.1 to 40 (w/v)%, preferably 1 to 20 (w/v)% [C6 The method described in ].
[C8] The carbonyl compound is added to the culture solution at a concentration of 0.001 to 10 (w/v)%, preferably 0.01 to 1 (w/v)% [C6] Or the method described in [C7].
[C9] The method according to any one of [C1] to [C8], wherein the pyruvate-supplying compound is a saccharide.
[C10] The saccharide may be a monosaccharide such as fructose or glucose; a disaccharide such as sucrose; a polysaccharide such as a polysaccharide containing glucose as a constituent monosaccharide; The method according to any one of [C1] to [C9], which is a saccharified liquid obtained by processing.
[C11] The method according to any one of [C1] to [C10], wherein the saccharide is glucose.
[C12] The method according to any one of [C1] to [C11], wherein the pyruvic acid supplying compound is pyruvic acid.
[C13] The method according to any one of [C1] to [C12], wherein the culturing is performed under anaerobic conditions or microaerobic conditions.
[C14] The method according to [C13], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[C15] The anaerobic condition or the microaerobic condition is such that the dissolved oxygen concentration in the culture solution is within the range of 0 to 2 ppm, preferably within the range of 0 to 1 ppm, and more preferably 0 to 1 ppm. The method according to [C14], wherein the conditions are within the range of 0.5 ppm.
[C16] The method according to any one of [C1] to [C15], wherein the culturing is performed with the microorganism suspended in a culture solution at high density.
[C17] The method according to [C16], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[C18] The state in which the microorganism is suspended in the culture solution at a high density is the state in which the microorganism is suspended in the culture solution so that the wet weight percentage of the microorganism is 1 to 50 (w/v)%. Preferably, the microorganism is suspended in the culture medium so that the wet weight percentage of the microorganism is within the range of 3 to 30 (w/v)%. The method according to [C17], which is in a cloudy state.
[C19] The method according to any one of [C1] to [C18], wherein the first gene encodes aldolase.
[C20] The method according to any one of [C1] to [C19], wherein the first gene encodes type I aldolase.
[C21] The method according to any one of [C1] to [C29], wherein the first gene encodes type II aldolase.
[C22] The first gene encodes an aldolase selected from the group consisting of:
(a1) 2-dehydro-3-deoxy-phosphogluconate aldolase (2-dehydro-3-deoxy-phosphogluconate aldolase),
(a2) 2-dehydro-3-deoxy-6-phosphogluconate aldolase (2-dehydro-3-deoxy-6-phosphogluconate aldolase),
(a3) 4-hydroxy-2-oxoglutarate aldolase,
(a4) (4S)-4-hydroxyl-2-oxoglutarate aldolase (4S)-4-hydroxyl-2-oxoglutarate aldolase,
(a5) 2-dehydro-3-deoxy-D-pentonate aldolase (2-dehydro-3-deoxy-D-pentonate aldolase),
(a6) 2-dehydro-3-deoxy-D-gluconate aldolase,
(a7) 3-deoxy-D-manno-octulosonate aldolase (3-deoxy-D-manno-octulosonate aldolase),
(a8) 4-hydroxyl-2-oxovalerate aldolase (4-hydroxyl-2-oxovalerate aldolase),
(a9) 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase),
(a10) 4-hydroxy-2-oxoheptanedioate aldolase (4-hydroxy-2-oxoheptanedioate aldolase),
(a11) 2-dehydro-3-deoxyglucarate aldolase (2-dehydro-3-deoxyglucarate aldolase),
(a12) 2-keto-3-deoxy-L-rhamnonate aldolase,
(a13) 4-hydroxyl-4-methyl-2-oxoglutarate aldolase, and (a14) 4-hydroxyl-2-oxohexanonate aldolase (4-hydroxyl-4-methyl-2-oxoglutarate aldolase). hydroxy-2-oxohexanonate aldolase);
The method according to any one of [C1] to [C21].
[C23] The first gene is an eda protein, preferably an E. coli-derived eda protein; a dgoA protein, preferably an E. coli-derived dgoA protein; a yjhH protein, preferably an E. coli-derived yjhH protein; a yagE protein, preferably , yagE protein from E. coli; nanA protein, preferably nanA protein from E. coli; mhpE protein, preferably mhpE protein from E. coli; hpaI protein, preferably hpaI protein from E. coli; bphI protein, preferably Burke bphI protein from Holderia xenovorans; phdJ protein, preferably phdJ protein from Nocardioides; garL protein, preferably garL protein from E. coli; rhmA protein, preferably rhmA protein from E. coli; and galC The method according to any one of [C1] to [C22], which encodes an aldolase selected from the group consisting of a protein, preferably galC protein derived from Pseudomonas putida.
[C24] The first gene is selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6,
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-7) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10,
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12,
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-22) 1 or 1 in SEQ ID NO: 22. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
The method according to any one of [C1] to [C23].
[C25] The first gene is selected from the group consisting of:
A gene consisting of the base sequence set forth in SEQ ID NO: 1,
A gene consisting of the base sequence set forth in SEQ ID NO: 3,
A gene consisting of the base sequence set forth in SEQ ID NO: 5,
A gene consisting of the base sequence set forth in SEQ ID NO: 7,
A gene consisting of the base sequence set forth in SEQ ID NO: 9,
A gene consisting of the base sequence set forth in SEQ ID NO: 11,
A gene consisting of the base sequence set forth in SEQ ID NO: 13,
A gene consisting of the base sequence set forth in SEQ ID NO: 15,
A gene consisting of the base sequence set forth in SEQ ID NO: 17,
A gene consisting of the base sequence set forth in SEQ ID NO: 19, and a gene consisting of the base sequence set forth in SEQ ID NO: 21;
The method according to any one of [C1] to [C24].
[C26] The fourth gene encodes the enzyme according to any one of [C1] to [C23], which encodes the enzyme that catalyzes the reduction reaction of α-keto acid obtained by the aldol reaction. Method.
[C27] The method according to any one of [C1] to [C26], wherein the fourth gene encodes dehydrogenase.
[C28] The method according to any one of [C1] to [C27], wherein the fourth gene encodes lactate dehydrogenase.
[C29] The fourth gene is ldhA protein, preferably ldhA protein derived from Lactococcus lactis, more preferably ldhA protein derived from Lactococcus lactis NBRC100676 strain, and even more preferably ldhA protein derived from Lactococcus lactis NBRC100676 strain. The method according to any one of [C1] to [C28], which encodes a protein in which the 85th glutamine residue from the N-terminus is replaced with a cysteine residue.
[C30] The fourth gene is selected from the group consisting of:
(4-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 28, which catalyzes the reduction reaction of α-keto acid; and (4-2) one or several genes in SEQ ID NO: 28. A gene encoding a protein that is a protein consisting of an amino acid sequence in which an amino acid is deleted, substituted, or added, and has the activity of catalyzing the reduction reaction of α-keto acid;
The method according to any one of [C1] to [C29].
[C31] The protein consists of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 28, and encodes the protein that has the activity of catalyzing the reduction reaction of α-keto acid. The method according to any one of [C1] to [C30], wherein the gene encodes a protein that maintains a cysteine residue at the 85th residue from the N-terminus.
[C32] The method according to any one of [C1] to [C31], wherein the fourth gene is a gene consisting of the base sequence set forth in SEQ ID NO: 27.
[C33] The microorganism is an aerobic bacterium, preferably a coryneform bacterium, more preferably a Corynebacterium genus, more preferably a Corynebacterium glutamicum [C1] to [ C32].
[C34] The method according to any one of [C1] to [C33], wherein the microorganism is a microorganism transformed by the first and fourth genes.
[C35] The microorganism has a lactate dehydrogenase gene, a phosphoenolpyrvate carboxylase gene, a pyruvate carboxylase gene, a pyruvate dehydrogenase gene, and a glutamate dehydrogenase gene. pyruvate aminotransferase gene, acetolactate synthase gene, N-succinyldiaminopimelate aminotransferase gene, S-(hydroxymethyl)mycothiol dehydrogenase (S-(hydroxymethyl)mycothiol dehydrogenase ) mycothiol dehydrogenase) gene, and the acetaldehyde dehydrogenase (acetaldehyde dehydrogenase) gene.
[C36] The method according to any one of [C1] to [C35], further comprising culturing the microorganism under aerobic conditions before the culturing.
[C37] The method according to any one of [C1] to [C36], further comprising recovering the produced 2,4-dihydroxycarboxylic acid.

[D1] The following genes:
(a) A microorganism comprising a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (e) a fifth gene encoding an enzyme that catalyzes a reductive amination reaction of an α-keto acid. a pyruvate-providing compound selected from pyruvate and sugars;
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
A method for producing 2-amino-4-hydroxycarboxylic acid, comprising the step of producing 2-amino-4-hydroxycarboxylic acid represented by
[D2] The method according to [D1], wherein R ₁₀₂ in the general formulas (1) and (6) is hydrogen.
[D3] In the general formulas (1) and (6), R ₁₀₁ is hydrogen, methyl group, ethyl group, propyl group or pentyl group, and R ₁₀₂ is hydrogen [D1] or [D2] The method described in.
[D4] The method according to any one of [D1] to [D3], wherein R ₁₀₁ and R ₁₀₂ in the general formulas (1) and (6) are hydrogen.
[D5] The method according to any one of [D1] to [D4], further comprising recovering the produced 2-amino-4-hydroxycarboxylic acid.
[D6] The following genes:
(a) A microorganism comprising a first gene encoding an enzyme that catalyzes an aldol reaction between pyruvate and a carbonyl compound, and (e) a fifth gene encoding an enzyme that catalyzes a reductive amination reaction of an α-keto acid. in the presence of pyruvate and a pyruvate-providing compound selected from saccharides and formaldehyde (which is a carbonyl compound) to produce at least one of homoserine, threonine and 2-aminobutyric acid. A method for producing at least one of homoserine, threonine and 2-aminobutyric acid, comprising:
[D7] The method according to [D6], further comprising recovering at least one of produced homoserine, threonine, and 2-aminobutyric acid.
[D8] The method according to any one of [D1] to [D7], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[D9] The method according to [D8], wherein the culture solution is a culture solution to which the pyruvate-supplying compound and carbonyl compound are added.
[D10] The pyruvate-supplying compound is added to the culture medium at a concentration of 0.1 to 40 (w/v)%, preferably 1 to 20 (w/v)% [D9 The method described in ].
[D11] The carbonyl compound is added to the culture solution at a concentration of 0.001 to 10 (w/v)%, preferably 0.01 to 1 (w/v)% [D9] Or the method described in [D10].
[D12] The method according to any one of [D1] to [D11], wherein the pyruvic acid supplying compound is a saccharide.
[D13] The saccharide may be a monosaccharide, such as fructose or glucose; a disaccharide, such as sucrose; a polysaccharide, such as a polysaccharide containing glucose as a constituent monosaccharide; The method according to any one of [D1] to [D12], which is a saccharified liquid obtained by processing.
[D14] The method according to any one of [D1] to [D13], wherein the saccharide is glucose.
[D15] The method according to any one of [D1] to [D14], wherein the pyruvic acid supplying compound is pyruvic acid.
[D16] The method according to any one of [D1] to [D15], wherein the culturing is performed under anaerobic conditions or microaerobic conditions.
[D17] The method according to [D16], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[D18] The anaerobic condition or the microaerobic condition is such that the dissolved oxygen concentration in the culture solution is within the range of 0 to 2 ppm, preferably within the range of 0 to 1 ppm, and more preferably 0 to 1 ppm. The method according to [D17], wherein the condition is within the range of 0.5 ppm.
[D19] The method according to any one of [D1] to [D18], wherein the culturing is performed with the microorganism suspended in a culture solution at high density.
[D20] The method according to [D19], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound and the carbonyl compound.
[D21] The state in which the microorganism is suspended in the culture solution at a high density is the state in which the microorganism is suspended in the culture solution so that the wet weight percentage of the microorganism is 1 to 50 (w/v)%. Preferably, the microorganism is suspended in the culture medium so that the wet weight percentage of the microorganism is within the range of 3 to 30 (w/v)%. The method according to [D20], which is in a cloudy state.
[D22] The method according to any one of [D1] to [D21], wherein the first gene encodes aldolase.
[D23] The method according to any one of [D1] to [D22], wherein the first gene encodes type I aldolase.
[D24] The method according to any one of [D1] to [D22], wherein the first gene encodes type II aldolase.
[D25] The first gene encodes an aldolase selected from the group consisting of:
(a1) 2-dehydro-3-deoxy-phosphogluconate aldolase (2-dehydro-3-deoxy-phosphogluconate aldolase),
(a2) 2-dehydro-3-deoxy-6-phosphogluconate aldolase (2-dehydro-3-deoxy-6-phosphogluconate aldolase),
(a3) 4-hydroxy-2-oxoglutarate aldolase,
(a4) (4S)-4-hydroxyl-2-oxoglutarate aldolase (4S)-4-hydroxyl-2-oxoglutarate aldolase,
(a5) 2-dehydro-3-deoxy-D-pentonate aldolase (2-dehydro-3-deoxy-D-pentonate aldolase),
(a6) 2-dehydro-3-deoxy-D-gluconate aldolase,
(a7) 3-deoxy-D-manno-octulosonate aldolase (3-deoxy-D-manno-octulosonate aldolase),
(a8) 4-hydroxyl-2-oxovalerate aldolase (4-hydroxyl-2-oxovalerate aldolase),
(a9) 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase),
(a10) 4-hydroxy-2-oxoheptanedioate aldolase (4-hydroxy-2-oxoheptanedioate aldolase),
(a11) 2-dehydro-3-deoxyglucarate aldolase (2-dehydro-3-deoxyglucarate aldolase),
(a12) 2-keto-3-deoxy-L-rhamnonate aldolase,
(a13) 4-hydroxyl-4-methyl-2-oxoglutarate aldolase, and (a14) 4-hydroxyl-2-oxohexanonate aldolase (4-hydroxyl-4-methyl-2-oxoglutarate aldolase). hydroxy-2-oxohexanonate aldolase);
The method according to any one of [D1] to [D24].
[D26] The first gene is an eda protein, preferably an E. coli-derived eda protein; a dgoA protein, preferably an E. coli-derived dgoA protein; a yjhH protein, preferably an E. coli-derived yjhH protein; a yagE protein, preferably , yagE protein from E. coli; nanA protein, preferably nanA protein from E. coli; mhpE protein, preferably mhpE protein from E. coli; hpaI protein, preferably hpaI protein from E. coli; bphI protein, preferably Burke bphI protein from Holderia xenovorans; phdJ protein, preferably phdJ protein from Nocardioides; garL protein, preferably garL protein from E. coli; rhmA protein, preferably rhmA protein from E. coli; and galC The method according to any one of [D1] to [D25], which encodes an aldolase selected from the group consisting of a protein, preferably galC protein derived from Pseudomonas putida.
[D27] The first gene is selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6,
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-7) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10,
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12,
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-22) 1 or 1 in SEQ ID NO: 22. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
The method according to any one of [D1] to [D26].
[D28] The first gene is selected from the group consisting of:
A gene consisting of the base sequence set forth in SEQ ID NO: 1,
A gene consisting of the base sequence set forth in SEQ ID NO: 3,
A gene consisting of the base sequence set forth in SEQ ID NO: 5,
A gene consisting of the base sequence set forth in SEQ ID NO: 7,
A gene consisting of the base sequence set forth in SEQ ID NO: 9,
A gene consisting of the base sequence set forth in SEQ ID NO: 11,
A gene consisting of the base sequence set forth in SEQ ID NO: 13,
A gene consisting of the base sequence set forth in SEQ ID NO: 15,
A gene consisting of the base sequence set forth in SEQ ID NO: 17,
A gene consisting of the base sequence set forth in SEQ ID NO: 19, and a gene consisting of the base sequence set forth in SEQ ID NO: 21;
The method according to any one of [D1] to [D27].
[D29] The method according to any one of [D1] to [D28], wherein the fifth gene encodes the enzyme that catalyzes the reductive amination reaction of the α-keto acid obtained by the aldol reaction.
[D30] The method according to any one of [D1] to [D29], wherein the fifth gene encodes glutamic acid/phenylalanine/leucine/valine dehydrogenase.
[D31] The method according to any one of [D1] to [D30], wherein the fifth gene encodes at least one dehydrogenase selected from the group consisting of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase, and valine dehydrogenase.
[D32] The fifth gene encodes a glutamic acid/phenylalanine/leucine/valine dehydrogenase selected from the group consisting of:
(d1) leucine dehydrogenase (EC number 1.4.1.9),
(d2) phenylalanine dehydrogenase (EC number 1.4.1.20), and (d3) valine dehydrogenase (EC number 1.4.1.8 and 1.4.1.23);
The method according to any one of [D1] to [D31].
[D33] The fifth gene is a leuDH protein, preferably a leuDH protein derived from Ricinibacillus sphaericus, a leuDH protein derived from Bacillus cereus, or a leuDH protein derived from Geobacillus stearothermophilus; a phedh protein, preferably derived from Bacillus badius The method according to any one of [D1] to [D32], which encodes an aldolase selected from the group consisting of: a phedh protein derived from Streptomyces frasiae;
[D34] The fifth gene is selected from the group consisting of:
(5-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, and (5-6) 1 in SEQ ID NO: 34. or a gene encoding a protein that is a protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted, or added, and that has the activity of catalyzing the reductive amination reaction of α-keto acids;
The method according to any one of [D1] to [D33].
[D35] The fifth gene is selected from the group consisting of:
A gene consisting of the nucleotide sequence set forth in SEQ ID NO: 29,
A gene consisting of the base sequence set forth in SEQ ID NO: 31, and a valdh gene consisting of the base sequence set forth in SEQ ID NO: 33;
The method according to any one of [D1] to [D34].
[D36] The microorganism is an aerobic bacterium, preferably a coryneform bacterium, more preferably a Corynebacterium genus, more preferably a Corynebacterium glutamicum [D1] to [ D35].
[D37] The method according to any one of [D1] to [D36], wherein the microorganism is a microorganism transformed by the first and fifth genes.
[D38] The microorganism has a lactate dehydrogenase gene, a phosphoenolpyrvate carboxylase gene, a pyruvate carboxylase gene, a pyruvate dehydrogenase gene, and a glutamic acid- pyruvate aminotransferase gene, acetolactate synthase gene, N-succinyldiaminopimelate aminotransferase gene, S-(hydroxymethyl)mycothiol dehydrogenase (S-(hydroxymethyl)mycothiol dehydrogenase ) mycothiol dehydrogenase) gene, and the acetaldehyde dehydrogenase (acetaldehyde dehydrogenase) gene.
[D39] The method according to any one of [D1] to [D38], further comprising culturing the microorganism under aerobic conditions before the culturing.

[E1] The following genes:
(a) a first gene encoding an enzyme that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound;
(e) the fifth gene encoding an enzyme that catalyzes the reductive amination reaction of α-keto acids, and (f) the methyl mercaptan that has the phosphate group that 2-amino-4-phosphonooxycarboxylic acid has. A microorganism containing the sixth gene encoding an enzyme that catalyzes the reaction of substitution with a mercapto group is treated with a pyruvate-supplying compound selected from pyruvate and sugars, and methyl mercaptan.
General formula (1) below:
R ₁₀₁ C(O)R ₁₀₂
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
by culturing in the presence of a carbonyl compound represented by
General formula (7) below:
R ₁₀₁ C(R ₁₀₂ )(SCH ₃ )CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (7) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (1), respectively)
A method for producing 2-amino-4-methylthiocarboxylic acid, comprising the step of producing 2-amino-4-methylthiocarboxylic acid represented by
[E2] The method according to [E1], wherein R ₁₀₂ in the general formulas (1) and (7) is hydrogen.
[E3] In the general formulas (1) and (7), R ₁₀₁ is hydrogen, methyl group, ethyl group, propyl group or pentyl group, and R ₁₀₂ is hydrogen [E1] or [E2] The method described in.
[E4] The method according to any one of [E1] to [E3], wherein R ₁₀₁ and R ₁₀₂ in the general formulas (1) and (7) are hydrogen.
[E5] The method according to any one of [E1] to [E4], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvate-supplying compound, the carbonyl compound, and methyl mercaptan. .
[E6] The method according to [E5], wherein the culture solution is a culture solution to which the pyruvic acid supplying compound, the carbonyl compound, and methyl mercaptan are added.
[E7] The pyruvate-supplying compound is added to the culture medium at a concentration of 0.1 to 40 (w/v)%, preferably 1 to 20 (w/v)% [E6 The method described in ].
[E8] The carbonyl compound is added to the culture medium at a concentration of 0.001 to 10 (w/v)%, preferably 0.01 to 1 (w/v)% [E6] Or the method described in [E7].
[E9] The method according to any one of [E6] to [E8], wherein methyl mercaptan is added to the culture medium at a concentration of 1mM to 1M, preferably 10mM to 300mM.
[E10] The method according to any one of [E1] to [E9], wherein the pyruvic acid supplying compound is a saccharide.
[E11] The saccharide may be a monosaccharide, such as fructose or glucose; a disaccharide, such as sucrose; a polysaccharide, such as a polysaccharide containing glucose as a constituent monosaccharide; The method according to any one of [E1] to [E10], which is a saccharified liquid obtained by processing.
[E12] The method according to any one of [E1] to [E11], wherein the saccharide is glucose.
[E13] The method according to any one of [E1] to [E9], wherein the pyruvic acid supplying compound is pyruvic acid.
[E14] The method according to any one of [E1] to [E13], wherein the culturing is performed under anaerobic conditions or microaerobic conditions.
[E15] The method according to [E17], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvic acid supplying compound, the carbonyl compound, and methyl mercaptan.
[E16] The anaerobic condition or the microaerobic condition is such that the dissolved oxygen concentration in the culture solution is within the range of 0 to 2 ppm, preferably 0 to 1 ppm, and more preferably 0 to 1 ppm. The method according to [E15], wherein the condition is within the range of 0.5 ppm.
[E17] The method according to any one of [E1] to [E16], wherein the culturing is performed with the microorganism suspended in a culture solution at high density.
[E18] The method according to [E17], wherein the culturing is performed by culturing the microorganism in a culture solution containing the pyruvic acid supplying compound, the carbonyl compound, and methyl mercaptan.
[E19] The state in which the microorganism is suspended in the culture solution at a high density is the state in which the microorganism is suspended in the culture solution so that the wet bacterial weight% of the microorganism is 1 to 50 (w/v)%. Preferably, the microorganism is suspended in the culture medium so that the wet weight percentage of the microorganism is within the range of 3 to 30 (w/v)%. The method according to [E18], which is in a cloudy state.
[E20] The method according to any one of [E1] to [E19], wherein the first gene encodes aldolase.
[E21] The method according to any one of [E1] to [E20], wherein the first gene encodes type I aldolase.
[E22] The method according to any one of [E1] to [E20], wherein the first gene encodes type II aldolase.
[E23] The first gene encodes an aldolase selected from the group consisting of:
(a1) 2-dehydro-3-deoxy-phosphogluconate aldolase (2-dehydro-3-deoxy-phosphogluconate aldolase),
(a2) 2-dehydro-3-deoxy-6-phosphogluconate aldolase (2-dehydro-3-deoxy-6-phosphogluconate aldolase),
(a3) 4-hydroxy-2-oxoglutarate aldolase,
(a4) (4S)-4-hydroxyl-2-oxoglutarate aldolase (4S)-4-hydroxyl-2-oxoglutarate aldolase,
(a5) 2-dehydro-3-deoxy-D-pentonate aldolase (2-dehydro-3-deoxy-D-pentonate aldolase),
(a6) 2-dehydro-3-deoxy-D-gluconate aldolase,
(a7) 3-deoxy-D-manno-octulosonate aldolase (3-deoxy-D-manno-octulosonate aldolase),
(a8) 4-hydroxyl-2-oxovalerate aldolase (4-hydroxyl-2-oxovalerate aldolase),
(a9) 4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase (4-(2-carboxyphenyl)-2-oxobut-3-enoate aldolase),
(a10) 4-hydroxy-2-oxoheptanedioate aldolase (4-hydroxy-2-oxoheptanedioate aldolase),
(a11) 2-dehydro-3-deoxyglucarate aldolase (2-dehydro-3-deoxyglucarate aldolase),
(a12) 2-keto-3-deoxy-L-rhamnonate aldolase,
(a13) 4-hydroxyl-4-methyl-2-oxoglutarate aldolase, and (a14) 4-hydroxyl-2-oxohexanonate aldolase (4-hydroxyl-4-methyl-2-oxoglutarate aldolase). hydroxy-2-oxohexanonate aldolase);
The method according to any one of [E1] to [E22].
[E24] The first gene is an eda protein, preferably an E. coli-derived eda protein; a dgoA protein, preferably an E. coli-derived dgoA protein; a yjhH protein, preferably an E. coli-derived yjhH protein; a yagE protein, preferably , yagE protein from E. coli; nanA protein, preferably nanA protein from E. coli; mhpE protein, preferably mhpE protein from E. coli; hpaI protein, preferably hpaI protein from E. coli; bphI protein, preferably Burke bphI protein from Holderia xenovorans; phdJ protein, preferably phdJ protein from Nocardioides; garL protein, preferably garL protein from E. coli; rhmA protein, preferably rhmA protein from E. coli; and galC The method according to any one of [E1] to [E23], which encodes an aldolase selected from the group consisting of a protein, preferably galC protein derived from Pseudomonas putida.
[E25] The first gene is selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6,
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-7) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10,
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12,
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-22) 1 or 1 in SEQ ID NO: 22. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
The method according to any one of [E1] to [E24].
[E26] The first gene is selected from the group consisting of:
A gene consisting of the base sequence set forth in SEQ ID NO: 1,
A gene consisting of the base sequence set forth in SEQ ID NO: 3,
A gene consisting of the base sequence set forth in SEQ ID NO: 5,
A gene consisting of the base sequence set forth in SEQ ID NO: 7,
A gene consisting of the base sequence set forth in SEQ ID NO: 9,
A gene consisting of the base sequence set forth in SEQ ID NO: 11,
A gene consisting of the base sequence set forth in SEQ ID NO: 13,
A gene consisting of the base sequence set forth in SEQ ID NO: 15,
A gene consisting of the base sequence set forth in SEQ ID NO: 17,
A gene consisting of the base sequence set forth in SEQ ID NO: 19, and a gene consisting of the base sequence set forth in SEQ ID NO: 21;
The method according to any one of [E1] to [E25].
[E27] The method according to any one of [E1] to [E26], wherein the fifth gene encodes the enzyme that catalyzes the reductive amination reaction of the α-keto acid obtained by the aldol reaction.
[E28] The method according to any one of [E1] to [E27], wherein the fifth gene encodes glutamic acid/phenylalanine/leucine/valine dehydrogenase.
[E29] The method according to any one of [E1] to [E28], wherein the fifth gene encodes at least one dehydrogenase selected from the group consisting of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase, and valine dehydrogenase.
[E30] The fifth gene encodes a glutamic acid/phenylalanine/leucine/valine dehydrogenase selected from the group consisting of:
(d1) leucine dehydrogenase (EC number 1.4.1.9),
(d2) phenylalanine dehydrogenase (EC number 1.4.1.20), and (d3) valine dehydrogenase (EC number 1.4.1.8 and 1.4.1.23);
The method according to any one of [E1] to [E29].
[E31] The fifth gene is a leuDH protein, preferably a leuDH protein derived from Ricinibacillus sphaericus, a leuDH protein derived from Bacillus cereus, or a leuDH protein derived from Geobacillus stearothermophilus; a phedh protein, preferably derived from Bacillus badius. The method according to any one of [E1] to [E30], which encodes an aldolase selected from the group consisting of: phedh protein derived from Streptomyces frasiae;
[E32] The fifth gene is selected from the group consisting of:
(5-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, and (5-6) 1 in SEQ ID NO: 34. or a gene encoding a protein that is a protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted, or added, and that has the activity of catalyzing the reductive amination reaction of α-keto acids;
The method according to any one of [E1] to [E31].
[E33] The fifth gene is selected from the group consisting of:
A gene consisting of the base sequence set forth in SEQ ID NO: 30,
A gene consisting of the base sequence set forth in SEQ ID NO: 32, and a gene consisting of the base sequence set forth in SEQ ID NO: 34;
The method according to any one of [E1] to [E32].
[E34] The method according to any one of [B1] to [B33], wherein the sixth gene encodes a transsulfuration enzyme.
[E35] The method according to any one of [E1] to [E34], wherein the sixth gene encodes cystathionine γ-synthase.
[E36] The method according to any one of [E1] to [E35], wherein the sixth gene encodes a CGS1 protein, preferably an Arabidopsis-derived CGS1 protein, more preferably an Arabidopsis-derived CGS1 mature protein.
[E37] The sixth gene is selected from the group consisting of:
(6-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 36, which catalyzes the reaction of replacing the phosphate group of 2-amino-4-phosphonooxycarboxylic acid with the methylmercapto group of methylmercaptan. (6-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 36, which is 2-amino-4-phosphonooxy A gene encoding a protein having an activity of catalyzing a reaction that replaces the phosphate group of carboxylic acid with the methyl mercapto group of methyl mercaptan;
The method according to any one of [E1] to [E36].
[E38] The method according to any one of [E1] to [E37], wherein the sixth gene is a gene consisting of the base sequence set forth in SEQ ID NO: 35.
[E39] The microorganism is an aerobic bacterium, preferably a coryneform bacterium, more preferably a Corynebacterium genus, more preferably a Corynebacterium glutamicum [E1] to [ E38].
[E40] The method according to any one of [E1] to [E39], wherein the microorganism is a microorganism transformed by the first, fifth, and sixth genes.
[E41] The microorganism has a lactate dehydrogenase gene, a phosphoenolpyrvate carboxylase gene, a pyruvate carboxylase gene, a pyruvate dehydrogenase gene, and a glutamate dehydrogenase gene. pyruvate aminotransferase gene, acetolactate synthase gene, N-succinyldiaminopimelate aminotransferase gene, S-(hydroxymethyl)mycothiol dehydrogenase (S-(hydroxymethyl)mycothiol dehydrogenase ) mycothiol dehydrogenase) gene, and the acetaldehyde dehydrogenase (acetaldehyde dehydrogenase) gene.
[E42] The method according to any one of [E1] to [E41], further comprising culturing the microorganism under aerobic conditions before the culturing.
[E43] The method according to any one of [E1] to [E42], further comprising recovering the produced 2-amino-4-methylthiocarboxylic acid.

[F1] General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
α-keto acid, nitrogen source and reducing agent represented by
React in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase,
General formula (6) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (6) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (2), respectively)
A method for producing 2-amino-4-hydroxycarboxylic acid, comprising the step of producing 2-amino-4-hydroxycarboxylic acid represented by
[F2] The method according to [F1], wherein R ₁₀₂ in the general formulas (2) and (6) is hydrogen.
[F3] The method according to [F1] or [F2], wherein R ₁₀₁ and R ₁₀₂ in the general formulas (2) and (6) are hydrogen.
[F4] The reaction is carried out in a reaction solution containing the α-keto acid represented by the general formula (2), the nitrogen source, the reducing agent, and glutamic acid/phenylalanine/leucine/valine dehydrogenase [F1 ] to [F3].
[F5] The reaction solution is a reaction solution to which the α-keto acid represented by the general formula (2), the nitrogen source, the reducing agent, and glutamic acid/phenylalanine/leucine/valine dehydrogenase are added [F4 The method described in ].
[F6] The α-keto acid represented by the general formula (2) is added to [F5] at a concentration of 0.1mM to 1M, preferably 1mM to 300mM in the reaction solution. Method described.
[F7] The method according to [F5] or [F6], wherein the nitrogen source is added to the reaction solution at a concentration of 0.1mM to 1M, preferably 1mM to 300mM.
[F8] The reducing agent according to any one of [F5] to [F7], wherein the reducing agent is added to the reaction solution at a concentration of 0.01 mM to 100 mM, preferably 0.1 mM to 10 mM. Method.
[F9] Glutamic acid/phenylalanine/leucine/valine dehydrogenase is added to the reaction solution at a concentration of 0.01 ug/mL to 1 mg/mL, preferably 0.1 to 100 ug/mL [F5] to [F8] ] The method described in any one of the above.
[F10] The method according to any one of [F1] to [F19], wherein the nitrogen source is an ammonium salt.
[F11] The method according to any one of [F1] to [F10], wherein the nitrogen source is at least one ammonium salt selected from the group consisting of ammonium chloride, ammonium sulfate, and ammonium carbonate.
[F12] The method according to any one of [F1] to [F11], wherein the reducing agent is at least one selected from the group consisting of NADH and NADPH.
[F13] Glutamate/phenylalanine/leucine/valine dehydrogenase is at least one dehydrogenase selected from the group consisting of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase, and valine dehydrogenase according to any one of [F1] to [F12]. the method of.
[F14] Glutamic acid/phenylalanine/leucine/valine dehydrogenase is selected from the group consisting of:
(d1) leucine dehydrogenase,
(d2) phenylalanine dehydrogenase, and (d3) valine dehydrogenase;
The method according to any one of [F1] to [F13].
[F15] Glutamic acid/phenylalanine/leucine/valine dehydrogenase is a leuDH protein, preferably a leuDH protein derived from Lysinibacillus sphaericus, a leuDH protein derived from Bacillus cereus, or a leuDH protein derived from Geobacillus stearothermophilus; a phedh protein, preferably, The method according to any one of [F1] to [F14], wherein the dehydrogenase is selected from the group consisting of phedh protein derived from Bacillus badius; and valdh protein, preferably valdh protein derived from Streptomyces Frazie.
[F16] Glutamic acid/phenylalanine/leucine/valine dehydrogenase is selected from the group consisting of:
(5'-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5'-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added to SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. protein,
(5'-3) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5'-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. protein,
(5'-5) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes a reductive amination reaction of α-keto acid, and (5'-6) 1 or a number in SEQ ID NO: 34. A protein consisting of an amino acid sequence in which 5 amino acids are deleted, substituted, or added, and has the activity of catalyzing the reductive amination reaction of α-keto acids;
The method according to any one of [F1] to [F15].

[G1] The following general formula (6):
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ CH(NH ₂ )COOH
(wherein R ₁₀₁ and R ₁₀₂ are independently hydrogen or an alkyl group having 1 to 5 carbon atoms)
2-amino-4-hydroxycarboxylic acid represented by and an oxidizing agent,
React in the presence of glutamic acid/phenylalanine/leucine/valine dehydrogenase,
General formula (2) below:
R ₁₀₁ C(R ₁₀₂ )(OH)CH ₂ COCOOH
(R ₁₀₁ and R ₁₀₂ in the above general formula (2) are the same groups as R ₁₀₁ and R ₁₀₂ in the above general formula (6), respectively)
A method for producing an α-keto acid, the method comprising the step of producing an α-keto acid represented by:
[G2] The method according to [G1], wherein in the general formulas (6) and (2), R ₁₀₂ is hydrogen.
[G3] The method according to [G1] or [G2], wherein R ₁₀₁ and R ₁₀₂ in the general formulas (6) and (2) are hydrogen.
[G4] The reaction is carried out in a reaction solution containing the 2-amino-4-hydroxycarboxylic acid represented by the general formula (6), the oxidizing agent, and glutamic acid/phenylalanine/leucine/valine dehydrogenase [ G1] to [G3].
[G5] The reaction solution is a reaction solution containing the 2-amino-4-hydroxycarboxylic acid represented by the general formula (6), the oxidizing agent, and glutamic acid/phenylalanine/leucine/valine dehydrogenase [ G4].
[G6] The 2-amino-4-hydroxycarboxylic acid represented by the general formula (6) is added to the reaction solution at a concentration of 0.1mM to 1M, preferably 1mM to 300mM. The method described in [G5].
[G7] The method according to [G5] or [G6], wherein the oxidizing agent is added to the reaction solution at a concentration of 0.01mM to 100mM, preferably 0.1mM to 10mM.
[G8] Glutamic acid/phenylalanine/leucine/valine dehydrogenase is added to the reaction solution at a concentration of 0.01 ug/mL to 1 mg/mL, preferably 0.1 to 100 ug/mL [G5] to [G7] ] The method described in any one of .
[G9] The method according to any one of [G1] to [G8], wherein the oxidizing agent is at least one selected from the group consisting of NAD ⁺ and NADP ⁺ .
[G10] Glutamate/phenylalanine/leucine/valine dehydrogenase is at least one dehydrogenase selected from the group consisting of glutamate dehydrogenase, phenylalanine dehydrogenase, leucine dehydrogenase, and valine dehydrogenase, according to any one of [G1] to [G9]. the method of.
[G11] Glutamic acid/phenylalanine/leucine/valine dehydrogenase is selected from the group consisting of:
(d1) leucine dehydrogenase,
(d2) phenylalanine dehydrogenase, and (d3) valine dehydrogenase;
The method according to any one of [G1] to [G10].
[G12] Glutamate/phenylalanine/leucine/valine dehydrogenase is a leuDH protein, preferably a leuDH protein derived from Lysinibacillus sphaericus, a leuDH protein derived from Bacillus cereus, or a leuDH protein derived from Geobacillus stearothermophilus; a phedh protein, preferably, The method according to any one of [G1] to [G11], wherein the dehydrogenase is selected from the group consisting of phedh protein derived from Bacillus badius; and valdh protein, preferably valdh protein derived from Streptomyces frasiae.
[G13] The glutamate/phenylalanine/leucine/valine dehydrogenase is selected from the group consisting of:
(5'-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5'-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added to SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. protein,
(5'-3) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5'-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. protein,
(5'-5) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes a reductive amination reaction of α-keto acid, and (5'-6) 1 or a number in SEQ ID NO: 34. A protein consisting of an amino acid sequence in which 5 amino acids are deleted, substituted, or added, and has the activity of catalyzing the reductive amination reaction of α-keto acids;
The method according to any one of [G1] to [G12].

[H1] First gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-6) 1 or 1 in SEQ ID NO: 6. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
coryneform bacteria, including
[H2] The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6,
(1-6) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 6, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. Genes that encode;
(1-7) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8,
(1-8) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 8, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-9) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 10,
(1-10) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 10, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-11) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 12,
(1-12) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 12, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-13) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 14,
(1-14) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 14, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-15) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 16,
(1-16) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 16, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-17) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 18,
(1-18) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 18, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-19) A gene encoding a protein that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, which is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 20,
(1-20) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in SEQ ID NO: 20, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-21) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, and (1-22) 1 or 1 in SEQ ID NO: 22. A gene that encodes a protein that is composed of an amino acid sequence in which several amino acids are deleted, substituted, or added, and that has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
coryneform bacteria, including
[H3] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 1. The coryneform bacterium according to gene [H1] or [H2].
[H4] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 3. The coryneform bacterium according to any one of genes [H1] to [H3].
[H5] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 5. The coryneform bacterium according to any one of genes [H1] to [H4].
[H6] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 8, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 7. The coryneform bacterium according to any one of genes [H1] to [H5].
[H7] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 9. The coryneform bacterium according to any one of genes [H1] to [H6].
[H8] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 12, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 11. The coryneform bacterium according to any one of genes [H1] to [H7].
[H9] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 14, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 13. The coryneform bacterium according to any one of genes [H1] to [H8].
[H10] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 16, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 15. The coryneform bacterium according to any one of genes [H1] to [H9].
[H11] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 18, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 17. The coryneform bacterium according to any one of genes [H1] to [H10].
[H12] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 20, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 19. The coryneform bacterium according to any one of genes [H1] to [H11].
[H13] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 22, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound, consists of the base sequence set forth in SEQ ID NO: 21. The coryneform bacterium according to any one of genes [H1] to [H12].
[H14] The coryneform bacterium is a Corynebacterium genus, preferably Corynebacterium glutamicum, and more preferably a Corynebacterium glutamicum with accession number NITE BP-02780, NITE BP-02781, NITE BP- The coryneform bacterium according to any one of [H1] to [H13], which is NITE BP-02782, NITE BP-02783, or NITE BP-02784.
[H15] The method according to any one of [H1] to [H14], wherein the coryneform bacterium is a coryneform bacterium transformed by the first gene.
[H16] Second gene selected from the group consisting of:
(2-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 24, which catalyzes the decarboxylation reaction of α-keto acids; and (2-2) 1 or several genes in SEQ ID NO: 24. A gene that encodes a protein consisting of an amino acid sequence in which five amino acids are deleted, substituted, or added, and that has the activity of catalyzing the decarboxylation reaction of α-keto acids; and a gene selected from the group consisting of: Third gene:
(3-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 26, which catalyzes the reduction reaction of aldehyde, and (3-2) A gene in which one or several amino acids in SEQ ID NO: 26 The coryneform protein according to any one of [H1] to [H15], which is a protein consisting of a deleted, substituted, or added amino acid sequence, and further comprises a gene encoding a protein having an activity of catalyzing an aldehyde reduction reaction. type bacteria.
[H17] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 24, which catalyzes the decarboxylation reaction of α-keto acids, is a gene consisting of the base sequence set forth in SEQ ID NO: 23. The coryneform bacterium according to [H16].
[H18] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 26, which catalyzes the reduction reaction of aldehyde, is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 25.[H16] ] or the coryneform bacterium according to [H17].
[H19] The coryneform bacterium is a Corynebacterium bacterium, preferably Corynebacterium glutamicum, more preferably a Coryneform bacterium whose accession number is NITE BP-02780 or NITE BP-02781. The coryneform bacterium according to any one of [H16] to [H18], which is a bacterium.
[H20] The method according to any one of [H16] to [H19], wherein the coryneform bacterium is a coryneform bacterium transformed by the first, second, and third genes.
[H21] Fourth gene selected from the group consisting of:
(4-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 28, which catalyzes the reduction reaction of α-keto acid; and (4-2) one or several genes in SEQ ID NO: 28. Any of [H1] to [H20], which further comprises a gene encoding a protein having the activity of catalyzing the reduction reaction of α-keto acids, which is a protein consisting of an amino acid sequence in which an amino acid is deleted, substituted, or added. The coryneform bacterium according to (1).
[H21] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 28, which catalyzes the reduction reaction of α-keto acid, is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 27. The coryneform bacterium according to [H20].
[H22] The coryneform bacterium is a Corynebacterium genus, preferably Corynebacterium glutamicum, more preferably a coryneform bacterium whose accession number is NITE BP-02782 [H20 ] or the coryneform bacterium according to [H21].
[H23] The method according to any one of [H20] to [H22], wherein the coryneform bacterium is a coryneform bacterium transformed by the first and fourth genes.
[H24] Fifth gene selected from the group consisting of:
(5-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, and (5-6) 1 in SEQ ID NO: 34. or a protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted, or added, further comprising a gene encoding a protein having the activity of catalyzing the reductive amination reaction of α-keto acids [H1] - The coryneform bacterium according to any one of [H23].
[H25] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids, is composed of the base sequence set forth in SEQ ID NO: 29. The coryneform bacterium according to [H24], which is a gene.
[H26] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids, is composed of the base sequence set forth in SEQ ID NO: 31. The coryneform bacterium according to [H24] or [H25], which is a gene.
[H27] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, is composed of the base sequence set forth in SEQ ID NO: 33. The coryneform bacterium according to any one of [H24] to [H26], which is a gene.
[H28] The coryneform bacterium is a Corynebacterium genus, preferably Corynebacterium glutamicum, more preferably a coryneform bacterium whose accession number is NITE BP-02783 [H24 The coryneform bacterium according to any one of ] to [H27].
[H29] The method according to any one of [H24] to [H28], wherein the coryneform bacterium is a coryneform bacterium transformed by the first and fifth genes.
[H30] The fifth gene selected from the group consisting of:
(5-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids;
(5-2) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 30, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-3) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids;
(5-4) A protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in SEQ ID NO: 32, and has an activity of catalyzing the reductive amination reaction of α-keto acids. the gene encoding the
(5-5) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, and (5-6) 1 in SEQ ID NO: 34. or a gene encoding a protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted, or added, and which has the activity of catalyzing the reductive amination reaction of α-keto acids; and a group consisting of the following: The 6th gene selected from:
(6-1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 36, which catalyzes the reaction of replacing the phosphate group of 2-amino-4-phosphonooxycarboxylic acid with the methylmercapto group of methylmercaptan. (6-2) A protein consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added to SEQ ID NO: 36, which is 2-amino-4-phosphonooxy The coryneform bacterium according to any one of [H1] to [H29], further comprising a gene encoding a protein having an activity of catalyzing a reaction in which a phosphate group of a carboxylic acid is replaced with a methyl mercapto group of a methyl mercaptan. .
[H31] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 30, which catalyzes the reductive amination reaction of α-keto acids, is composed of the base sequence set forth in SEQ ID NO: 29. The coryneform bacterium according to [H30], which is a gene.
[H32] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 32, which catalyzes the reductive amination reaction of α-keto acids, is composed of the base sequence set forth in SEQ ID NO: 31. The coryneform bacterium according to [H30] or [H31], which is a gene.
[H33] The gene encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 34, which catalyzes the reductive amination reaction of α-keto acids, is composed of the base sequence set forth in SEQ ID NO: 33. The coryneform bacterium according to any one of [H30] to [H32], which is a gene.
[H34] The protein consisting of the amino acid sequence set forth in SEQ ID NO: 36, which catalyzes the reaction of substituting the phosphoric acid group of 2-amino-4-phosphonooxycarboxylic acid with the methylmercapto group of methylmercaptan. The coryneform bacterium according to any one of [H30] to [H33], wherein the gene encoding the protein is a gene consisting of the base sequence set forth in SEQ ID NO: 35.
[H35] The coryneform bacterium is a Corynebacterium genus, preferably Corynebacterium glutamicum, more preferably a coryneform bacterium whose accession number is NITE BP-02784 [H30 The coryneform bacterium according to any one of ] to [H34].
[H36] The method according to any one of [H30] to [H35], wherein the coryneform bacterium is a coryneform bacterium transformed by the first and fifth genes.

1. Materials and Methods (1-1) Culture reagents were purchased from Fuji Film Wako Pure Chemical Industries, Ltd. unless otherwise specified.

(A medium)
2.0 g of urea, 7.0 g of ammonium sulfate, 0.50 g of potassium dihydrogen phosphate, 0.50 g of dipotassium hydrogen phosphate, 0.50 g of magnesium sulfate heptahydrate, 2.0 g of yeast extract (manufactured by Difco), Casamino Acid Vitamin Assay (Difco) 7.0g, iron(II) sulfate heptahydrate 6.0mg, manganese(II) sulfate monohydrate 4.2mg, D(+)-biotin 0.20mg, thiamine hydrochloride 0.20 mg was dissolved in 0.92 L of water, and the pH was adjusted to 7.0 using a 5M aqueous potassium hydroxide solution. A liquid medium was prepared by autoclaving this at 121° C. for 20 minutes, cooling it to room temperature, and then adding 80 mL of an autoclaved 50% (w/v) glucose aqueous solution.

(A agar medium)
2.0 g of urea, 7.0 g of ammonium sulfate, 0.50 g of potassium dihydrogen phosphate, 0.50 g of dipotassium hydrogen phosphate, 0.50 g of magnesium sulfate heptahydrate, 2.0 g of yeast extract (manufactured by Difco), Casamino Acid Vitamin Assay (Difco) 7.0g, iron(II) sulfate heptahydrate 6.0mg, manganese(II) sulfate monohydrate 4.2mg, D(+)-biotin 0.20mg, thiamine hydrochloride 0.20 mg and 15 g of agar were dissolved and suspended in 0.92 L of water, and the pH was adjusted to 7.0 using a 5M aqueous potassium hydroxide solution. After sterilizing this in an autoclave at 121°C for 20 minutes and cooling it to 50°C, add 80 mL of an autoclaved 50% (w/v) glucose aqueous solution, dispense it into a Petri dish, and cool and solidify. solid medium.

(BA medium)
2.0 g of urea, 7.0 g of ammonium sulfate, 0.50 g of potassium dihydrogen phosphate, 0.50 g of dipotassium hydrogen phosphate, 0.50 g of magnesium sulfate heptahydrate, 2.0 g of yeast extract (manufactured by Difco), Casamino Acid Vitamin Assay (Difco) 7.0g, iron(II) sulfate heptahydrate 6.0mg, manganese(II) sulfate monohydrate 4.2mg, D(+)-biotin 0.20mg, thiamine hydrochloride 0.20 mg, 21 g of 4-morpholinopropanesulfonic acid was dissolved in 0.92 L of water, and the pH was adjusted to 7.0 using a 5M aqueous potassium hydroxide solution. A liquid medium was prepared by autoclaving this at 121° C. for 20 minutes, cooling it to room temperature, and then adding 80 mL of an autoclaved 50% (w/v) glucose aqueous solution.

(LB medium)
After dissolving 10 g of Bactotryptone (manufactured by Difco), 5.0 g of yeast extract (manufactured by Difco), and 10 g of sodium chloride in 1 L of water, and adjusting the pH to 7.0 using a 5M aqueous sodium hydroxide solution, Liquid medium prepared by autoclaving at 121°C for 20 minutes.

(LB agar medium)
10 g of Bactotryptone (manufactured by Difco), 5.0 g of yeast extract (manufactured by Difco), 10 g of sodium chloride, and 15 g of agar were dissolved and suspended in 1 L of water, and the pH was adjusted to 7.0 using a 5M aqueous sodium hydroxide solution. A solid medium prepared by adjusting the medium to 121°C, sterilizing it in an autoclave at 121°C for 20 minutes, cooling it to 50°C, dispensing it into a Petri dish, and solidifying it by cooling.

(Suc agar medium)
10 g of Bactotryptone (manufactured by Difco), 5.0 g of yeast extract (manufactured by Difco), 10 g of sodium chloride, and 15 g of agar were dissolved and suspended in 0.5 L of water, and the pH was adjusted to 7 using a 5M aqueous sodium hydroxide solution. Adjusted to .0. After sterilizing this in an autoclave at 121°C for 20 minutes and cooling it to 50°C, add 0.5 L of an autoclaved 20% (w/v) sucrose aqueous solution, dispense it into a Petri dish, and cool and solidify. A solid medium prepared in

(BHIS medium)
By dissolving 36g of Brain Heart Infusion (manufactured by Difco) and 91g of sorbitol in 1L of water, adjusting the pH to 7.0 using a 5M aqueous sodium hydroxide solution, and sterilizing it in an autoclave at 121°C for 20 minutes. Prepared liquid medium.

(BHIS-GIT medium)
36 g of Brain Heart Infusion (manufactured by Difco), 8.0 g of glycine, 1.3 g of isoniazid, 3 mL of Tween 80, and 91 g of sorbitol were dissolved in 1 L of water, and the pH was adjusted to 7.0 using a 5M aqueous sodium hydroxide solution. Then, the liquid medium was prepared by autoclaving at 121°C for 20 minutes.

(1-2) Buffer and reagent (TE buffer)
1.2 g of trishydroxymethylaminomethane and 0.37 g of ethylenediamine-N,N,N',N'-tetraacetic acid disodium salt dihydrate were dissolved in 1 L of water, and the pH was adjusted to 8.0 using 6M hydrochloric acid. Buffer solution prepared by autoclaving at 121°C for 20 minutes after adjusting to 0.

(TG buffer)
A buffer solution prepared by dissolving 0.12 g of trishydroxymethylaminomethane and 10 g of glycerol in 1 L of water, adjusting the pH to 7.5 using 6M hydrochloric acid, and then autoclaving at 121°C for 20 minutes. .

(10% glycerol aqueous solution)
An aqueous solution prepared by dissolving 100 g of glycerol in 1 L of water and sterilizing it in an autoclave at 121° C. for 20 minutes.

(BR buffer)
Potassium dihydrogen phosphate 0.50 g, dipotassium hydrogen phosphate 0.50 g, magnesium sulfate heptahydrate 0.50 g, iron (II) sulfate heptahydrate 6.0 mg, manganese (II) sulfate monohydrate A buffer solution obtained by dissolving 4.2 mg in 1.0 L of water and adjusting the pH to 7.0 using a 5M aqueous potassium hydroxide solution.

(BS buffer)
Potassium dihydrogen phosphate 0.50 g, dipotassium hydrogen phosphate 0.50 g, magnesium sulfate heptahydrate 0.50 g, iron (II) sulfate heptahydrate 6.0 mg, manganese (II) sulfate monohydrate A buffer solution obtained by dissolving 4.2 mg of 4-morpholinopropanesulfonic acid and 21 g of 4-morpholinopropanesulfonic acid in 1.0 L of water and adjusting the pH to 7.0 using a 5M aqueous potassium hydroxide solution.

(BT buffer)
Ammonium sulfate 7.0g, potassium dihydrogen phosphate 0.50g, dipotassium hydrogen phosphate 0.50g, magnesium sulfate heptahydrate 0.50g, iron(II) sulfate heptahydrate 6.0mg, manganese(II) sulfate ) A buffer solution obtained by dissolving 4.2 mg of monohydrate in 1.0 L of water and adjusting the pH to 7.0 using a 5 M aqueous potassium hydroxide solution.

(1-3) Definition of analytical conditions and experimental method (extraction and purification of DNA fragments after agarose gel electrophoresis)
DNA fragments were extracted and purified using NucleoSpin (registered trademark) Gel and PCR Clean-up (manufactured by Machleri-Nagel) according to its instructions.

(Plasmid extraction)
Plasmids were extracted and purified using NucleoSpin (registered trademark) Plasmid EasyPure (manufactured by Machleri-Nagel) according to its instructions.

(PCR method)
PCR was performed using any of the following DNA polymerases and their associated reagents according to their instructions: PrimeSTAR (registered trademark) HS DNA Polymerase (Takara Bio Inc.), PrimeSTAR (registered trademark) Max DNA Polymerase (Takara Bio Inc.). Tks Gflex ^TM DNA Polymerase (manufactured by Takara Bio).

(Liquid chromatography for organic acid analysis)
An organic acid analysis system made by Shimadzu Corporation and consisting of the following equipment was used. System controller: CBM-20A, liquid feeding unit: LC-20AB, online degassing unit: DGU-20A3R, autoinjector: SIL-20AC, column oven: CTO-20AC, photodiode array detector: SPD-M20A.

The analysis conditions are as follows.
Guard column: TSKgel OApak-P (manufactured by Tosoh Corporation)
Analytical column: TSKgel OApak-A (manufactured by Tosoh Corporation)
Column oven temperature: 40℃
Mobile phase: 0.75mM sulfuric acid Flow rate: 1.0mL/min.

(Liquid chromatography for sugar analysis)
A sugar analysis system made by Shimadzu Corporation and consisting of the following equipment was used. System controller: CBM-20A, liquid feeding unit: LC-20AB, online degassing unit: DGU-20A3R, autoinjector: SIL-20AC, column oven: CTO-20AC, refractive index detector: RID-10A.

The analysis conditions are as follows.
Pretreatment: Desalination cartridge (manufactured by Bio-Rad)
Analytical column: Aminex HPX-87P (manufactured by Bio-Rad)
Column oven temperature: 85℃
Mobile phase: water flow rate: 0.6 mL/min.

(Liquid chromatography for amino acid analysis)
An amino acid analysis system made by Shimadzu Corporation and consisting of the following equipment was used. System controller: CBM-20A, Liquid feeding unit: 1 LC-20AB, 2 LC-20AD, Flow path switching valve: FCV-11AL, Online degassing unit: DGU-20A3R, Autoinjector: SIL-20AC, Column oven: CTO -20AC, fluorescence detector: RF-20AXs.

The analysis conditions were in accordance with the Na-type rapid conditions shown in the Shimadzu high-performance liquid chromatograph Prominence amino acid analysis system instruction manual, using an ammonia trap column ISC-30/S 0504 NA (manufactured by Shimadzu Corporation) and an analytical column Shim-pack AMINO-NA. (manufactured by Shimadzu Corporation) was used for analysis.

(Gas Chromatography Mass Spectrometry, GCMS)
GCMS was performed using a gas chromatogram quadrupole mass spectrometer (7890B GC/5977A MSD, manufactured by Agilent Technologies) and a multifunctional autosampler (MPS2-xt, manufactured by Gestel) under the following conditions.
<Thermal desorption sampling conditions>
Heating conditions: 50 ℃ (0.5 min) → 50 ℃ / min → 300 ℃ (10 min)
Trap temperature: -100℃ → 12℃/s → 300℃
<Gas chromatograph conditions>
Separation column: DB-5ht [30 m × 0.25 mm ID, 0.10 μm, manufactured by Agilent Technologies]
Temperature increase condition: 40 ℃ (5 min) → 10 ℃ / min → 320 ℃ (12 min)
<Mass spectrometry conditions>
Ionization method: Electron ionization (EI) Measurement mass range: m/z = 29-550

(Genomic DNA extraction)
Microorganisms cultured in liquid according to the manufacturer's instructions were collected by centrifugation and frozen at -80°C. The bacterial cell pellet was thawed at room temperature, 537 μL of a 10 mg/mL lysozyme aqueous solution was added, and the mixture was shaken at 37° C. for 1 hour. Next, 30 μL of 0.5M EDTA aqueous solution, 30 μL of 10% SDS aqueous solution, and 3 μL of 20 mg/mL proteinase K aqueous solution were added, and the mixture was shaken at 37° C. for 1 hour. Thereafter, 100 μL of a 5M aqueous sodium chloride solution and 80 μL of a 10% aqueous hexadecyltrimethylammonium bromide solution (in 0.7M sodium chloride) were added, and the mixture was heated at 65° C. for 10 minutes. After cooling to room temperature, 780 μL of chloroform-isoamyl alcohol (24:1) was added and mixed gently. Centrifugation was performed at 15,000×g and 4° C. for 5 minutes, and 600 μL of the aqueous layer was collected. To this was added 600 μL of phenol-chloroform-isoamyl alcohol (25:24:1) and mixed gently. Centrifugation was performed at 15,000×g and 4° C. for 5 minutes, and 500 μL of the aqueous layer was collected. After adding 300 μL of isopropanol to this and centrifuging at 15,000×g and 4° C. for 5 minutes, the supernatant was removed. The genomic DNA obtained as a precipitate was washed with 500 μL of 70% ethanol, and centrifuged at 15,000×g and 4° C. for 5 minutes. After repeating this washing twice and drying at room temperature, the obtained genomic DNA was dissolved in 100 μL of TE buffer.

(Electroporation method for transforming Corynebacterium glutamicum ATCC13032-derived strain)
A glycerol stock of a strain derived from Corynebacterium glutamicum ATCC13032 to be transformed was inoculated onto an A agar medium under aseptic conditions and cultured at 33°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of medium A, and cultured with shaking at 33°C to obtain a seed culture solution. This seed culture was inoculated into 100 mL of BHIS medium prepared in a 500 mL flask so that the turbidity was 0.2. The mixture was shaken at 33°C and cultured until the turbidity reached 0.5-0.7. This culture solution was centrifuged at 4° C. and 5500×g for 20 minutes, and the supernatant was removed. The collected cells were washed with 10 mL of TG buffer, centrifuged at 5,500×g at 4° C. for 5 minutes, and the supernatant was removed. After performing this washing twice, it was washed with 10 mL of a 10% glycerol aqueous solution, centrifuged at 4° C. and 5500×g for 5 minutes, and the supernatant liquid was removed. The obtained bacterial cell pellet was suspended in 1 mL of 10% glycerol aqueous solution, dispensed into 150 μL portions into sterilized 1.5 mL tubes, frozen using liquid nitrogen, and stored at -80°C as competent cells. .

For gene deletion and gene replacement on genomic DNA: 150 μL of competent cells were thawed on ice, and 500 ng of plasmid was added. This was transferred to a sterilized 0.2 cm gap electroporation cuvette (manufactured by Bio-Rad), and electroporation was performed at 25 μF, 200 Ω, and 2500 V using a Gene Pulser Xcell ^TM electroporation system (manufactured by Bio-Rad). Ta. The bacterial cell suspension was diluted with 1 mL of BHIS medium, incubated at 46°C for 6 minutes, and then incubated at 33°C for 1 hour. This cell suspension was inoculated onto an A agar medium containing an appropriate antibiotic, and the desired transformants were selected.

When introducing a plasmid for gene overexpression: 50 μL of competent cells were thawed on ice, and 200 ng of plasmid was added. This was transferred to a sterilized 0.2 cm gap electroporation cuvette (manufactured by Bio-Rad), and electroporation was performed at 25 μF, 200 Ω, and 2500 V using a Gene Pulser Xcell ^TM electroporation system (manufactured by Bio-Rad). Ta. The bacterial cell suspension was diluted with 1 mL of BHIS medium, incubated at 46°C for 6 minutes, and then incubated at 33°C for 1 hour. This cell suspension was inoculated onto an A agar medium containing an appropriate antibiotic, and the desired transformants were selected.

(Colony PCR)
Using a combination of primers capable of amplifying the desired DNA sequence, reagents other than the template DNA were mixed according to the instructions for TaKaRa Ex Taq (registered trademark) (manufactured by Takara Bio Inc.). A small amount of colonies of the bacterial strain that had formed on the agar medium were suspended in this, and the DNA contained therein was used as a template, and PCR was performed according to the instructions. The PCR product was analyzed by agarose gel electrophoresis, and it was confirmed that the target plasmid was retained or that the corresponding DNA sequence on the genome was deleted or replaced.

(Turbidity measurement)
The turbidity of the culture solution of E. coli and Corynebacterium glutamicum was measured at a wavelength of 610 nm using Ultraspec 8000 (manufactured by GE Healthcare).

(1-4) Details of primer DNA, bacterial cells, and plasmids Details of primer DNA, bacterial cells, and plasmids are listed in Tables 37 to 67 below.

2. Plasmid construction (2-1) Gene deletion version 2-1-(1) Construction of plasmid for gene disruption Using pNIC28-Bsa4 (manufactured by Source BioScience) as a template, GEP007 (SEQ ID NO: 37) and GEP008 (SEQ ID NO: 38) A DNA fragment containing the sacB gene derived from Bacillus subtilis [Mol. Microbiol. , 6, 1195 (1992)] was amplified by PCR method. This PCR product was treated with BamHI and PstI, and after agarose gel electrophoresis, the DNA fragment was extracted and purified. In addition, plasmid pHSG299 (manufactured by Takara Bio Inc.) containing a gene conferring resistance to kanamycin was treated with BamHI and PstI, and after agarose gel electrophoresis, DNA fragments were extracted and purified. These two DNA fragments were ligated using DNA Ligation Kit <Mighty Mix> (manufactured by Takara Bio Inc.) according to its instructions to obtain plasmid pGE015.

For the purpose of deleting the KpnI recognition site that pGE015 has on sacB, PrimeSTAR (registered trademark) was prepared using pGE015 as a template and DNA with the base sequences shown by GEP808 (SEQ ID NO: 39) and GEP809 (SEQ ID NO: 40). Mutagenesis and plasmid amplification were performed using Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) according to its instructions to obtain pGE209.

2-1-(2) Construction of plasmid for creating ppc gene disrupted strain Corynebacterium glutamicum ATCC13032 strain was obtained as NBRC12168 strain from the Biotechnology Center of the National Institute of Technology and Evaluation, and was cultured according to the attached instructions. After that, genomic DNA was extracted.

Using the genomic DNA of ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP132 (SEQ ID NO: 41) and GEP133 (SEQ ID NO: 42), and the bases shown by GEP134 (SEQ ID NO: 43) and GEP135 (SEQ ID NO: 44) Two types of PCR products were obtained by PCR using the combination of DNA sequences, and these were extracted and purified after agarose gel electrophoresis. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE020.

2-1-(3) Creation of ppc gene deletion strain Corynebacterium glutamicum ATCC13032 strain was transformed by electroporation using pGE020, and kanamycin-resistant was transformed on A medium containing 25 μg/ml kanamycin at 33°C. Selected stocks. Next, the obtained kanamycin-resistant strain was spread on a Suc agar medium and cultured at 33°C. The grown colonies were further cultured in medium A, and strains lacking the ppc gene were selected by colony PCR. This was named GES007.

2-1-(4) Construction of plasmid for creating ldhA gene-disrupted strain Using the genomic DNA of ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP169 (SEQ ID NO: 45) and GEP170 (SEQ ID NO: 46), and GEP215 (SEQ ID NO: 47) and GEP216 (SEQ ID NO: 48), two types of PCR products were obtained by PCR method, and these were extracted and purified after agarose gel electrophoresis. went. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE033.

2-1-(5) Creation of ldhA gene deletion strain GES007 was transformed by electroporation using pGE033, and the selected strain was named GES048 by the same operation as in the generation of GES007.

2-1-(6) Construction of a plasmid for creating a pyc gene-disrupted strain Using the genomic DNA of the ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP615 (SEQ ID NO: 49) and GEP616 (SEQ ID NO: 50), and GEP617 (SEQ ID NO: 51) and GEP618 (SEQ ID NO: 52), two types of PCR products were obtained by PCR method, and these were extracted and purified after agarose gel electrophoresis. went. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE177.

2-1-(7) Creation of pyc gene deletion strain GES048 was transformed by electroporation using pGE177, and the selected strain was named GES385 using the same procedure as in the generation of GES007.

2-1-(8) Construction of a plasmid for creating a poxB gene-disrupted strain Using the genomic DNA of the ATCC13032 strain as a template, a combination of DNA with the nucleotide sequences shown by GEP406 (SEQ ID NO: 53) and GEP407 (SEQ ID NO: 54), and GEP698 (SEQ ID NO: 55) and GEP409 (SEQ ID NO: 56), two types of PCR products were obtained by the PCR method, and after agarose gel electrophoresis, they were extracted and purified. went. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed, and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE191.

2-1-(9) Creation of poxB gene deletion strain GES385 was transformed by electroporation using pGE191, and the selected strain was named GES388 using the same procedure as in the generation of GES007.

2-1-(10) Construction of a plasmid for creating an alaA gene-disrupted strain Using the genomic DNA of the ATCC13032 strain as a template, a combination of DNA with the nucleotide sequences shown by GEP810 (SEQ ID NO: 57) and GEP811 (SEQ ID NO: 58), and GEP812 (SEQ ID NO: 59) and GEP813 (SEQ ID NO: 60), two types of PCR products were obtained by the PCR method, and after agarose gel electrophoresis, they were extracted and purified. went. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed and the resulting plasmid was designated as pGE210 in the same manner as in the construction of pGE020.

2-1-(11) Creation of alaA gene deletion strain GES388 was transformed by electroporation using pGE210, and the selected strain was named GES393 using the same procedure as in the generation of GES007.

2-1-(12) Construction of plasmid for creating ilvB gene-disrupted strain Using the genomic DNA of ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP956 (SEQ ID NO: 61) and GEP957 (SEQ ID NO: 62), and GEP958 (SEQ ID NO: 63) and GEP959 (SEQ ID NO: 64), two types of PCR products were obtained by PCR method, and these were extracted and purified after agarose gel electrophoresis. went. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed and the resulting plasmid was named pGE228 in the same manner as in the preparation of pGE020.

2-1-(13) Creation of ilvB gene deletion strain GES393 was transformed by electroporation using pGE228, and the selected strain was named GES405 using the same procedure as in the generation of GES007.

2-1-(14) Construction of a plasmid for creating a cg0931 gene-disrupted strain Using the genomic DNA of the ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP1132 (SEQ ID NO: 65) and GEP1133 (SEQ ID NO: 66), and GEP1134 (SEQ ID NO: 67) and GEP1135 (SEQ ID NO: 68), two types of PCR products were obtained by PCR method, and these were extracted and purified after agarose gel electrophoresis. went. Furthermore, pGE015 was treated with EcoRI, subjected to agarose gel electrophoresis, and then extracted and purified. These three DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE253.

2-1-(15) Creation of cg0931 gene deletion strain GES405 was transformed by electroporation using pGE253, and the strain selected by the same operation as in the generation of GES007 was named GES435.

2-1-(16) Construction of plasmid for creating adhE gene-disrupted strain Using the genomic DNA of ATCC13032 strain as a template, a combination of DNA with the nucleotide sequences shown by GEP2606 (SEQ ID NO: 69) and GEP2607 (SEQ ID NO: 70), and GEP2608 (SEQ ID NO: 71) and GEP2609 (SEQ ID NO: 72), two types of PCR products were obtained by PCR method, and these were extracted and purified after agarose gel electrophoresis. went. Furthermore, pGE209 was treated with KpnI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These three DNA fragments were mixed and the resulting plasmid was designated pGE1171 in the same manner as in the preparation of pGE020.

Using the genomic DNA of ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP2688 (SEQ ID NO: 73) and GEP2607 (SEQ ID NO: 70), and the bases shown by GEP2608 (SEQ ID NO: 71) and GEP2689 (SEQ ID NO: 74) Two types of PCR products were obtained by PCR using the combination of DNA sequences, and these were extracted and purified after agarose gel electrophoresis. Furthermore, pGE209 was treated with KpnI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These three DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE1218.

2-1-(17) Creation of adhE gene deletion strain GES048 was transformed by electroporation using pGE1171, and the selected strain was named GES1041 using the same procedure as in the generation of GES007.

GES435 was transformed using pGE1171 by electroporation, and the selected strain was named GES1042 using the same procedure as when creating GES007.

2-1-(18) Construction of a plasmid for creating an ald gene-disrupted strain Using the genomic DNA of the ATCC13032 strain as a template, a combination of DNA with the nucleotide sequences shown by GEP2610 (SEQ ID NO: 75) and GEP2611 (SEQ ID NO: 76), and GEP2612 (SEQ ID NO: 77) and GEP2613 (SEQ ID NO: 78), two types of PCR products were obtained by PCR method, and these were extracted and purified after agarose gel electrophoresis. went. Furthermore, pGE209 was treated with KpnI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These three DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE1172.

Using the genomic DNA of ATCC13032 strain as a template, a combination of DNA with the base sequences shown by GEP2692 (SEQ ID NO: 79) and GEP2611 (SEQ ID NO: 76), and the bases shown by GEP2612 (SEQ ID NO: 77) and GEP2693 (SEQ ID NO: 80) Two types of PCR products were obtained by PCR using the combination of DNA sequences, and these were extracted and purified after agarose gel electrophoresis. Furthermore, pGE209 was treated with KpnI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These three DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE020 was designated as pGE1221.

2-1-(19) Creation of ald gene deletion strain GES1041 was transformed by electroporation using pGE1172, and the selected strain was named GES1070 using the same procedure as in the generation of GES007.

GES1042 was transformed using pGE1172 by electroporation, and the selected strain was named GES1211 using the same procedure as in the production of GES007.

(2-2) Gene replacement version 2-2-(1) Construction of pGE409 Using pHSG298 (manufactured by Takara Bio Inc.) as a template, construct the DNA with the base sequences shown by GEP433 (SEQ ID NO: 81) and GEP434 (SEQ ID NO: 82). PCR products were obtained using the PCR method using the combination of the combination and the DNA with the base sequences shown by GEP437 (SEQ ID NO: 83) and GEP438 (SEQ ID NO: 84), and these were extracted and purified after agarose gel electrophoresis. I did it. These are DNA fragments containing the kanamycin resistance gene and the replication origin pUCori for E. coli, respectively.

Corynebacterium glutamicum NBRC12169 strain was obtained from the Biotechnology Center of the National Institute of Technology and Evaluation, an independent administrative agency, and cultured according to the attached instructions, and the bacterial cells were collected by centrifugation. The bacterial pellet was washed with 1 mL of 10 mM Cyclohexylenedinitrilotetraacetic acid, 25 mM Tris-HCl (pH 8.0) buffer, centrifuged at 6,000×g for 5 minutes at 4° C., and collected again. The bacterial cell pellet was suspended in 300 μL of Buffer A1 (supplied with NucleoSpin (registered trademark) Plasmid EasyPure manufactured by Machleri-Nagel) containing 15 mg/mL lysozyme, and shaken at 37° C. for 2 hours. Thereafter, the pCG1 plasmid was extracted and purified according to the NucleoSpin (registered trademark) Plasmid EasyPure instructions. Using this as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP445 (SEQ ID NO: 85) and GEP446 (SEQ ID NO: 86), and this was extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing the Corynebacterium replication origin pCG1ori.

The above three DNA fragments were mixed and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE403.

Using the genomic DNA of ATCC13032 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP472 (SEQ ID NO: 87) and GEP478 (SEQ ID NO: 88), and these were subjected to agarose gel electrophoresis. After that, extraction and purification were performed. In addition, a PCR product was obtained by PCR using pFLAG-CTC (manufactured by Sigma-Aldrich) as a template and a combination of DNA having the base sequence shown by GEP467 (SEQ ID NO: 89) and GEP468 (SEQ ID NO: 90). These were subjected to agarose gel electrophoresis, followed by extraction and purification. These are DNA fragments containing the gapA promoter (PgapA) and E. coli ribosomal RNA transcription terminator (TrrnB) of ATCC13032 strain, respectively. Further, pGE403 was treated with BamHI and EcoRV, and subjected to agarose gel electrophoresis, followed by extraction and purification. These three DNA fragments were mixed and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE409.

2-2-(2) Construction of pGE1185 (plasmid for creating ΔadhE::hpaI replacement strain) ECOS ^TM Competent E. E. coli BL21 (DE3) (manufactured by Nippon Gene) was purchased, inoculated onto an LB agar medium under aseptic conditions, and cultured at 37°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of LB medium, the grown bacterial cells were collected, and the genomic DNA was extracted. Using the genomic DNA of the BL21 (DE3) strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequences shown by GEP2268 (SEQ ID NO: 91) and GEP2269 (SEQ ID NO: 92), and these were mixed with agarose. After gel electrophoresis, extraction and purification were performed. This is a DNA fragment containing the E. coli BL21 (DE3) strain hpaI gene (Ec_hpaI). Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed, and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 100 μg/ml ampicillin. This transformed strain was cultured in LB medium containing 100 μg/ml ampicillin, and plasmid extraction was performed using the resulting culture suspension. Plasmid pGE1031 in which Ec_hpaI was cloned into pET-15b was thus obtained.

A PCR product was obtained by PCR using pGE1031 as a template and a combination of DNA with the base sequence shown by GEP2519 (SEQ ID NO: 93) and GEP2518 (SEQ ID NO: 94), which was extracted after agarose gel electrophoresis. , purification was performed. Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed, and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. In this way, plasmid pGE1143 in which Ec_hpaI was subcloned into pGE409 was obtained.

PCR products were obtained by PCR using pGE1143 as a template and a combination of DNA with the base sequences shown by GEP2637 (SEQ ID NO: 95) and GEP2638 (SEQ ID NO: 96), and these were extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing PgapA-hpaI-TrrnB. Furthermore, pGE1171 was treated with XhoI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed, and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE1185.

2-2-(3) Construction of pGE1186 (plasmid for creating a Δald::hpaI replacement strain) Using pGE1143 as a template, use the combination of DNA with the base sequence shown by GEP2639 (SEQ ID NO: 97) and GEP2640 (SEQ ID NO: 98). Then, PCR products were obtained by PCR method, and these were subjected to agarose gel electrophoresis, followed by extraction and purification. This is a DNA fragment containing PgapA-hpaI-TrrnB. Furthermore, pGE1172 was treated with XhoI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE1185 was designated as pGE1186.

2-2-(4) Creation of ΔadhE::hpaI Δald::hpaI replacement strain GES1041 was transformed by electroporation using pGE1186, and kanamycin-resistant was transformed on A medium containing 25 μg/ml kanamycin at 33°C. Selected stocks. Next, the obtained kanamycin-resistant strain was spread on a Suc agar medium and cultured at 33°C. The grown colonies were further cultured in medium A, and strains in which the ald gene was replaced with PgapA-hpaI-TrrnB were selected by colony PCR. This was named GES1052. Furthermore, GES1052 was transformed by electroporation using pGE1185, and a strain in which PgapA-hpaI-TrrnB was inserted at the adhE gene position was selected by the same operation as when creating GES1052. This was named GES1063.

GES1042 was transformed using pGE1186 by electroporation, and a strain in which the ald gene was replaced with PgapA-hpaI-TrrnB was selected by the same operation as in the production of GES1052. This was named GES1053. GES1053 was transformed using pGE1185 by electroporation, and a strain in which PgapA-hpaI-TrrnB was inserted at the adhE gene position was selected by the same operation as when creating GES1052. This was named GES1064.

2-2-(5) Construction of pGE1304 (plasmid for creating ΔadhE::rhmA replacement strain) E. E. coli DH5α Competent Cells (manufactured by Takara Bio Inc.) were purchased, inoculated onto an LB agar medium under aseptic conditions, and cultured at 37°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of LB medium, the grown bacterial cells were collected, and the genomic DNA was extracted. Using the genomic DNA of the DH5α strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2765 (SEQ ID NO: 99) and GEP2766 (SEQ ID NO: 100), and these were subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the E. coli DH5α strain rhmA gene (Ec_rhmA). Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed and a plasmid pGE1258 in which Ec_rhmA was cloned into pGE409 was obtained by the same operation as in the production of GE1143.

PCR products were obtained by PCR using pGE1258 as a template and a combination of DNA with the base sequences shown by GEP2637 (SEQ ID NO: 95) and GEP2638 (SEQ ID NO: 96), and these were extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing PgapA-rhmA-TrrnB. Furthermore, pGE1218 was treated with XhoI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE1185 was designated as pGE1304.

2-2-(6) Construction of pGE1302 (plasmid for creating a Δald::rhmA replacement strain) Using pGE1258 as a template, use the combination of DNA with the base sequence shown by GEP2639 (SEQ ID NO: 97) and GEP2640 (SEQ ID NO: 98). Then, PCR products were obtained by PCR method, and these were subjected to agarose gel electrophoresis, followed by extraction and purification. Furthermore, pGE1221 was treated with XhoI, subjected to agarose gel electrophoresis, and then extracted and purified. This is a DNA fragment containing PgapA-rhmA-TrrnB. These two DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE1185 was designated as pGE1302.

2-2-(7) Creation of ΔadhE::rhmA Δald::rhmA replacement strain GES1041 was transformed by electroporation using pGE1302, and the ald gene was transformed into PgapA-rhmA-TrrnB by the same operation as when creating GES1052. The strain being replaced was selected. This was named GES1215. Furthermore, GES1215 was transformed by electroporation using pGE1304, and a strain in which PgapA-rhmA-TrrnB was inserted at the adhE gene position was selected by the same operation as in the generation of GES1052. This was named GES1242.

2-2-(8) Construction of pGE1272 (plasmid for creating ΔadhE::nanA replacement strain) E. E. coli DH5α Competent Cells (manufactured by Takara Bio Inc.) were purchased, inoculated onto an LB agar medium under aseptic conditions, and cultured at 37°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of LB medium, the grown bacterial cells were collected, and the genomic DNA was extracted. Using the genomic DNA of the DH5α strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequences shown by GEP2722 (SEQ ID NO: 101) and GEP2723 (SEQ ID NO: 102), and these were subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the Escherichia coli DH5α strain nanA gene (Ec_nanA). Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed and a plasmid pGE1226 in which Ec_nanA was cloned into pGE409 was obtained by the same operation as in the construction of pGE1143.

PCR products were obtained by PCR using pGE1226 as a template and a combination of DNA with the base sequences shown by GEP2637 (SEQ ID NO: 95) and GEP2638 (SEQ ID NO: 96), and these were extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing PgapA-nanA-TrrnB. Furthermore, pGE1218 was treated with XhoI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid obtained by the same operation as in the construction of pGE1185 was designated as pGE1272.

2-2-(9) Construction of pGE1273 (plasmid for creating a Δald::nanA replacement strain) Using pGE1226 as a template, use the combination of DNA with the base sequence shown by GEP2639 (SEQ ID NO: 97) and GEP2640 (SEQ ID NO: 98). Then, PCR products were obtained by PCR method, and these were subjected to agarose gel electrophoresis, followed by extraction and purification. Furthermore, pGE1221 was treated with XhoI, subjected to agarose gel electrophoresis, and then extracted and purified. This is a DNA fragment containing PgapA-nanA-TrrnB. These two DNA fragments were mixed and the resulting plasmid was named pGE1273 in the same manner as in the preparation of pGE1185.

2-2-(10) Creation of ΔadhE::nanA Δald::nanA replacement strain GES1041 was transformed by electroporation using pGE1273, and the ald gene was transformed into PgapA-nanA-TrrnB by the same operation as when creating GES1052. The strain being replaced was selected. This was named GES1188. Furthermore, GES1188 was transformed by electroporation using pGE1272, and a strain in which PgapA-nanA-TrrnB was inserted at the adhE gene position was selected by the same operation as in the generation of GES1052. This was named GES1223.

GES1042 was transformed using pGE1273 by electroporation, and a strain in which the ald gene was replaced with PgapA-nanA-TrrnB was selected by the same operation as in the production of GES1052. This was named GES1189. Furthermore, pGE1272 was used to transform GES1189 by electroporation, and a strain in which PgapA-nanA-TrrnB was inserted at the adhE gene position was selected by the same operation as in the production of GES1052. This was named GES1233.

2-2-(11) Creation of ΔadhE Δald strain Transform GES1041 by electroporation using pGE1273, and select a strain in which the ald gene has been replaced with PgapA-nanA-TrrnB by the same operation as in the creation of GES1052. did. This was named GES1188. Furthermore, GES1188 was transformed by electroporation using pGE1272, and a strain in which PgapA-nanA-TrrnB was inserted at the adhE gene position was selected by the same operation as in the generation of GES1052. This was named GES1223.

GES1042 was transformed using pGE1273 by electroporation, and a strain in which the ald gene was replaced with PgapA-nanA-TrrnB was selected by the same operation as in the production of GES1052. This was named GES1189. Furthermore, pGE1272 was used to transform GES1189 by electroporation, and a strain in which PgapA-nanA-TrrnB was inserted at the adhE gene position was selected by the same operation as in the production of GES1052. This was named GES1233.

(2-3) Plasmid for overexpression 2-3-(1) Construction of pGE411 Using pHSG298 (manufactured by Takara Bio Inc.) as a template, the nucleotide sequences shown by GEP433 (SEQ ID NO: 81) and GEP434 (SEQ ID NO: 82) were PCR products were obtained by the PCR method using the combination of DNA and the DNA combination of the base sequences shown by GEP437 (SEQ ID NO: 83) and GEP438 (SEQ ID NO: 84), and these were extracted after agarose gel electrophoresis. , purification was performed. These are DNA fragments containing the kanamycin resistance gene and the replication origin pUCori for E. coli, respectively.

Corynebacterium casei strain JCM12072 was obtained from the RIKEN BioResource Research Center, cultured according to the attached instructions, and the bacterial cells were collected by centrifugation. The bacterial cell pellet was suspended in 1 mL of 10 mM Cyclohexylenedinitrilotetraacetic acid, 25 mM Tris-HCl (pH 8.0) buffer, centrifuged at 6,000×g for 5 minutes at 4° C., and collected again. The bacterial pellet was suspended in 300 μL of Buffer A1 (attached to NucleoSpin (registered trademark) Plasmid EasyPure manufactured by Machleri-Nagel) containing 15 mg/mL lysozyme, and shaken at 37° C. for 2 hours. Thereafter, the pCASE1 plasmid was extracted and purified according to the NucleoSpin (registered trademark) Plasmid EasyPure instructions. Using this as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP443 (SEQ ID NO: 103) and GEP444 (SEQ ID NO: 104), and this was extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing the Corynebacterium replication origin pCASE1ori.

The above three DNA fragments were mixed and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE402.

Using the genomic DNA of ATCC13032 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP474 (SEQ ID NO: 105) and GEP477 (SEQ ID NO: 106), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. In addition, a PCR product was obtained by PCR using pFLAG-CTC (manufactured by Sigma-Aldrich) as a template and a combination of DNA having the base sequence shown by GEP467 (SEQ ID NO: 89) and GEP468 (SEQ ID NO: 90). This was subjected to agarose gel electrophoresis, followed by extraction and purification. These are DNA fragments containing the ldhA promoter (PldhA) and E. coli ribosomal RNA transcription terminator (TrrnB) of ATCC13032 strain, respectively. Further, pGE402 was treated with BamHI and EcoRV, and subjected to agarose gel electrophoresis, followed by extraction and purification. These three DNA fragments were mixed and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE411.

2-3-(2) Construction of pGE945-2 Using pCR8/GW/TOPO (manufactured by Invitrogen) as a template, a combination of DNA with the base sequence shown by GEP435 (SEQ ID NO: 107) and GEP436 (SEQ ID NO: 108) was used. A PCR product was obtained using the PCR method, which was subjected to agarose gel electrophoresis, followed by extraction and purification. In addition, a PCR product was obtained by PCR using pHSG298 (manufactured by Takara Bio) as a template and a combination of DNA having the base sequences shown by GEP437 (SEQ ID NO: 83) and GEP438 (SEQ ID NO: 84), and this was transferred to agarose.・After gel electrophoresis, extraction and purification were performed. These are DNA fragments containing the spectinomycin resistance gene and the replication origin pUCori for E. coli, respectively.

The above two DNA fragments and the DNA fragment containing pCASE1ori obtained during the creation of pGE402 were mixed, and a binding reaction of the DNA fragments was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. . Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformant strain was selected by culturing on an LB agar medium containing 100 μg/ml spectinomycin. This transformed strain was cultured in LB medium containing 100 μg/ml spectinomycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE619.

Using the genomic DNA of ATCC13032 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP474 (SEQ ID NO: 105) and GEP477 (SEQ ID NO: 106), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. In addition, a PCR product was obtained by PCR using pFLAG-CTC (manufactured by Sigma-Aldrich) as a template and a combination of DNA having the base sequence shown by GEP467 (SEQ ID NO: 89) and GEP2897 (SEQ ID NO: 109). This was subjected to agarose gel electrophoresis, followed by extraction and purification. These are DNA fragments containing the ldhA promoter (PldhA) and E. coli ribosomal RNA transcription terminator (TrrnB) of ATCC13032 strain, respectively. Furthermore, pGE619 was treated with BamHI and EcoRV, and extracted and purified after agarose gel electrophoresis. These three DNA fragments were mixed and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformant strain was selected by culturing on an LB agar medium containing 100 μg/ml spectinomycin. This transformed strain was cultured in LB medium containing 100 μg/ml spectinomycin, and plasmid extraction was performed using the resulting culture suspension. The plasmid thus obtained was named pGE945-2.

2-3-(3) Construction of plasmid for Pp_mdlC overexpression Pseudomonas putida NBRC14164 strain was obtained from the Biotechnology Center of the National Institute of Technology and Evaluation, an independent administrative agency, and after culturing according to the attached instructions, genomic DNA was extracted. went. Using the genomic DNA of the NBRC14164 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequences shown by GEP2550 (SEQ ID NO: 110) and GEP2551 (SEQ ID NO: 111), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the mdlC gene (Pp_mdlC) of Pseudomonas putida NBRC14164 strain. Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1161 in which Pp_mdlC was cloned into pET-15b was obtained by the same operation as in the construction of pGE1031.

A PCR product was obtained by PCR using pGE1161 as a template and a combination of DNA with the base sequence shown by GEP2653 (SEQ ID NO: 112) and GEP2654 (SEQ ID NO: 113), which was extracted after agarose gel electrophoresis. , purification was performed. Furthermore, pGE409 was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1197 in which Pp_mdlC was subcloned into pGE409 was obtained by the same operation as in the construction of pGE1185.

2-3-(4) Construction of Kp_dhaT overexpression plasmid The plasmid pHA12-dhaBT containing the gene derived from Klebsiella pneumoniae (DDBJ accession number: LC412902) was kindly provided by Dr. Takahisa Tajima of Hiroshima University. Using this as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2529 (SEQ ID NO: 114) and GEP2530 (SEQ ID NO: 115), and this was extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing the Klebsiella pneumoniae dhaT gene (Kp_dhaT). Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1150 in which Kp_dhaT was cloned into pET-15b was obtained by the same operation as in the construction of pGE1161.

pGE1150 was treated with NdeI and BamHI, and a 1.2 kb DNA fragment was extracted and purified after agarose gel electrophoresis. Further, pGE945-2 was treated with NdeI and BamHI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and Ligation high Ver. 2 (manufactured by Toyobo Co., Ltd.) according to the instructions to perform a binding reaction of DNA fragments. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformant strain was selected by culturing on an LB agar medium containing 100 μg/ml spectinomycin. This transformed strain was cultured in LB medium containing 100 μg/ml spectinomycin, and plasmid extraction was performed using the resulting culture suspension. In this way, plasmid pGE1190 in which Kp_dhaT was subcloned into pGE945-2 was obtained.

2-3-(5) Construction of plasmid for overexpression of Ll_ldhA Q85C Lactococcus lactis NBRC100676 strain was obtained from the Biotechnology Center of the National Institute of Technology and Evaluation, an independent administrative agency, and after culturing according to the attached instructions, the genomic DNA was Extraction was performed. Using the genomic DNA of the NBRC100676 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2799 (SEQ ID NO: 116) and GEP2800 (SEQ ID NO: 117), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the ldhA gene (Ll_ldhA) of Lactococcus lactis NBRC100676 strain. Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1274 in which Ll_ldhA was cloned into pET-15b was obtained by the same operation as in the construction of pGE1031.

For the purpose of introducing mutations into lactate dehydrogenase derived from Lactococcus lactis strain NBRC100676, PrimeSTAR (registered trademark) Mutagenesis and plasmid amplification were performed using the Mutagenesis Basal Kit (manufactured by Takara Bio Inc.) according to its instructions, and plasmid pGE1275 having the sequence of the Ll_ldhA mutant gene Ll_ldhA Q85C was obtained.

Using pGE1275 as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2801 (SEQ ID NO: 120) and GEP2802 (SEQ ID NO: 121), and this was extracted after agarose gel electrophoresis. , purification was performed. Further, pGE945-2 was treated with NdeI and BamHI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed, and a DNA fragment binding reaction was performed using In-Fusion (registered trademark) HD Cloning Kit according to its instructions. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformant strain was selected by culturing on an LB agar medium containing 100 μg/ml spectinomycin. This transformed strain was cultured in LB medium containing 100 μg/ml spectinomycin, and plasmid extraction was performed using the resulting culture suspension. In this way, plasmid pGE1286 in which Ll_ldhA Q85C was subcloned into pGE945-2 was obtained.

2-3-(6) Construction of plasmid for Ls_leudh overexpression Ricinibacillus sphaericus NBRC3525 strain was obtained from the Biotechnology Center of the National Institute of Technology and Evaluation, and was cultured according to the attached instructions, followed by genomic DNA extraction. went. Using the genomic DNA of the NBRC3525 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2525 (SEQ ID NO: 122) and GEP2526 (SEQ ID NO: 123), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the leudh gene (Ls_leudh) of Lysinibacillus sphaericus NBRC3525 strain. Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1148 in which Ls_leudh was cloned into pET-15b was obtained by the same operation as in the construction of pGE1031.

A PCR product was obtained by PCR using pGE1148 as a template and a combination of DNA with the base sequence shown by GEP2655 (SEQ ID NO: 124) and GEP2650 (SEQ ID NO: 125), which was extracted after agarose gel electrophoresis. , purification was performed. Further, pGE945-2 was treated with NdeI and BamHI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1203 in which Ls_leudh was subcloned into pGE945-2 was obtained by the same operation as in the construction of pGE1286.

2-3-(7) Construction of plasmid for overexpression of Sf_valdh Streptomyces Frazie ATCC19609 strain was obtained from American Type Culture Collection, and after culturing according to the attached instructions, genomic DNA was extracted. Using the genomic DNA of ATCC19609 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2600 (SEQ ID NO: 126) and GEP2601 (SEQ ID NO: 127), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the Streptomyces Frazie ATCC19609 valdh gene (Sf_valdh). Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed, and plasmid pGE1175 in which Sf_valdh was cloned into pET-15b was obtained by the same operation as in the construction of pGE1031.

2-3-(8) Construction of plasmid for Bb_phedh overexpression Bacillus badius strain NBRC15713 was obtained from the National Institute of Technology and Evaluation (NIT) Biotechnology Center, and after culturing according to the attached instructions, genomic DNA was extracted. went. Using the genomic DNA of strain NBRC15713 as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequences shown by GEP2678 (SEQ ID NO: 128) and GEP2679 (SEQ ID NO: 129), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the phedh gene (Bb_phedh) of Bacillus badius strain NBRC15713. Furthermore, pET-15b (manufactured by Novagen) was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE1237 in which Bb_phedh was cloned into pET-15b was obtained by the same operation as in the construction of pGE1031.

2-3-(9) Construction of AtCGS1 overexpression plasmid pMK-AtCGS was purchased from Thermo Fisher. pMK-AtCGS is an amino acid sequence encoded by the Arabidopsis CGS1 gene (Biochem. J. 331, 639-648 (1998), DDBJ accession number: U43709), and the sequence starting from the 69th amino acid in Corynebacterium glutamicum. This is a plasmid containing synthetic DNA optimized for expression. Using this as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP1053 (SEQ ID NO: 130) and GEP1054 (SEQ ID NO: 131), and this was extracted after agarose gel electrophoresis. , purification was performed. This is a DNA fragment containing the Arabidopsis CGS1 gene (AtCGS1) that has been optimized for expression in Corynebacterium glutamicum. Furthermore, pGE411 was treated with NdeI, subjected to agarose gel electrophoresis, and then extracted and purified. These two DNA fragments were mixed and a plasmid pGE479 in which AtCGS1 was subcloned into pGE411 was obtained by the same operation as in the construction of pGE1185.

pGE479 was treated with NdeI and BamHI, and a 1.5 kb DNA fragment was extracted and purified after agarose gel electrophoresis. Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed and Ligation high Ver. 2 (manufactured by Toyobo Co., Ltd.) according to the instructions to perform a binding reaction of DNA fragments. Using the reaction product, E. E. coli DH5α Competent Cells (manufactured by Takara Bio) were transformed according to the instructions. The desired transformed strain was selected by culturing on an LB agar medium containing 50 μg/ml kanamycin. This transformed strain was cultured in LB medium containing 50 μg/ml kanamycin, and plasmid extraction was performed using the resulting culture suspension. In this way, plasmid pGE513 in which AtCGS1 was subcloned into pGE409 was obtained.

2-3-(10) Construction of plasmids for overexpressing various aldolase enzymes Using the genomic DNA of the DH5α strain as a template, the DNA combination of the base sequences shown by GEP2712 (SEQ ID NO: 132) and GEP2713 (SEQ ID NO: 133), GEP2714 ( A combination of DNA with the nucleotide sequence shown by SEQ ID NO: 134) and GEP2715 (SEQ ID NO: 135), a combination of DNA with the nucleotide sequence shown by GEP2720 (SEQ ID NO: 136) and GEP2721 (SEQ ID NO: 137), GEP2726 (SEQ ID NO: 138) A PCR product was obtained by the PCR method using a combination of DNA with the base sequence shown as These were extracted and purified after agarose gel electrophoresis. These are DNA fragments containing the E. coli DH5α strain eda gene (Ec_eda), yjjJ gene (Ec_yjhH), dgoA gene (Ec_dgoA), mhpFE gene (Ec_mhpFE), and garL gene (Ec_garL), respectively. Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. Plasmids pGE1223, pGE1224, pGE1225, pGE409 in which Ec_eda, Ec_yjhH, Ec_dgoA, Ec_mhpFE, and Ec_garL were cloned into pGE409 by mixing DNA fragments having various genes and a DNA fragment derived from pGE409 and performing the same operations as when creating pGE1185. GE1227, pGE1259 was obtained.

Using the genomic DNA of Pseudomonas putida strain NBRC14164 as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2769 (SEQ ID NO: 142) and GEP2770 (SEQ ID NO: 143), and these were transferred to an agarose gel. After electrophoresis, extraction and purification were performed. This is a DNA fragment containing the galC gene (Pp_galC) of Pseudomonas putida NBRC14164 strain. Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed and a plasmid pGE1260 in which Pp_galC was cloned into pGE409 was obtained by the same operation as in the production of pGE1185.

Nocardioides NBRC109351 strain was obtained from the Biotechnology Center of the National Institute of Technology and Evaluation, an independent administrative institution, and after culturing according to the attached instructions, genomic DNA was extracted. Using the genomic DNA of the NBRC109351 strain as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequences shown by GEP2716 (SEQ ID NO: 144) and GEP2717 (SEQ ID NO: 145), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the phdJ gene (Ns_phdJ) of Nocardioides NBRC109351 strain. Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed and a plasmid pGE1239 in which Ns_phdJ was cloned into pGE409 was obtained by the same operation as in the construction of pGE1185.

Burkholderia xenovorans strain DSMZ17367 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, and after culturing according to the attached instructions, genomic DNA was extracted. Using the genomic DNA of strain DSMZ17367 as a template, a PCR product was obtained by PCR using a combination of DNA with the base sequence shown by GEP2771 (SEQ ID NO: 146) and GEP2772 (SEQ ID NO: 147), and this was subjected to agarose gel electrophoresis. After that, extraction and purification were performed. This is a DNA fragment containing the Burkholderia xenovorans strain DSMZ17367 bphJI gene (Bx_bphJI). Furthermore, pGE409 was treated with NdeI and BamHI, and subjected to agarose gel electrophoresis, followed by extraction and purification. These two DNA fragments were mixed and a plasmid pGE1262 in which Bx_bphJI was cloned into pGE409 was obtained by the same operation as in the construction of pGE1185.

3. Bacterial cell construction (3-1) Creation of strain introduced with Pp_mdlC and Kp_dhaT overexpression plasmids GES1070 was transformed by electroporation using pGE1197 and pGE1190, and 25 μg/ml kanamycin and 100 μg/ml spectinomycin were added at 33°C. Kanamycin spectinomycin resistant strains were selected on agar medium containing Kanamycin spectinomycin. This was named GES1206.

GES1063 was transformed by electroporation using pGE1197 and pGE1190, and Kanamycin spectinomycin resistant strains were selected on A agar medium containing 25 μg/ml kanamycin and 100 μg/ml spectinomycin at 33°C. This was named GES1077.

GES1064 was transformed by electroporation using pGE1197 and pGE1190, and Kanamycin spectinomycin resistant strains were selected on A agar medium containing 25 μg/ml kanamycin and 100 μg/ml spectinomycin at 33°C. This was named GES1068.

Corynebacterium glutamicum GES1068 was deposited at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818). Date: September 13, 2018, Accession number: NITE BP-02780)
GES1223 was transformed by electroporation using pGE1197 and pGE1190, and Kanamycin spectinomycin resistant strains were selected on A agar medium containing 25 μg/ml kanamycin and 100 μg/ml spectinomycin at 33°C. This was named GES1253.

GES1233 was transformed by electroporation using pGE1197 and pGE1190, and kanamycin spectinomycin resistant strains were selected on A agar medium containing 25 μg/ml kanamycin and 100 μg/ml spectinomycin at 33°C. This was named GES1254.

Corynebacterium glutamicum GES1254 was deposited at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818). Date: September 13, 2018, Accession number: NITE BP-02781)
GES1242 was transformed by electroporation using pGE1197 and pGE1190, and Kanamycin spectinomycin resistant strains were selected on A agar medium containing 25 μg/ml kanamycin and 100 μg/ml spectinomycin at 33°C. This was named GES1282.

(3-2) Creation of strain introduced with Ll_ldhA Q85C overexpression plasmid GES1064 was transformed by electroporation using pGE1286, and spectinomycete-resistant strains were selected on A agar medium containing 100 μg/ml spectinomycetes at 33°C. . This was named GES1232.

Corynebacterium glutamicum GES1232 was deposited at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818). Date: September 13, 2018, Accession number: NITE BP-02782)
GES1211 was transformed using pGE1286 by electroporation, and spectinomycin-resistant strains were selected on A agar medium containing 100 μg/ml spectinomycin at 33°C. This was named GES1294.

(3-3) Creation of strain introduced with Ls_leudh overexpression plasmid GES1064 was transformed by electroporation using pGE1203, and a spectinomycete-resistant strain was selected on medium A containing 100 μg/ml spectinomycetes at 33°C. This was named GES1073.

Corynebacterium glutamicum GES1073 was deposited at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818). Date: September 13, 2018, Accession number: NITE BP-02783)
pGE1203 was used to transform GES1211 by electroporation, and spectinomycin-resistant strains were selected on A medium containing 100 μg/ml spectinomycin at 33°C. This was named GES1295.

(3-4) Creation of Ls_leudh and AtCGS1 expression plasmid-introduced strain GES1064 was transformed by electroporation using pGE1203 and pGE513, and A agar medium containing 25 μg/ml kanamycin and 100 μg/ml spectinomycin was incubated at 33°C. Kanamycin spectinomycin resistant strains were selected above. This was named GES1097.

Corynebacterium glutamicum GES1097 was deposited at the Patent Microorganism Depositary, National Institute of Technology and Evaluation, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan (zip code 292-0818). Date: September 13, 2018, Accession number: NITE BP-02784)

(3-5) Creation of various aldolase expression plasmid introduced strains Transform GES435 by electroporation using pGE1143, pGE1223, pGE1224, pGE1225, pGE1226, pGE1227, pGE1239, pGE1258, pGE1259, pGE1260, and pGE1262, respectively. 33℃ Kanamycin-resistant strains were selected on A agar medium containing 25 μg/ml kanamycin. These were named GES1009, GES1119, GES1120, GES1121, GES1122, GES1123, GES1127, GES1154, GES1155, GES1156, and GES1157, respectively.

4. Substance production experiment (4-1) Production of 4-hydroxy-2-oxobutyric acid (HOB) 4-1-(1) Cultivation of GES1077 Glycerol stock of GES1077 was added to A containing 25 μg/mL of kanamycin and 100 μg/mL of spectinomycin. It was inoculated onto an agar medium under aseptic conditions and cultured at 33°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of BA medium containing 25 μg/mL of kanamycin and 100 μg/mL of spectinomycin, and cultured with shaking at 33° C. to obtain a seed culture. This seed culture was inoculated into 500 mL of BA medium containing 25 μg/mL of kanamycin and 100 μg/mL of spectinomycin prepared in a 2 L flask so that the turbidity was 0.1. The cells were shaken and cultured overnight at 33°C. This main culture solution was centrifuged at 4° C. and 5500×g for 15 minutes, and the supernatant was removed. The wet bacterial cells thus obtained were used in the following reactions.

4-1-(2) Production of HOB using GES1077 The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution and a 1.2M formaldehyde aqueous solution were added to this bacterial cell suspension so that the concentrations were 50mM and 40mM, respectively, and the pH was adjusted to 6.0 using a 1.0M potassium hydroxide aqueous solution. Culture was performed at 33°C while maintaining the temperature. At this wet bacterial cell concentration, anaerobic conditions or microaerobic conditions will naturally be achieved if agitation is suppressed to the extent that the aqueous potassium hydroxide solution is efficiently mixed and aeration is not performed. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and it was found that 0.65 mM of HOB had been produced.

4-1-(3) Comparative example 1
The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution was added to this cell suspension to give a concentration of 50 mM, and the reaction was carried out at 33°C while maintaining the pH at 6.0 using a 1.0 M potassium hydroxide aqueous solution. went. After 3 hours, the supernatant of the reaction suspension was analyzed by liquid chromatography for organic acid analysis, and no HOB was detected.

4-1-(4) Comparative example 2
Regarding GES1206, experiments similar to those in 4-1-(1) and 4-1-(2) above were conducted. After 3 hours of reaction, the supernatant of the reaction suspension was analyzed by liquid chromatography for organic acid analysis, and no HOB was detected.

From the results of 4-1-(2), 4-1-(3), and 4-1-(4), it is clear that bacterial cells with a gene encoding class 2 aldolase, such as GES1077, do not react well in the presence of sugars and aldehydes. It was found that by culturing, pyruvate is produced from sugars, and the enzyme catalyzes the aldol reaction between this and aldehyde, producing aldol compounds such as HOB.

4-1-(5) Cultivation of GES1282 Wet bacterial cells obtained by culturing GES1282 glycerol stock in the same manner as in 4-1-(1) were used in the following reactions.

4-1-(6) Production of HOB using GES1282 The wet bacterial cells prepared as described above were cultured in the same manner as in 4-1-(2). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and it was found that 0.36 mM of HOB had been produced.

4-1-(7) Comparative example 1
The wet bacterial cells prepared as described above were cultured in the same manner as in 4-1-(3). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction suspension was analyzed by liquid chromatography for organic acid analysis, and no HOB was detected.

From the results of 4-1-(6), 4-1-(7), and 4-1-(4), it is clear that bacterial cells with a gene encoding a class 2 aldolase, which is different from GES1077, such as GES1282, also produce sugars and It was found that culturing in the presence of aldehyde produces pyruvic acid from sugars, and that the enzyme catalyzes the aldol reaction between this and aldehyde, producing aldol compounds such as HOB. .

4-1-(8) Cultivation of GES1253 Wet bacterial cells obtained by culturing GES1253 glycerol stock in the same manner as in 4-1-(1) were used in the following reactions.

4-1-(9) Production of HOB using GES1253 The wet bacterial cells prepared as described above were cultured in the same manner as in 4-1-(2). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and it was found that 0.34 mM of HOB was produced.

4-1-(10) Comparative example 1
The wet bacterial cells prepared as described above were cultured in the same manner as in 4-1-(2). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction suspension was analyzed by liquid chromatography for organic acid analysis, and no HOB was detected.

From the results of 4-1-(9), 4-1-(10), and 4-1-(4), it is clear that bacterial cells with genes encoding class 1 aldolases such as GES1253 do not react well in the presence of sugars and aldehydes. It was found that by culturing, pyruvate is produced from sugars, and the enzyme catalyzes the aldol reaction between this and aldehyde, producing aldol compounds such as HOB.

4-1-(11) Cultivation of GES1068 and GES1254 Glycerol stocks of GES1068 and GES1254 were cultured in the same manner as in 4-1-(1), and the resulting wet bacterial cells were used in the following reactions.

4-1-(12) Production of HOB using GES1068 and GES1254 The wet bacterial cells prepared as described above were cultured in the same manner as in 4-1-(2). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and it was found that HOB was produced at 1.2 mM in the reaction using GES1068 and 0.42 mM in the reaction using GES1254.

From the results of 4-1-(12), even if the microorganism has a genotype different from GES1077 and GES1253, such as GES1068 and GES1254, if it has a gene encoding class 2 aldolase, it can produce sugars and aldehydes. It was found that by culturing in the presence of pyruvic acid, pyruvic acid is produced from sugars, and the enzyme catalyzes the aldol reaction between this and aldehyde, thereby producing aldol compounds such as HOB.

(4-2) Production of 1,3-propanediol (PDO) 4-2-(1) Cultivation of GES1077, GES1068 Glycerol stock of GES1077, GES1068 was transferred to A agar containing 25 μg/mL of kanamycin and 100 μg/mL of spectinomycin. The culture medium was inoculated under sterile conditions and cultured at 33°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of BA medium containing 25 μg/mL of kanamycin and 100 μg/mL of spectinomycin, and cultured with shaking at 33° C. to obtain a seed culture. This seed culture was inoculated into 500 mL of BA medium containing 25 μg/mL of kanamycin and 100 μg/mL of spectinomycin prepared in a 2 L flask so that the turbidity was 0.1. The cells were shaken and cultured overnight at 33°C. This main culture solution was centrifuged at 4° C. and 5500×g for 15 minutes, and the supernatant was removed. The wet bacterial cells thus obtained were used in the following reactions.

4-2-(2) Production of PDO using GES1077 and GES1068 The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution and a 1.2M formaldehyde aqueous solution were added to this bacterial cell suspension so that the concentrations were 50mM and 40mM, respectively, and the pH was adjusted to 6.0 using a 1.0M potassium hydroxide aqueous solution. Culture was performed at 33°C while maintaining the temperature. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for sugar analysis, and it was found that PDO was produced at 7.4 mM in the reaction using GES1077 and 11.4 mM in the reaction using GES1068.

From the above results, if a bacterial cell that has genes encoding class 2 aldolases such as GES1077 and GES1068 and can produce HOB when cultured in the presence of sugars and formaldehyde also has genes encoding decarboxylase and alcohol dehydrogenase. , it was found that HOB could be further converted to PDO to produce PDO.

4-2-(3) Cultivation of GES1282 Wet bacterial cells obtained by culturing GES1282 glycerol stock in the same manner as in 4-2-(1) were used in the following reactions.

4-2-(4) Production of PDO using GES1282 The wet bacterial cells prepared as described above were cultured in the same manner as in 4-2-(2). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 6 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for sugar analysis, and it was found that 11.4 mM of PDO had been produced.

From the above results, bacterial cells such as GES1282 that have a gene encoding a class 2 aldolase different from GES1077 and GES1068 and can produce HOB when cultured in the presence of sugars and formaldehyde also encode decarboxylase and alcohol dehydrogenase. It was found that when the gene is present, HOB can be further converted to PDO and PDO can be manufactured.

4-2-(5) Cultivation of GES1253 and GES1254 Wet cells obtained by culturing GES1254 glycerol stock in the same manner as in 4-2-(1) were used in the following reactions.

4-2-(6) Production of PDO using GES1253 and GES1254 The wet bacterial cells prepared as described above were cultured in the same manner as in 4-2-(2). During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 6 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for sugar analysis, and it was found that PDO was produced at 4.1 mM in the reaction using GES1253 and 5.8 mM in the reaction using GES1254.

From the above results, if a bacterial cell that has genes encoding class 1 aldolases such as GES1253 and GES1254 and can produce HOB when cultured in the presence of sugars and formaldehyde also has genes encoding decarboxylase and alcohol dehydrogenase. , it was found that HOB could be further converted to PDO to produce PDO.

(4-3) Production of 1,3-butanediol (BDO) 4-3-(1) Cultivation of GES1068 Glycerol stock of GES1068 was cultured in the same manner as in 4-2-(1). It was used in the following reaction.

4-3-(2) Production of BDO using GES1068 The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution and a 1.2M acetaldehyde aqueous solution were added to this bacterial cell suspension so that the concentrations were 50mM and 40mM, respectively, and the pH was adjusted to 6.0 using a 1.0M potassium hydroxide aqueous solution. Culture was performed at 33°C while maintaining the temperature. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for sugar analysis, and it was found that 8.2 mM of BDO had been produced.

From the above results, it was found that BDO can be produced by culturing a bacterial cell having genes encoding aldolase, decarboxylase, and alcohol dehydrogenase such as GES1068 in the presence of sugars and acetaldehyde.

In addition, in 4-2-(2), if the reaction is continued even after the reaction time exceeds 3 hours and the formaldehyde concentration decreases, after 22 hours BDO will be 3.5mM in the reaction using GES1077 and 3.5mM in the reaction using GES1068. In the previous reaction, 2.7mM was produced. This is because when formaldehyde is reduced to a concentration that cannot participate in the aldolase reaction, acetaldehyde is generated by the catalytic action of mdlC (decarboxylase) on pyruvate, which then undergoes an aldol reaction with another molecule of pyruvate. -hydroxy-2-oxovalerate (HOV) is produced, which is further decarboxylated and reduced by the catalytic action of mdlC and dhaT, thereby producing BDO. Therefore, a bacterial cell that has a gene encoding aldolase and also genes encoding decarboxylase and alcohol dehydrogenase can be cultured using only sugars as a carbon source to produce BDO. In fact, when GES1077 was cultured under the conditions described in 4-2-(2) without the addition of formaldehyde, 0.60 mM of BDO was produced after 22 hours.

(4-4) Production of various 1,3-diols 4-4-(1) Cultivation of GES1068 GES1068 glycerol stock was cultured in the same manner as in 4-2-(1), and the resulting wet bacterial cells were subjected to the following reaction. It was used for.

4-4-(2) Production of various 1,3-diols using GES1068 The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution and aldehydes (propionaldehyde, n-butyraldehyde, isobutyraldehyde, n-valeraldehyde) were added to this bacterial cell suspension to give a concentration of 50mM and 40mM, respectively, and 1.0M hydroxyl Culture was performed at 33° C. while maintaining the pH at 6.0 using an aqueous potassium solution. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by GCMS, and it was found that propionaldehyde, n-butyraldehyde, isobutyraldehyde, and 1,3-pentanediol, 1,3-hexanediol, and -Methyl-1,3-pentanediol and 1,3-heptanediol were confirmed. FIG. 6 shows the results of gas chromatography mass spectrometry using propionaldehyde. FIG. 7 shows the results of gas chromatography mass spectrometry in the case of using n-butyraldehyde. FIG. 8 shows the results of gas chromatography mass spectrometry in the case of using isobutyraldehyde. FIG. 8 shows the results of gas chromatography mass spectrometry using normal valeraldehyde.

From the above results, it is possible to culture bacteria having genes encoding aldolase, decarboxylase, and alcohol dehydrogenase such as GES1068 in the presence of sugars and propionaldehyde, normal butyraldehyde, isobutyraldehyde, or normal valeraldehyde. It was found that 1,3-pentanediol, 1,3-hexanediol, 4-methyl-1,3-pentanediol, and 1,3-heptanediol can be produced using this method.

In addition, generally, by culturing a bacterial cell having genes encoding aldolase, decarboxylase, and alcohol dehydrogenase such as GES1068 in the presence of sugars and aldehydes, pyruvic acid is produced from sugars, and this and aldehyde 4-hydroxy-2-oxocarboxylic acid, which is an aldol compound, is produced from the above reaction, which is converted to a 3-hydroxyaldehyde compound, and then to 1,3-diol, and finally 1,3-diol is produced. It is hoped that this will be possible.

(4-5) Production of 2,4-dihydroxybutyric acid (DHB) 4-5-(1) Cultivation of GES1232 Glycerol stock of GES1232 was inoculated onto A agar medium containing 100 μg/mL of spectinomycin under aseptic conditions. Cultured at 33°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of BA medium containing 100 μg/mL of spectinomycin, and cultured with shaking at 33° C. to obtain a seed culture. This seed culture was inoculated into 500 mL of BA medium containing 100 μg/mL of spectinomycin prepared in a 2 L flask so that the turbidity was 0.1. The cells were shaken and cultured overnight at 33°C. This main culture solution was centrifuged at 4° C. and 5500×g for 15 minutes, and the supernatant was removed. The wet bacterial cells thus obtained were used in the following reactions.

4-5-(2) Production of DHB using GES1232 The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution and a 1.2M formaldehyde aqueous solution were added to this bacterial cell suspension so that the concentrations were 100mM and 40mM, respectively, and the pH was adjusted to 7.0 using a 1.0M potassium hydroxide aqueous solution. Culture was performed at 33°C while maintaining the temperature. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerophilic conditions are achieved at the moist bacterial cell concentration. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and it was found that 11 mM of DHB had been produced.

4-5-(3) Comparative example 1
The wet bacterial cells prepared as described above were suspended in BR buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution was added to this cell suspension to give a concentration of 100 mM, and the reaction was carried out at 33°C while maintaining the pH at 7.0 using a 1.0 M potassium hydroxide aqueous solution. went. After 3 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and no DHB was detected.

4-5-(4) Comparative example 2
Regarding GES1294, experiments similar to those in (1) and (2) above were conducted. After 3 hours of reaction, the supernatant of the reaction suspension was analyzed by liquid chromatography for organic acid analysis, and no DHB was detected.

From the above results, if a bacterial cell that has a gene encoding an aldolase like GES1232 and can produce HOB when cultured in the presence of sugars and formaldehyde also has a gene encoding a dehydrogenase with HOB reduction activity, It was found that by culturing in the presence of sugars and formaldehyde, HOB was further converted to DHB, and DHB could be produced.

(4-6) Confirmation of L-homoserine N-dehydrogenase activity 4-6-(1) Measurement of expression of leucine dehydrogenase and oxidative deamination reaction activity Using pGE1148, ECOSTM Competent E. E. coli BL21 (DE3) (manufactured by Nippon Gene) was transformed according to the instructions, and the resulting strain was named GES1007. GES1007 was inoculated onto an LB agar medium containing 100 μg/mL ampicillin under aseptic conditions and cultured at 37°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of LB medium containing 100 μg/mL of ampicillin, and cultured with shaking at 37° C. to obtain a seed culture. This seed culture was inoculated into 100 mL of LB medium containing 100 μg/mL of ampicillin prepared in a 500 mL flask so that the turbidity was 0.1. The mixture was shaken at 37°C and cultured until the turbidity reached 0.7. IPTG was added to a final concentration of 0.2mM, and cultured with shaking at 16°C overnight. This main culture solution was centrifuged at 4° C. and 6000×g for 5 minutes, and the supernatant was removed. The wet bacterial cells thus obtained were frozen once at -80°C and then thawed at room temperature. Suspend in 4.5 mL of 50 mM sodium phosphate buffer (pH 8.0), 500 mM sodium chloride, 10% glycerol, add 2.0 g of glass beads YGB01 Φ0.1 mm (manufactured by Yasui Kikai Co., Ltd.), and mix the multi beads. The bacterial cells were disrupted using Shocker MB1001C (S) (manufactured by Yasui Kikai Co., Ltd.) at 2500 rpm and 4°C. Centrifugation was performed at 18,000×g and 4° C. for 3 minutes, and the supernatant was collected. The protein concentration of this crushed solution was measured and found to be 12 mg/mL.

Using this cell suspension, the oxidative deamination reaction activity against L-homoserine was measured. The reaction conditions were as follows: An appropriately diluted bacterial cell suspension was mixed with 100mM glycine-KCl-KOH (pH 10.0), 2.5mM NAD ⁺ , and 50mM L-homoserine. It was measured using LUX (manufactured by Thermo Fisher). As a result, the specific activity of this bacterial cell lysate against L-homoserine was 0.21 U/mg protein.

4-6-(2) Measurement of expression of valine dehydrogenase and oxidative deamination reaction activity Using pGE1175, ECOSTM Competent E. E. coli BL21 (DE3) (manufactured by Nippon Gene) was transformed according to the instructions, and the resulting strain was named GES1057. Regarding GES1057, the protein concentration of the crushed liquid obtained by the same operation as GES1007 was measured and found to be 4.2 mg/mL.

Using this cell suspension, the oxidative deamination reaction activity against L-homoserine was measured in the same manner as in 4-6-(1). As a result, the specific activity of this cell suspension against L-homoserine was 0. It was 21U/mg protein.

4-6-(3) Measurement of expression of phenylalanine dehydrogenase and oxidative deamination reaction activity Using pGE1237, ECOSTM Competent E. E. coli BL21 (DE3) (manufactured by Nippon Gene) was transformed according to the instructions, and the resulting strain was named GES1136. Regarding GES1136, the protein concentration of the crushed solution obtained by the same operation as GES1007 was measured and found to be 11 mg/mL.

Using this cell suspension, the oxidative deamination reaction activity against L-homoserine was measured in the same manner as in 4-6-(1). As a result, the specific activity of this cell suspension against L-homoserine was 0. It was 045 U/mg protein.

From the results of 4-6-(1), 4-6-(2), and 4-6-(3) above, leucine dehydrogenase, valine dehydrogenase, and phenylalanine dehydrogenase all have the activity of oxidizing L-homoserine to HOB. I found out that it was. Generally, amino acid dehydrogenase catalyzes both oxidative deamination reactions and reductive amination reactions, and the activity strengths of these reactions are correlated. (For example, Biochim. Biophys. Acta 96, 248-262 (1965), J. Biol. Chem. 253, 5719-5725 (1978), Eur. J. Biochem. 100, 29-39 (1979)). Therefore, leucine dehydrogenase, valine dehydrogenase, and phenylalanine dehydrogenase are all expected to catalyze the reductive amination reaction of HOB to L-homoserine. For confirmation, leucine dehydrogenase was purified and enzyme kinetic analysis was performed on the reductive amination reaction of HOB.

4-6-(4) Purification of leucine dehydrogenase and measurement of reductive amination activity A filtration process was performed. Using the AKTA purifier system (manufactured by GE Healthcare) connected to a HisTrap ^TM FF 1mL column, 50mM sodium phosphate buffer (pH 8.0), 500mM sodium chloride, and 10% glycerol were used as adsorption buffers. Buffer (pH 8.0), 500mM sodium chloride, 10% glycerol, 500mM imidazole was used as an elution buffer for fractionation. Fractions containing leucine dehydrogenase confirmed by SDS-PAGE were collected, concentrated using ultrafiltration, and then treated with 50 mM potassium phosphate (pH 7.4) using a PD-10 column (manufactured by GE Healthcare). The buffer was replaced with 100 mM sodium chloride, 1 mM EDTA, and 10% glycerol, and concentrated again using ultrafiltration to obtain a 5.9 mg/mL leucine dehydrogenase aqueous solution.

Using the purified leucine dehydrogenase thus obtained, enzyme reaction kinetic analysis was performed based on the Michaelis-Menten equation using HOB as a substrate. The reaction conditions were 100mM HEPES/KOH (pH 7.5), 200mM ammonium chloride, 0.2mM NADH, 3.7μg/mL leucine dehydrogenase, and HOB concentrations were 3.13, 6.25, 12.5, and 25.0. , 50.0, and 100 mM were measured. The reaction rate was calculated by measuring the continuous decrease in absorption by NADH at 340 nm using Varioskan LUX (manufactured by Thermo Fisher). The enzyme reaction kinetic constants were calculated by plotting the reaction rate at each concentration and fitting to the Michaelis-Menten equation.As a result, the Michaelis constant for HOB was _7.7mM , and the catalytic rotation speed _kcat was 2.7sec. ⁻¹ , and therefore the specificity constant k _cat /K _M was 0.35 mM ⁻¹ sec ⁻¹ .

From this result, it was found that leucine dehydrogenase exhibits catalytic activity for the reductive amination reaction of HOB as expected. Furthermore, from this result, it is considered that valine dehydrogenase and phenylalanine dehydrogenase also exhibit catalytic activity for reductive amination reactions.

(4-7) Production of amino acids 4-7-(1) Cultivation of GES1073 Wet bacterial cells obtained by culturing GES1073 glycerol stock in the same manner as in 4-5-(1) were used in the following reactions.

4-7-(2) Production of amino acids using GES1073 The wet bacterial cells prepared as described above were suspended in BT buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution and a 2.0M formaldehyde aqueous solution were added to this bacterial cell suspension to give a concentration of 2% (w/v) and 100mM, respectively, and the pH was adjusted using a 5.0M ammonia aqueous solution. Culture was performed at 33° C. while maintaining the temperature at 7.0. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 6 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for amino acid analysis, and it was found that 15.7mM of L-homoserine, 1.3mM of L-threonine, and 0.15mM of 2-aminobutyric acid were produced.

4-7-(3) Comparative example 1
The wet bacterial cells prepared as described above were suspended in BT buffer at a concentration of 10% (w/v). A 50% (w/v) glucose aqueous solution was added to this bacterial suspension to give a concentration of 2% (w/v), and the pH was maintained at 7.0 using a 5.0M ammonia aqueous solution at 33°C. The reaction was carried out at After 6 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for amino acid analysis, and L-homoserine, L-threonine, and 2-aminobutyric acid were all below the limit of quantification.

4-7-(4) Comparative example 2
Regarding GES1295, experiments similar to those in 4-7-(1) and 4-7-(2) above were conducted. After 6 hours of reaction, the supernatant of the reaction suspension was analyzed by liquid chromatography for amino acid analysis, and L-homoserine, L-threonine, and 2-aminobutyric acid were all below the limit of quantification.

From the above results, it is clear that a bacterial cell that has a gene encoding an aldolase such as GES1073 and can produce HOB when cultured in the presence of sugars and formaldehyde also has a gene encoding a dehydrogenase that has the activity of reductively aminating HOB. It was found that when HOB is present, by culturing it in the presence of sugars and formaldehyde, HOB can be further converted to L-homoserine and L-homoserine can be produced. It was also found that L-homoserine can be produced because part of it is converted to L-threonine via O-phospho-L-homoserine (OPH) by an endogenous metabolic system. Furthermore, L-threonine is dehydrated by the endogenous metabolic system to become 2-oxobutyric acid, which is then converted to 2-aminobutyric acid by endogenous amino acid dehydrogenase or leucine dehydrogenase that GES1073 has specifically. It turns out that it can be manufactured.

(4-8) Production of L-methionine 4-8-(1) Cultivation of GES1097 The wet bacterial cells obtained by culturing the glycerol stock of GES1097 in the same manner as in 4-2-(1) were used in the following reactions. .

4-8-(2) Production of amino acids using GES1097 The wet bacterial cells prepared as described above were suspended in BT buffer at a concentration of 20% (w/v). A 50% (w/v) glucose aqueous solution, a 2.0 M formaldehyde aqueous solution, and a 0.2 M methyl mercaptan aqueous solution (15% methyl mercaptan sodium aqueous solution (manufactured by Tokyo Kasei Kogyo Co., Ltd.) were added to this bacterial cell suspension in BT buffer. (adjusted to pH 8.0 using concentrated hydrochloric acid) and further diluted with BT buffer to adjust the concentrations to 2% (w/v), 100 mM, and 50 mM, respectively. Further, at this time, the final wet bacterial cell concentration was set to 10% (w/v). Culture was performed at 33° C. while maintaining the pH at 7.0 using a 5.0 M ammonia aqueous solution. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 21 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for amino acid analysis, and it was found that 2.1 mM of L-methionine had been produced. Furthermore, 8.6 mM of L-homoserine, 3.0 mM of L-threonine, and 0.86 mM of 2-aminobutyric acid were produced.

4-8-(3) Comparative example 1
The wet bacterial cells prepared as described above were suspended in BT buffer at a concentration of 20% (w/v). A 50% (w/v) glucose aqueous solution and a 2.0M formaldehyde aqueous solution were added to this cell suspension, and further diluted with BT buffer to give a concentration of 2% (w/v) and 100mM, respectively. It was adjusted. Further, at this time, the final wet bacterial cell concentration was set to 10% (w/v). The reaction was carried out at 33° C. while maintaining the pH at 7.0 using a 5N ammonia aqueous solution. After 21 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for amino acid analysis, and L-methionine was found to be below the limit of quantification. On the other hand, 5.4mM of L-homoserine, 5.6mM of L-threonine, and 0.98mM of 2-aminobutyric acid were produced.

4-8-(4) Comparative example 2
Regarding GES1073, experiments similar to those in 4-8-(1) and 4-8-(2) above were conducted. After 21 hours of reaction, the supernatant of the reaction suspension was analyzed by liquid chromatography for amino acid analysis, and the production of L-methionine was below the limit of quantification. On the other hand, 12.8mM of L-homoserine, 1.6mM of L-threonine, and 0.40mM of 2-aminobutyric acid were produced.

From the above results, it was found that bacterial cells that have a gene encoding an aldolase such as GES1097 and a gene encoding a dehydrogenase that has the activity of reductively aminating HOB, and can produce L-homoserine when cultured in the presence of sugars and formaldehyde. Furthermore, if a gene encoding a synthase with the activity of combining OPH and methyl mercaptan is present, L-homoserine is converted to L-methionine via OPH by culturing in the presence of sugars, formaldehyde, and methyl mercaptan. It was found that L-methionine can be produced.

5. Production of HOB from pyruvate (5-1) Culture of GES1009, GES1119, GES1120, GES1121, GES1122, GES1123, GES1127, GES1154, GES1155, GES1156, GES1157 GES1009, GES1119, G ES1120, GES1121, GES1122, GES1123, GES1127, GES1154, Glycerol stocks of GES1155, GES1156, and GES1157 were inoculated onto A agar medium containing 25 μg/mL of kanamycin under aseptic conditions and cultured at 33°C. The bacterial cells grown on the agar medium were inoculated into 10 mL of BA medium containing 25 μg/mL of kanamycin, shaken at 33° C., and cultured overnight. This culture solution was centrifuged at 4° C. and 5500×g for 15 minutes, and the supernatant was removed. The wet bacterial cells thus obtained were used in the following reactions.

(5-2) Production of HOB from pyruvate using GES1009, GES1119, GES1120, GES1121, GES1122, GES1123, GES1127, GES1154, GES1155, GES1156, GES1157 Wet bacterial cells prepared as described above were added to 10% (w/ v) Suspended in BS buffer. A 1.0 M pyruvate aqueous solution and a 1.2 M formaldehyde aqueous solution were added to this bacterial cell suspension so that both were 40 mM, and cultured at 33°C. During the culture, stirring was performed to efficiently mix the potassium hydroxide aqueous solution, and no aeration was performed. In addition, by this operation, anaerobic conditions or microaerobic conditions are achieved at the moist bacterial cell concentration. After 6 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and it was found that HOB was not found in the reactions using GES1009, GES1119, GES1120, GES1121, GES1122, GES1123, GES1127, GES1154, GES1155, GES1156, and GES1157. , 3.6, 4.1, 2.1, 3.4, 30.5, 1.9, 18.3, 19.5, 6.5, 4.5, and 2.0 mM, respectively.
(5-3) Comparative example 1
The wet bacterial cells prepared as described above were suspended in BS buffer at a concentration of 10% (w/v). A 1.0 M pyruvate aqueous solution was added to this cell suspension to a concentration of 40 mM, and the reaction was carried out at 33°C. After 6 hours, the supernatant of the reaction solution was analyzed by liquid chromatography for organic acid analysis, and no HOB was detected in any case.

Based on the results above, there is a gene that corded aldolase like GES1009, GES1119, GES1120, GES1121, GES1122, GES1123, GES1127, GES1154, GES1155, GES1156, GES1156. Both bacteria are all between pilbic acid and formaldehyde. It was found that aldol compounds such as HOB are produced by the enzyme catalyzing the aldol reaction.

Reference materials

Claims (1)

The first gene selected from the group consisting of:
(1-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-2) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 2, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-3) A gene encoding a protein that is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 and that catalyzes the aldol reaction between pyruvic acid and a carbonyl compound,
(1-4) A protein consisting of an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4, which has the activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound. the gene that encodes
(1-5) a gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6, which catalyzes the aldol reaction between pyruvic acid and a carbonyl compound; A gene encoding a protein having an amino acid sequence having an identity of 90% or more to the amino acid sequence and having an activity of catalyzing the aldol reaction between pyruvic acid and a carbonyl compound;
A second gene selected from the group consisting of:
(2-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 24, which catalyzes the decarboxylation reaction of α-keto acids, and (2-2) the amino acid set forth in SEQ ID NO: 24. A gene encoding a protein consisting of an amino acid sequence having 90% or more identity to the sequence and having the activity of catalyzing the decarboxylation reaction of α-keto acids; and a gene selected from the group consisting of: Third gene:
(3-1) A gene encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 26, which catalyzes the reduction reaction of aldehyde, and (3-2) For the amino acid sequence set forth in SEQ ID NO: 26. A coryneform bacterium containing a gene encoding a protein having an amino acid sequence having an identity of 90% or more and having an activity of catalyzing an aldehyde reduction reaction.

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