Method for producing 2-methylbutyric acid by bacterial fermentation

Abstract

The present invention provides a method for producing 2-methylbutyric acid by fermentation using a bacterium belonging to the order Enterobacteriaceae that has been modified to attenuate expression of the tyrB gene, which encodes a protein having tyrosine aminotransferase activity. The method also makes it possible to reduce the production of by-products of 2-methylbutyric acid during fermentation by a bacterium belonging to the order Enterobacteriaceae that is capable of producing 2-methylbutyric acid.

Description

[Technical Field]

[0001] The present invention relates to the microbial industry, in particular to the attenuation of the expression of a gene encoding a protein with tyrosine aminotransferase activity, and the production of 2-methyl- ... The present invention relates to a method for producing 2-methylbutyric acid by fermentation with bacteria belonging to the order Enterobacterales that have been modified to reduce the production of the by-product 2-methylbutyric acid. [Background technology]

[0002] 2-Methylbutyric acid differs significantly from closely related unbranched and branched fatty acids, such as pentanoic acid (also called "valeric acid") and 3-methylbutyric acid (also called "isovaleric acid"). 2-Methylbutyric acid has a mild, soft, dried-fruit-like character, whereas related fatty acids have a stronger, but much more cheese-like character. 2-Methylbutyric acid is widespread in nature and is widely used in the flavor industry (Wright J. 2-Methyl butyric acid. Use in berry, fruit, brown, fermented, and savory flavors, Perfumer and Flavorist, 2011, 36(7):18-19). Lower alcohol esters of 2-methylbutyric acid are used in the production of fragrances, and polyol esters of 2-methylbutyric acid are used in the production of synthetic lubricants.

[0003] Certain applications, such as the manufacture of perfumes and some lubricants, require 2-methylbutyric acid of high purity, with residual by-products (e.g., 3-methylbutyric acid) of 0.2% by weight or less. Therefore, there is a need for a method to produce 2-methylbutyric acid with reduced by-product content.

[0004] 2-Methylbutyric acid can be produced by chemical synthesis or by microbial fermentation in a suitable nutrient medium. Commercially, 2-methylbutyric acid is typically produced chemically by the oxidation of 2-methylbutyraldehyde, which is generally produced by the dehydrogenation of alcohols in the presence of a catalyst (Ullmann's Encyclopedia of industrial chemistry, 7 th (Edition, 2011, Wiley-VCH). Alcohols are often available inexpensively and in high purity. The synthesis of 2-methylbutyric acid by the Oxo process (also known as "hydroformylation"), which involves reacting butene with a mixture of carbon monoxide and hydrogen (so-called "synthesis gas") in the presence of a transition metal compound, is not particularly suitable because the resulting product is often not of sufficient purity. To increase the purity of 2-methylbutyric acid, a method for producing 2-methylbutyric acid with a reduced content of 3-methylbutyric acid has been developed (US20160264503 A1). This method involves controlling the composition of the butene feed mixture, optimizing the hydroformylation conditions, followed by catalytic hydrogenation reduction under high temperature and pressure, and treatment with an oxidizing agent. The resulting product contains 2-methylbutyric acid with a 3-methylbutyric acid content of 0.2 wt% or less. .

[0005] One example of a method for producing 2-methylbutyric acid by bacterial fermentation is the fermentation production of (S)-2-methylbutyric acid in an L-isoleucine-containing medium using bacteria belonging to the genus Bacillus (JP5271606 B2). In another example, a 2-methylbutyric acid-producing bacterium was constructed by inactivating the threonine exporter and alcohol dehydrogenase, encoded by the rhtA and yqhD genes, respectively, from the L-threonine-producing E. coli strain ATCC 98082, and introducing the native thrABC operon and an artificial ilvAGMCD operon under the control of the PLlacO1 promoter on a low-copy plasmid. The ability to produce 2-methylbutyric acid was enhanced in the bacterium by enhancing the expression of genes encoding ketoacid decarboxylase and aldehyde dehydrogenase, which are responsible for the final two steps of the 2-methylbutyric acid biosynthetic pathway. Granted (US20150132813 A1).

[0006] The tyrB gene encodes tyrosine aminotransferase (TyrB), which catalyzes the final step in the biosynthesis of tyrosine, leucine, and phenylalanine. This enzyme transfects leucine (Powell JT and Morrison JF, Role of the Escherichia coli aromatic amino acid aminotransferase in leucine biosynthesis, J. Bacteriol., 1978, 136(1):1-4), tyrosine (Collier RH and Kohlhaw G., Nonidentity of the aspartate and the aromatic aminotransferase components of transaminase A in Escherichia coli, J. Bacteriol., 1972, 112(1):365-371), and 2-ketoisovaleric acid (CN109402034 A; Vartak NB et al., A functional leuABCD operon is required for leucine synthesis by the tyrosine-repressible transaminase in Escherichia coli K-12, J. Bacteriol., 1991, 173(12):3864-3871).

[0007] L-tyrosine production strains using tyrosine aminotransferase TyrB with enhanced activity (US9540652 B2, KR101869869 B1, CN109266592 A, US20080102499 A1, US7700328 B2; Lutke-Eversloh T. and Stephanopoulos G., Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression, Metabolic engineering, 2008, 10(2):69-77 ), L-phenylalanine-producing strains (CN104531597 B, CN100352928 C; Zhang C. et al., Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum, J. Ind. Microbiol. Biotechnol., 2015, 42(5):787-797; Liu SP et al., A systems-level engineered E. coli capable of efficiently producing L-phenylalanine, Proc. Biochem., 2014, 49(5):751-757; Wo YQ et al., Co-expression of five genes in E. coli for L-phenylalanine in Brevibacterium flavum, World J. Gastroenterol., 2003, 9(2):342-346), and L-tryptophan-producing strains (CN104531597 B, CN100352928 C; Zhang C. et al., Rational engineering of multiple module pathways for the production of L-phenylalanine in Corynebacterium glutamicum, J. Ind. Microbiol. Biotechnol., 2015, 42(5):787-797; Liu SP et al., A systems-level engineered E. coli capable of efficiently producing L-phenylalanine, Proc. Biochem., 2014, 49(5):751-757; Wo YQ et al., Co-expression of five genes in E. coli for L-phenylalanine in Brevibacterium flavum, World J. Gastroenterol., 2003, 9(2):342-346). strain (EP2147972 A1), L-homophenylalanine producing strain (US6146859 A), and L-2 A 2-aminobutyric acid-producing strain (CN106148259 A; Fotheringham IG et al., Engineering of a novel biochemical pathway for the biosynthesis of L-2-aminobutyric acid in Escherichia coli K12, Bioorg. Med. Chem., 1999, 7(10):2209-2213) was constructed.

[0008] A method for reducing the production of by-products of target compounds by deleting or weakening the tyrB gene has been reported (e.g., Li FF et al., Engineering Escherichia coli for production of 4-hydroxymandelic acid using glucose-xylose mixture, Microb. Cell Fact., 2016, 15:90; Zhu Y. et al., Metabolic engineering of indole pyruvic acid biosynthesis in Escherichia coli with tdiD, Microb. Cell Fact., 2017, 16(1):2; Liu SP et al., Heterologous pathway for the production of L-phenylglycine from glucose by E. coli, J. Biotechnol., 2014, 186:91-97).

[0009] In a series of reports, Escherichia coli strains with increased expression of genes in the leucine biosynthetic pathway that can be used to produce 2-ketoisocaproic acid and deleted ilvE and tyrB genes were used in methods for producing alcohols, particularly 3-methyl-1-butanol, by bacterial fermentation (US2009081746 A1, US20100209986 A1, WO2010045629 A2; Connor MR and Liao JC, Engineering an Escherichia coli strain for the production of 3-methyl-1-butanol, App. Env. Microbiol., 2008, 74(18):5769-5775). Removal of tyrB and tyrB was found to increase production of 2-ketoisocaproic acid, which can then be converted to 3-methyl-1-butanol by decarboxylation and reduction steps.

[0010] However, there are no data describing the effect of attenuated expression of the tyrB gene on the production of 2-methylbutyric acid and its by-products by fermentation in 2-methylbutyric acid-producing bacteria belonging to the order Enterobacteriaceae. From the viewpoint of industrial production, reducing the amount of by-products of 2-methylbutyric acid in the production method by fermentation with bacteria belonging to the order Enterobacteriaceae is extremely important because improved methods would enable the production of 2-methylbutyric acid at high purity and low cost. Summary of the Invention

[0011] This specification describes an improved method for producing 2-methylbutyric acid by fermentation using bacteria belonging to the order Enterobacteriale. According to the present invention, the production of 2-methylbutyric acid by fermentation using bacteria belonging to the order Enterobacteriale can be improved by attenuating the expression of the tyrB gene in the bacteria, thereby reducing the production of by-products of 2-methylbutyric acid (e.g., 3-methylbutyric acid, isobutyric acid, L-alloisoleucine, and / or D-alloisoleucine) by the modified bacteria compared to unmodified bacteria. When 2-methylbutyric acid is produced by culturing bacteria belonging to the order Enterobacteriale that have been modified to attenuate the expression of the tyrB gene in a medium, the leuABCD operon gene By attenuating the expression of one or more genes selected from the gene family, the amount of 2-methylbutyric acid by-product can be further reduced, resulting in higher purity and cheaper production of 2-methylbutyric acid compared to methods that do not use the engineered bacteria described herein.

[0012] The present invention provides the following:

[0013] One embodiment of the present invention is a method for producing 2-methylbutyric acid, comprising the steps of: (i) 2-methylbutyric acid-producing bacteria belonging to the order Enterobacterales were cultured in a medium. and producing and accumulating 2-methylbutyric acid in the medium or the bacterial cells of the bacterium, or both; and (ii) recovering 2-methylbutyric acid from the medium or the bacterial cells, or both. Including, The bacterium has been modified to attenuate expression of a gene encoding a protein having tyrosine aminotransferase activity.

[0014] It is another aspect of the present invention to provide the method as described above, wherein the gene encoding the protein having tyrosine aminotransferase activity is the tyrB gene.

[0015] It is another aspect of the present invention to provide the method as described above, wherein the protein having tyrosine aminotransferase activity is selected from the group consisting of: (A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (B) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, with substitution, deletion, insertion, and / or addition of 1 to 150 amino acid residues, and having tyrosine aminotransferase activity; and (C) an amino acid sequence having 60% or more identity to the entire amino acid sequence shown in SEQ ID NO: 2 1. A protein comprising the formula:

[0016] It is another aspect of the present invention to provide the method as described above, wherein the gene is selected from the group consisting of: (a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1; (b) a gene comprising a nucleotide sequence capable of hybridizing under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, and encoding a protein having tyrosine aminotransferase activity; (c) a gene encoding a protein comprising an amino acid sequence containing a substitution, deletion, insertion, and / or addition of 1 to 150 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, a gene, the protein of which has tyrosine aminotransferase activity; and (d) A gene comprising a mutant base sequence of SEQ ID NO: 1, wherein the mutant base sequence is due to the degeneracy of the genetic code.

[0017] It is another aspect of the present invention to provide the method as described above, wherein expression of a gene encoding a protein having tyrosine aminotransferase activity is attenuated by inactivating said gene.

[0018] It is another aspect of the present invention to provide the method as described above, wherein the gene encoding the protein having tyrosine aminotransferase activity is deleted.

[0019] It is another aspect of the present invention to provide the method as described above, wherein the bacterium belongs to the family Enterobacteriaceae or Erwiniaceae.

[0020] Another aspect of the present invention is a method for producing a bacterium, wherein the bacterium is a bacterium of the genus Escherichia or Pantoea. The present invention also provides the method, wherein the strain belongs to the genus Pantoea.

[0021] It is another aspect of the present invention to provide the method as described above, wherein the bacterium is Escherichia coli or Pantoea ananatis.

[0022] It is another aspect of the present invention to provide the method as described above, wherein the bacterium has been further modified to attenuate expression of one or more genes selected from the group consisting of leuA, leuB, leuC, and leuD genes.

[0023] It is another aspect of the present invention to provide the method as described above, wherein the amount of 2-methylbutyric acid by-product is reduced compared to the unmodified bacterium.

[0024] It is another aspect of the present invention to provide the method as described above, wherein the by-product is selected from the group consisting of 3-methylbutyric acid, isobutyric acid, L-alloisoleucine, D-alloisoleucine, and combinations thereof.

[0025] Still other objects, features and attendant advantages of the present invention will become apparent to those skilled in the art from the following detailed description of embodiments constructed in accordance therewith. [Brief explanation of the drawings]

[0026] [Figure 1]FIG. 1 shows the biosynthetic scheme of 2-methylbutyric acid and the by-product of 2-methylbutyric acid. KdcA (also indicated as "1" in a circle): 2-ketoacid decarboxylase (EC 4.1.1.72) encoded by the Lactococcus lactis kdcA gene optimized for expression in E. coli; AldH (also indicated as "2" in a circle): aldehyde dehydrogenase (EC 1.2.1.3); IlvE: branched-chain amino acid aminotransferase, aminotransferase B (EC 2.6.1.42); TyrB: aromatic amino acid aminotransferase (EC 2.6.1.57); LeuA: 2-isopropylmalate sythase (EC 2.3.3.13); LeuCD: 3-isopropylmalate dehydratase (EC 4.2.1.33), LeuB: 3-isopropylmalate dehydrogenase (EC 1.1.1.85), E-4-P: D-erythrose-4-phosphate, PEP: phosphoenolpyruvate, PhePyr: 3-phenylpyruvate, hPhePyr: 4-hydroxyphenylpyruvate, Pyr: pyruvate, 2-KB: 2-ketobutyrate, Prop: propionic acid, AHB: 2-aceto-2-hydroxybutyrate, (S)-KMV: (S)-2-keto-3-methylvalerate, (R)-KMV: (R)-2-keto-3-methylvalerate, alloIle: alloisoleucine, (S)-2-MB: (S)-2-methylbutyrate (R)-2-MB: (R)-2-methylbutyric acid, AL: 2-acetolactic acid, KIV: 2-ketoisovaleric acid, isoBut: isobutyric acid, KIC: 2-ketoisocaproic acid, 3-MB: 3-methylbutyric acid. [Figure 2]Figure 2 shows the DNA sequence of the Ptac promoter (SEQ ID NO: 69). The -35 and -10 sequences of the Ptac promoter are underlined. The lac repressor binding site is shown in uppercase. The sequence of the Ptac fragment containing half of the lac operator is shown in bold. [Figure 3] 3 shows the construction scheme of the 2-methylbutyric acid-producing E. coli strain L1201-1. SD1: modified Shine-Dalgarno sequence, *: mutant allele of the gene. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will now be described in more detail with reference to exemplary embodiments (by way of example only) and the accompanying drawings, in which:

[0028] 1. Bacteria The bacteria described herein have been modified to attenuate the expression of a gene encoding a protein having tyrosine aminotransferase activity. The bacterium is a 2-methylbutyric acid-producing bacterium belonging to the order Enterobacterales. The bacterium described herein can be used in the methods described herein. Therefore, the following description of the bacterium can be applied mutatis mutandis to any bacterium that can be used instead or equivalently in the methods described herein.

[0029] Attenuated expression of genes encoding proteins with tyrosine aminotransferase activity Any 2-methylbutyrate-producing bacterium belonging to the order Enterobacteriaceae that has been modified to produce 2-methylbutyrate can be used.

[0030] The term "2-methylbutyric acid-producing bacteria" may be used interchangeably or equivalently with the terms "bacteria capable of producing 2-methylbutyric acid," "bacteria having the ability to produce 2-methylbutyric acid," or "bacteria having the ability to produce 2-methylbutyric acid."

[0031] The term "2-methylbutyric acid-producing bacteria" may refer to bacteria belonging to the order Enterobacteriaceae that have the ability to produce, excrete or secrete, and / or accumulate 2-methylbutyric acid in a medium and / or in the bacterial cells (i.e., bacterial cells) when the bacteria are cultured in a medium.

[0032] The term "2-methylbutyric acid-producing bacteria" may also refer to bacteria that have the ability to produce, excrete or secrete, and / or accumulate 2-methylbutyric acid in a medium in greater amounts than, for example, unmodified bacteria. The term "unmodified bacteria" may be used interchangeably or equivalently with the term "unmodified strain." The term "unmodified strain" refers to bacteria that do not have tyrosine aminotransferase activity. The term "2-methylbutyric acid-producing bacteria" may refer to a control strain that has not been modified to attenuate the expression of a gene encoding a protein encoding the 2-methylbutyric acid-producing bacterium. Examples of unmodified bacteria include wild-type or parent strains such as E. coli K-12 strains (e.g., W3110 (ATCC 27325) and MG1655 (ATCC 47076)). The term "2-methylbutyric acid-producing bacteria" may refer to bacteria that produce 2-methylbutyric acid at a concentration of, for example, 0.1 g / L or more, 0.5 g / L or more, or 1.0 g / L or more. It may also refer to bacteria that are capable of accumulating butyric acid in a medium.

[0033] In addition, Enterobacteriaceae, which contains a protein with tyrosine aminotransferase activity, Bacteria capable of producing 2-methylbutyric acid that have been modified to attenuate the expression of a gene (such as the tyrB gene) that regulates 2-methylbutyric acid production can also be used. The bacterium may naturally have the ability to produce 2-methylbutyric acid, or may be modified to have the ability to produce 2-methylbutyric acid. Such modifications can be achieved, for example, by mutation or DNA recombinant technology. The bacterium may naturally have the ability to produce 2-methylbutyric acid. Proteins with tyrosine aminotransferase activity in bacteria capable of producing butylbutyric acid It can be obtained by attenuating the expression of a gene encoding the tyrB gene or the like. The bacterium contains a gene encoding a protein having tyrosine aminotransferase activity. The ability to produce 2-methylbutyrate can be obtained by imparting the ability to produce 2-methylbutyrate to a bacterium that has already been modified to attenuate the expression of a gene (such as the tyrB gene). Alternatively, the bacterium can be obtained by imparting the ability to produce 2-methylbutyrate to a bacterium that has already been modified to attenuate the expression of a gene (such as the tyrB gene) that encodes tyrosine aminotransferase. The bacterium may have acquired the ability to produce 2-methylbutyric acid by being modified to attenuate the expression of a gene (such as the tyrB gene) encoding a protein having such activity. Specifically, the bacterium described herein can be obtained, for example, by modifying the bacterial strains described below.

[0034] The term "2-methylbutyric acid-producing ability" may refer to the ability of a bacterium belonging to the order Enterobacteriaceae to produce, excrete or secrete, and / or accumulate 2-methylbutyric acid in a medium and / or bacterial cells. Specifically, the term "2-methylbutyric acid-producing ability" may refer to the ability of a bacterium to produce, excrete or secrete, and / or accumulate 2-methylbutyric acid in a medium and / or bacterial cells to an extent that 2-methylbutyric acid can be recovered from the medium and / or bacterial cells when the bacterium is cultured in a medium.

[0035] The term "cultured" when referring to bacteria grown according to the methods described herein may be used interchangeably or equivalently with terms such as "cultivated" and "grown," which are well known to those skilled in the art.

[0036] Bacteria can produce 2-methylbutyric acid as either the S- or R-enantiomer of 2-methylbutyric acid, or as a mixture of the S- and R-enantiomers in various ratios, especially the S-enantiomer.

[0037] Bacteria can produce 2-methylbutyric acid alone or as a mixture of 2-methylbutyric acid and one or more substances other than 2-methylbutyric acid. For example, bacteria can produce 2-methylbutyric acid alone or as a mixture of 2-methylbutyric acid and one or more amino acids (e.g., L-amino acids (also called L-amino acids)). Furthermore, for example, bacteria can produce 2-methylbutyric acid alone or as a mixture of 2-methylbutyric acid and one or more organic acids other than 2-methylbutyric acid (e.g., carboxylic acids).

[0038] In addition, Enterobacteriaceae, which contains a protein with tyrosine aminotransferase activity, Any 2-methylbutyric acid-producing bacterium that has been modified to attenuate expression of a gene (such as the tyrB gene) that regulates 2-methylbutyric acid production, thereby reducing the production of the by-product 2-methylbutyric acid compared to an unmodified strain (e.g., a wild-type strain or parent strain, as described below), can also be used. The by-product may consist of one, two, or more substances. Specifically, a tyrosine aminotransferase having tyrosine aminotransferase activity can be used. A bacterium can be used that has been modified to attenuate the expression of a gene encoding a protein (such as the tyrB gene), and that can produce one, two, or more by-products of 2-methylbutyric acid in reduced amounts compared to an unmodified strain, or that thereby reduces the production of one, two, or more by-products of 2-methylbutyric acid compared to an unmodified strain. The amount of 2-methylbutyric acid by-product may be expressed as an absolute value (e.g., grams / liter (g / L)) or a relative value (e.g., The amount of 2-methylbutyric acid produced by the modified bacterium may be expressed as a percentage (e.g., %). That is, the amount of 2-methylbutyric acid by-product produced may be reduced in absolute and / or relative terms. The phrase "the amount of 2-methylbutyric acid by-product expressed as a relative value" may mean that the amount of 2-methylbutyric acid by-product is expressed as a ratio of the amount of 2-methylbutyric acid by-product to the control substance, preferably multiplied by 100%. The "control substance" particularly includes 2-methylbutyric acid. Therefore, when using the method for producing 2-methylbutyric acid described herein, it is acceptable that while the relative amount of 2-methylbutyric acid by-product produced by the modified bacterium is reduced compared to unmodified bacteria, the absolute amount of the by-product may remain unchanged or even increase compared to unmodified bacteria, thereby allowing for the production of 2-methylbutyric acid with a higher purity compared to methods not using the modified bacteria described herein.

[0039] The term "capable of producing 2-methylbutyric acid by-products" used herein with respect to bacteria may refer to the ability of the bacterium belonging to the order Enterobacteriale to produce, excrete or secrete, and / or accumulate one, two, or more 2-methylbutyric acid by-products in a medium or bacterial cells. Specifically, the term "capable of producing 2-methylbutyric acid by-products" used herein with respect to bacteria may refer to the ability of the bacterium belonging to the order Enterobacteriale to produce, excrete or secrete, and / or accumulate one, two, or more 2-methylbutyric acid by-products in a medium or bacterial cells, or both, to such an extent that the one, two, or more 2-methylbutyric acid by-products can be recovered from the medium and / or bacterial cells when the bacterium is cultured in the medium. The term "2-methylbutyric acid by-products" is explained below.

[0040] L-amino acids include, but are not limited to, L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-citrulline, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine, and derivatives thereof.

[0041] Carboxylic acids include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, lactic acid, citric acid, pentanoic acid, and derivatives thereof.

[0042] The terms "L-amino acid" and "carboxylic acid" are not limited to amino acids and carboxylic acids in their free form, but may also refer to their derived forms (such as salts, hydrates, adducts, or combinations thereof). An adduct may be a compound formed between an amino acid or a carboxylic acid and another organic or inorganic compound. That is, the terms "L-amino acid" and "carboxylic acid" may refer, for example, to L-amino acids and carboxylic acids in their free form, their derived form, or a mixture thereof. The terms "L-amino acid" and "carboxylic acid" may refer, for example, to L-amino acids and carboxylic acids in their free form, their salts, or mixtures thereof, among others. The terms "L-amino acid" and "carboxylic acid" may refer, for example, to any of their salts, such as sodium salts, potassium salts, ammonium salts, monohydrates, dihydrates, trihydrates, monohydrochlorides, dihydrochlorides, etc. Unless otherwise specified, the terms "L-amino acid" and "carboxylic acid" that do not refer to the hydration state (e.g., the terms "free form of an L-amino acid or carboxylic acid" and "salt of an L-amino acid or carboxylic acid") can refer to non-hydrated L-amino acids and carboxylic acids, hydrated L-amino acids and carboxylic acids, or mixtures thereof.

[0043] The term "2-methylbutyric acid by-product" can refer to one, two, or more by-products of 2-methylbutyric acid, and can also refer to byproducts, co-products, or other by-products of the 2-methylbutyric acid production process, for example, by bacterial fermentation as described herein. can refer to a substance such as an organic compound other than 2-methylbutyric acid that is produced as a side product. The term "by-product of 2-methylbutyric acid" can also refer to a substance that can be produced, excreted, or secreted by a 2-methylbutyric acid-producing bacterium belonging to the order Enterobacteriale when the bacterium is cultured in a medium to produce 2-methylbutyric acid, and thus accumulates in the medium or the bacterial cells, or both, to an extent that the by-product can be recovered from the medium and / or the bacterial cells when the bacterium is cultured in the medium. The amount of the by-product of 2-methylbutyric acid in the medium and / or the bacterial cells can be less than, equal to, or greater than the amount of 2-methylbutyric acid produced by fermentation by a bacterium belonging to the order Enterobacteriale that is capable of producing 2-methylbutyric acid.

[0044] Since the biosynthetic pathway of 2-methylbutyric acid branches off from the biosynthetic pathway of L-isoleucine, specific examples of by-products of 2-methylbutyric acid include, but are not limited to, the following: Examples of intermediates include intermediates in the 2-methylbutyric acid biosynthetic pathway, intermediates in other biosynthetic pathways branched from the 2-methylbutyric acid biosynthetic pathway, and combinations thereof. The intermediates are not limited to intermediates in the 2-methylbutyric acid biosynthetic pathway, but may also be precursors, intermediates, or substrates in metabolic pathways of one, two, or more other substances, for example, precursors, intermediates, or substrates in the branched-chain L-amino acid biosynthetic pathway.

[0045] The term "branched-chain L-amino acid" can refer to L-amino acids such as L-valine, L-leucine, L-isoleucine, etc. Because pyruvate (also called "α-ketopropionic acid") is a precursor in the biosynthetic pathways of L-valine, L-leucine, and L-isoleucine, the by-product of 2-methylbutyrate may be a by-product of another biosynthetic pathway that branches off from pyruvate in the biosynthetic pathway of 2-methylbutyrate. Other by-products of the 2-methylbutyrate biosynthetic pathway that branch off from pyruvate include, among others, the by-products of 2-methylbutyrate, as well as 3-methylbutyrate and isobutyrate, which share 2-ketoisovalerate (also known as "α-oxoisovalerate") as a common precursor, and L-alloisoleucine and D-alloisoleucine, which share 2-keto-3-methylvalerate (also known as "α-oxomethylvalerate") as a common precursor, and 2-ketoisovalerate and 2-keto-3-methylvalerate share pyruvate as a common precursor (Figure 1).

[0046] Therefore, as will be described later, the protein encoding the tyrosine aminotransferase activity In the method for producing 2-methylbutyric acid by fermentation of a bacterium belonging to the order Enterobacteriaceae that has been modified to attenuate the expression of a gene related to 2-methylbutyric acid, the by-product substrate of 2-methylbutyric acid may be, but is not limited to, 3-methylbutyric acid, isobutyric acid, L-alloisoleucine, D-alloisoleucine, or a combination thereof.

[0047] In addition, bacteria belonging to the Enterobacteriaceae family have recently been classified into a number of subfamily groups, including a phylogenetic tree reconstruction based on 1548 core proteins, 53 ribosomal proteins, and 4 multilocus sequence-analyzed proteins. It has been reclassified based on comprehensive comparative genomic analysis (Adelou M. et al., Genome-based phylogeny and taxonomy of the 'Enterobacteriales': proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov., Int. J. Syst. Evol. Microbiol., 2016, 66:5575-5599).

[0048] Based on reclassification, bacteria previously classified in the family Enterobacteriaceae are now classified in different families within the order Enterobacteriale. Based on the above analysis, bacteria belonging to the order Enterobacteriale used in the methods described herein include Enterobacter, Escherichia, Klebsiella, Salmonella, Erwinia, Pantoea, Morganella, Photorhabdus ), Providencia, Yersinia, etc. Preferably, the strain can be capable of producing 2-methylbutyric acid. Specifically, bacteria classified into the order Enterobacteriaceae according to the classification used in the NCBI (National Center for Biotechnology Information) database (ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?id=91347) can be used. Strains belonging to the order Enterobacteriaceae that can be modified include bacteria of the family Enterobacteriaceae or Erwiniaceae, specifically bacteria of the genus Escherichia, Enterobacter, or Pantoea.

[0049] The species of bacteria belonging to the genus Escherichia are not particularly limited, and examples thereof include species classified into the genus Escherichia according to classification known to experts in microbiology. Examples of bacteria belonging to the genus Escherichia include those described in Neidhardt et al. (Bachmann, BJ, Derivations and genotypes of some mutant derivatives of E. coli K-12, pp. 2460-2488. In F.C. Neidhardt et al. (ed.), E. coli li and Salmonella: cellular and molecular biology, 2 nd ed. ASM Press, Washington, DC, 1996). Examples of Escherichia bacteria include Escherichia coli (E. coli). Specific examples of E. coli include: The prototype wild-type strain E. coli K-12 (E. coli W3110 (ATCC 27325) and E. coli MG1655 (ATCC 47076), etc.

[0050] The Enterobacter bacteria are not particularly limited, and examples thereof include species classified into the genus Enterobacter according to classification known to experts in microbiology. Examples of Enterobacter bacteria include Enterobacter agglomerans and Enterobacter aerogenes. Specific examples of Enterobacter agglomerans strains include Enterobacter agglomerans ATCC 12287. Specific examples of Enterobacter aerogenes strains include Enterobacter aerogenes ATCC 13048 and NBRC 12010 (Sakai S. and Yaqishita T., Microbial production of hydrogen and ethanol from glycerol-containing wastes discharged from a biodiesel fuel production plant in a bioelectrochemical reactor with thionine, Biotechnol. Bioeng., 2007, 98(2):340-348), AJ110637 (FERM BP-10955). Examples of Enterobacter strains include those described in European Patent Application Publication No. 0952221. The species of Pantoea agglomerans is classified as Pantoea agglomerans. This also includes some strains of

[0051] The Pantoea bacteria are not particularly limited, and include species classified into the genus Pantoea according to classification known to microbiologists. Examples of Pantoea bacteria include Pantoea ananatis (Pantoea ananatis), Pantoea stoolwort, and the like. Examples of Pantoea ananatis strains include Pantoea stewartii, Pantoea agglomerans, and Pantoea citrea. Specific examples of Pantoea ananatis strains include Pantoea ananatis LMG20103, AJ13355 (FERM BP-6614), AJ13356 (FERM BP-6615), AJ13601 (FERM BP-7207), SC17 (FERM BP-11091), and SC17(0) (VKPM B-9246). Some species of Enterobacter agglomerans have recently been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, etc., based on 16S rRNA sequence analysis and other findings (Mergaert J. et al., Transfer of Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the Genus Pantoea emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii (Smith 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov., Int. J. Syst. Evol. Microbiol., 1993, 43:162-173). The genus Pantoea also includes bacteria reclassified as such.

[0052] Erwinia bacteria include Erwinia amylovora, Erwinia Examples of bacteria in the genus Klebsiella include Erwinia carotovora and Klebsiella planticola.

[0053] These strains can be obtained, for example, from the American Type Culture Collection (Address: PO Box 1549, Manassas, VA 20108, United States of America). Each strain has a corresponding accession number, which can be used to order (see atcc.org / ). The accession number for each strain is listed in the American Type Culture Collection (Address: PO Box 1549, Manassas, VA 20108, United States of America). Listed in the Culture Collection catalogue.

[0054] The bacterium may be one that inherently has the ability to produce 2-methylbutyric acid, or may be one that has been modified to have the ability to produce 2-methylbutyric acid. The bacterium can be obtained, for example, by imparting 2-methylbutyric acid-producing ability to the above-mentioned bacterium, or by enhancing the 2-methylbutyric acid-producing ability of the above-mentioned bacterium.

[0055] The ability to produce 2-methylbutyric acid can be imparted or enhanced by methods that have been conventionally used for breeding amino acid-producing bacteria such as Escherichia bacteria (see Amino Acid Fermentation, Academic Press, first published May 30, 1986, pp. 77-100). Examples of such methods include obtaining auxotrophic mutants, obtaining strains resistant to 2-methylbutyric acid analogs, obtaining metabolically controlled mutants, and creating recombinant strains with enhanced activity of 2-methylbutyric acid biosynthetic enzymes. When breeding 2-methylbutyric acid-producing bacteria, the properties imparted, such as auxotrophy, analog resistance, and metabolic control mutation, may be one or more of the following: Furthermore, when breeding 2-methylbutyric acid-producing bacteria, the activity of the 2-methylbutyric acid biosynthetic enzymes to be enhanced may be one or more of the following: Furthermore, the impartation of properties such as auxotrophy, analog resistance, and metabolic control mutation may be combined with the enhancement of the activity of the biosynthetic enzymes.

[0056] Auxotrophic mutants, analog-resistant mutants, or metabolically controlled mutants capable of producing 2-methylbutyric acid can be obtained by subjecting a parent strain or wild-type strain to a typical mutagenesis treatment and selecting from the resulting mutants those that exhibit auxotrophy, analog-resistant, or metabolically controlled mutations and also have the ability to produce 2-methylbutyric acid. Typical mutagenesis treatments include irradiation with X-rays or ultraviolet light, and treatment with mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate (EMS), and methyl methanesulfonate (MMS).

[0057] Alternatively, the ability to produce 2-methylbutyric acid can be imparted or enhanced by enhancing the activity of an enzyme involved in the biosynthesis of the target 2-methylbutyric acid. Enhancing enzyme activity can be achieved, for example, by modifying the bacterium so that expression of the gene encoding the enzyme is enhanced. Methods for enhancing gene expression are described in WO0018935, EP1010755A, etc.

[0058] Alternatively, 2-methylbutyric acid-producing ability can be imparted or enhanced by reducing the activity of an enzyme that catalyzes a reaction that branches off from the 2-methylbutyric acid biosynthetic pathway to produce a compound other than 2-methylbutyric acid. The term "enzyme that catalyzes a reaction that branches off from the 2-methylbutyric acid biosynthetic pathway to produce a compound other than 2-methylbutyric acid" also encompasses enzymes involved in the decomposition of 2-methylbutyric acid. For example, enzyme activity can be reduced by modifying the bacterium so that the gene encoding the enzyme is inactivated. Methods for reducing enzyme activity are described below.

[0059] Specific examples of 2-methylbutyric acid-producing bacteria and methods for imparting or enhancing 2-methylbutyric acid-producing ability are provided below. Note that the properties of 2-methylbutyric acid-producing bacteria and the modifications for imparting or enhancing 2-methylbutyric acid-producing ability, as exemplified below, may be used alone or in appropriate combination.

[0060] 2-methylbutyric acid producing bacteria 2-Methylbutyric acid producers and parent strains that can be used to derive 2-methylbutyric acid producers include, but are not limited to, strains with enhanced expression of one or more genes encoding proteins responsible for the last two steps of the 2-methylbutyric acid biosynthetic pathway (Figure 1). The gene encoding the protein responsible for the penultimate step of the 2-methylbutyric acid biosynthetic pathway is the kivD gene encoding branched-chain 2-ketoacid decarboxylase (KivD), which is specific for, for example, Lactococcus lactis. (US20150132813 A1; de la Plaza et al., Biochemical and molecular lar characterization of alpha-keto-isovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by Lactococcus lactis, FEMS Microbiol Lett., 2004, 238(2):367-374), the kdcA gene encoding branched-chain 2-ketoacid decarboxylase (which may be unique to Lactococcus lactis B1157, for example, which shows 89.8% identity to KivD) (Smit BA et al., Identification, cloning, and characterization of a Lactococcus lactis Examples include the branched-chain α-ketoacid decarboxylase involved in flavor formation, Appl. Env. Microbiol., 2005, 71(1):303-311; Savrasova EA et al., Use of the valine biosynthesis pathway to convert glucose into isobutanol, J. Ind. Microbiol. Biotechnol., 2011, 38(9):1287-1294), and the ipdC gene encoding indolepyruvate decarboxylase, which may be endemic to, for example, Salmonella typhimurium (US20150132813 A1). Genes encoding proteins responsible for the final step of the synthesis pathway include the feaB gene (also known as the padA gene) encoding phenylacetaldehyde dehydrogenase (which may be endemic to E. coli, for example) (US20150132813 A1; Rodriguez-Zavala JS et al., Characterization of E. coli tetrameric aldehyde dehydrogenases with atypical properties compared to other aldehyde dehydrogenases, Protein Sci., 2006, 15(6):1387-1396), and the aldB gene encoding aldehyde dehydrogenase (which may be endemic to E. coli, for example) (US20150132813 A1; Ho KK and Weiner H., Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli, J. Bacteriol., 2005, 187(3):1067-1073), and the puuC gene (also known as the aldH gene) encoding γ-glutamyl-γ-aminobutyraldehyde dehydrogenase, which may be endemic to E. coli, for example (US20150132813 A1; Kurihara S. et al., A novel putrescine utilization pathway involves γ-glutamylated intermediates of Escherichia coli K-12, J. Biol. Chem., 2005, 280(6):4602-4608; Jo J.-E. et al., Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-hydroxypropionaldehyde as a substrate, Appl. Microbiol. Biotechnol., 2008, 81(1):51-60). Examples include:

[0061] Since the biosynthetic pathway of 2-methylbutyric acid branches off from 2-keto-3-methylvalerate in the biosynthetic pathway of L-isoleucine, 2-methylbutyric acid producing bacteria and parent strains that can be used to derive 2-methylbutyric acid producing bacteria include, but are not limited to, those that express threonine deaminase (JP 2-458 A) and acetohydroxy acid synthase (acetohydroxy acid synthase). synthase (EP0356739 A1) and other proteins involved in L-isoleucine biosynthesis. Other parent strains that can be used to derive L-isoleucine and 2-methylbutyrate producing bacteria include, but are not limited to, E. Examples of such strains include strains belonging to the genus Escherichia, such as E. coli AJ12919 (JP 8-47397 A), and E. coli VL1892 and KX141 strains (VKPM B-4781) (US Pat. No. 5,658,766).

[0062] Because the L-isoleucine biosynthesis pathway begins with L-threonine, 2-methylbutyrate-producing bacteria and parent strains that can be used to derive 2-methylbutyrate-producing bacteria include, but are not limited to, strains in which expression of one or more genes encoding L-threonine biosynthetic enzymes is enhanced. For example, to enhance expression of the threonine operon thrABC, it is desirable to remove the attenuator region from the operon, which affects transcription (WO2005049808 A1, WO2003097839 A1). Other parent strains that can be used to derive L-threonine and 2-methylbutyrate producers include, but are not limited to, E. coli TDH-6 / pVIC40 (VKPM B-3996; US Pat. No. 5,175,107 and US Pat. No. 5,705,371), E. coli 472T23 / pYN7 (ATCC 98081; US ​​Pat. No. 5,631,157), E. coli NRRL-21593 (US Pat. No. 5,939,307), E. coli MG442 (US Pat. No. 4,278,765), and E. coli MG442 (US Pat. No. 4,278,765). ; Gusyatiner MM et al., Study of relA gene function in the expression of amino Examples of strains that can be used include strains belonging to the genus Escherichia, such as E. coli VL643 and VL2055 (EP1149911 A2), and E. coli VKPM B-5318 (EP0593792 A1), and examples of strains that can be used include E. coli K-12 mutants resistant to β-hydroxynorvaline acid, Genetika (Russian), 1978, 14(6):957-968, E. coli VL643 and VL2055 (EP1149911 A2), and E. coli VKPM B-5318 (EP0593792 A1).

[0063] The L-isoleucine-producing strain AJ12919 was obtained by introducing the ilvGMEDA operon, which contains the ilvA gene encoding threonine deaminase, which is substantially desensitized to L-isoleucine inhibition and from which the region required for attenuation has been deleted, carried on the plasmid pMWD5, into the L-threonine-producing strain VKPM B-3996 (EP0685555 B1).

[0064] 2-Methylbutyric acid-producing bacteria can be obtained from any L-isoleucine-producing bacteria by inactivating the branched-chain amino acid aminotransferase encoded by the ilvE gene. Methods for gene inactivation are described herein.

[0065] The genes and proteins used in breeding 2-methylbutyric acid-producing bacteria may have, for example, the known nucleotide sequences and amino acid sequences of the above-exemplified genes and proteins, respectively. Furthermore, the genes and proteins used in breeding 2-methylbutyric acid-producing bacteria may be mutants (variants) of the above-exemplified genes and proteins (for example, variants of such known nucleotide sequences and amino acid sequences) as long as their original functions (for example, in the case of proteins, their respective enzymatic activities) are maintained. The gene and protein variants may be mutants of genes encoding proteins having tyrosine aminotransferase activity and mutants of tyrosine aminotransferases encoded thereby, as described herein.

[0066] The bacteria belonging to the order Enterobacteriaceae described herein have been modified so that at least a gene encoding a protein having tyrosine aminotransferase activity is weakened.

[0067] "Tyrosine aminotransferase activity The term "protein-encoding gene" refers to a gene encoding a protein with the enzymatic activity that catalyzes the following reactions: an aromatic amino acid + 2-ketoglutarate ⇔ an aromatic 2-ketoacid + L-glutamate (Enzyme Commission number, EC: 2.6.1.57) and L-leucine + 2-ketoglutarate ⇔ 4-methyl-2-keto-pentanoate + L-glutamate (EC: 2.6.1.6). As a gene encoding an enzyme having tyrosine aminotransferase activity, A specific example is the tyrB gene, which encodes tyrosine aminotransferase. The gene encoding an enzyme having tyrosine aminotransferase activity may be the tyrB gene, or a homolog or mutant nucleotide sequence thereof. tyrB and its homolog and mutant nucleotide sequences will be described in more detail below.

[0068] The tyrB gene encodes the tyrosine aminotransferase TyrB (also known as aromatic-amino-acid aminotransferase, leucine aminotransferase) (KEGG, Kyoto Encyclopedia of Genes and Genomes, entry No. b4054; Protein Knowledgebase, UniProtKB / Swiss-Prot, accession No. P04693).

[0069] The tyrB gene (GenBank, accession No. NC_000913.3; nucleotide positions: 4267114 to 4268307, complement; Gene ID: 948563) is located in the same chromosome of E. coli K-12. It is located between the alr gene on one strand and the yjbS gene on the opposite strand. The tyrB gene of E. coli K-12 The base sequence of the gene (SEQ ID NO: 1) and the TyrB gene specific to E. coli K-12 strains encoded by the gene were analyzed. The amino acid sequence of the protein (SEQ ID NO: 2) is known.

[0070] That is, the gene encoding a protein having tyrosine aminotransferase activity (such as the tyrB gene) may be a gene (for example, DNA) having the nucleotide sequence shown in SEQ ID NO: 1. A protein having tyrosine aminotransferase activity (such as TyrB protein) may be a protein having the amino acid sequence shown in SEQ ID NO: 2. Unless otherwise specified, the expression "a gene or protein has a nucleotide sequence or an amino acid sequence" may mean that a gene or protein contains the nucleotide sequence or the amino acid sequence in a longer sequence, or may mean that a gene or protein has only the nucleotide sequence or the amino acid sequence.

[0071] The production of 2-methylbutyrate by-product by bacteria that can be used in the methods described herein can be further reduced compared to unmodified bacteria by attenuating the expression of one or more genes involved in the L-leucine biosynthetic pathway, which branches off from pyruvate in the 2-methylbutyrate biosynthetic pathway. The first step in the L-leucine biosynthetic pathway is the production of 2-isopropylmalate from 2-ketoisovalerate, acetyl-CoA, and HO, which is catalyzed by 2-isopropylmalate synthase encoded by the leuA gene. The second step in the L-leucine biosynthetic pathway is the conversion of 2-isopropylmalate to 3-isopropylmalate, which is encoded by the leuCD gene. 3-isopropylmalate dehydratase, which The next reaction is the production of 2-isopropyl-3-ketosuccinate from 3-isopropylmalate, which is catalyzed by 3-isopropylmalate dehydrogenase, encoded by the leuB gene. 2-Isopropyl-3-ketosuccinate naturally converts to 2-ketoisocaproic acid, a precursor of 3-methylbutyric acid (a by-product of 2-methylbutyric acid production by fermentation with Enterobacteriaceae).

[0072] By attenuating expression of one or more genes in the L-leucine biosynthetic pathway, such as leuA, leuB, leuC, and / or leuD, bacteria that can be used in the methods described herein can be conferred with the property of producing one or more by-products in lower amounts compared to bacteria in which the leuA, leuB, leuC, and / or leuD genes are not attenuated. As a result of attenuating expression of the above genes, the production of one or more by-products (e.g., 3-methylbutyric acid) by the modified bacteria can be further reduced, and 2-methylbutyric acid can be produced in high purity.

[0073] The term "leuA gene" may refer to a gene encoding an enzyme having 2-isopropylmalate synthase activity. A specific example of a gene encoding an enzyme having 2-isopropylmalate synthase activity is the leuA gene encoding 2-isopropylmalate synthase. The gene encoding an enzyme having 2-isopropylmalate synthase activity may be the leuA gene or a homolog or mutant nucleotide sequence thereof. The nucleotide sequences of leuA and its mutants will be described in more detail below.

[0074] The leuA gene encodes the 2-isopropylmalate synthase protein LeuA (KEGG entry No. b0074; Protein Knowledgebase, UniProtKB / Swiss-Prot, accession No. P09151). The leuA gene (GenBank accession No. NC_000913.3; nucleotide positions: 81958 to 83529, complement; Gene ID: 947465) is located between the leuL and leuB genes on the same strand of the chromosome of E. coli K-12. The leuA gene is part of the leuABCD operon. The nucleotide sequence of the leuA gene and the amino acid sequence of the LeuA protein encoded by the leuA gene are shown in SEQ ID NOs: 3 and 4, respectively.

[0075] The term "2-isopropylmalate synthase activity" refers to the reaction: 3-methyl-2-keto-butanoate + acetyl-CoA + HO → (2S)-2-isopropylmalate + coenzyme A + H + (EC: 2.3.3.13)

[0076] The term "leuB gene" refers to a gene encoding an enzyme with 3-isopropylmalate dehydrogenase activity. It can refer to a gene encoding an enzyme with 3-isopropylmalate dehydrogenase activity. Specifically, the gene encoding 3-isopropylmalate dehydrogenase, leuB, is Genes encoding enzymes with 3-isopropylmalate dehydrogenase activity include: The gene may be the leuB gene and its homologues or mutant nucleotide sequences, which will be described in more detail below.

[0077] The leuB gene encodes the 3-isopropylmalate dehydrogenase protein LeuB (also known as IMDH, 3-carboxy-2-hydroxy-4-methylpentanoate: NAD + The leuB gene (GenBank accession number NC_000913.3; nucleotide positions: 80867 to 81958, complement; Gene ID: 944798) is located on the same strand of the chromosome of E. coli K-12, between the leuA and leuC genes. The leuB gene is part of the leuABCD operon. The nucleotide sequence of the leuB gene and the amino acid sequence of the LeuB protein encoded by the leuB gene are shown in SEQ ID NOs: 5 and 6, respectively.

[0078] 3-Isopropylmalate dehydrogenase The term "activity" refers to the reaction: (2R,3S)-3-isopropylmalate + NAD + → 4-methyl-2-keto-pentanoate + CO2+ This may refer to the enzyme activity that catalyzes NADH (EC: 1.1.1.85).

[0079] The term "leuCD gene" can refer to a gene encoding an enzyme having 3-isopropylmalate dehydratase activity. Specifically, the genes involved include the large subunit of 3-isopropylmalate dehydratase and The genes encoding the small subunits are the leuC and leuD genes, respectively. The genes encoding the enzymes with 3-isopropylmalate dehydratase activity are the leuC and leuD genes, and The nucleotide sequences of leuC, leuD, and their homologs or mutants will be described in more detail below.

[0080] The leuC gene encodes the large subunit LeuC of 3-isopropylmalate dehydratase (KEGG, entry No. b0072; Protein Knowledgebase, UniProtKB / Swiss-Prot, accession No. The leuC gene (GenBank accession No. NC_000913.3; nucleotide positions: 79464 to 80864, ​​complement; Gene ID: 945076) is located on the chromosome of E. coli K-12. , located between the leuB and leuD genes on the same strand. The leuC gene is part of the leuABCD operon The nucleotide sequence of the leuC gene and the amino acid sequence of the LeuC protein encoded by the leuC gene are shown in SEQ ID NOs: 7 and 8, respectively.

[0081] The leuD gene encodes the small subunit LeuD of 3-isopropylmalate dehydratase (KEGG, entry No. b0071; Protein Knowledgebase, UniProtKB / Swiss-Prot, accession No. P30126). The leuD gene (GenBank accession No. NC_000913.3; nucleotide positions: The leuD gene is part of the leuABCD operon. The nucleotide sequence of the leuD gene and the LeuD tag encoded by the leuD gene are shown in Table 1. The amino acid sequences of the proteins are shown in SEQ ID NOs: 9 and 10, respectively.

[0082] LeuC and LeuD are 3-isopropylmalate dehydratase LeuCD (also known as 3-isopropylmalate isomerase).

[0083] 3-Isopropylmalate dehydratase activity " refers to the following reaction: (2R,3S)-3-isopropylmalate ⇔ (2S)-2-isopropylmalate (EC: 4.2.1.33) can refer to the enzymatic activity that catalyzes

[0084] That is, the leuA, leuB, leuC, and leuD genes may be genes (e.g., DNA) having the nucleotide sequences shown in SEQ ID NOs: 3, 5, 7, and 9, and the LeuA, LeuB, LeuC, and LeuD genes may be genes (e.g., DNA) having the nucleotide sequences shown in SEQ ID NOs: 3, 5, 7, and 9. The protein has the amino acid sequence shown in SEQ ID NOs: 4, 6, 8, and 10. good.

[0085] Protein concentration can be determined by the Bradford protein assay using Coomassie dye with bovine serum albumin (BSA) as a standard, or by the Lowry method (Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 1976, 72:248-254; Lowry OH et al., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 1951, 193:265-275), or by Western blot analysis (Hirano S., Western blot analysis, Methods Mol. Biol., 2012, 926:87-97).

[0086] For example, the following description of attenuated gene expression, mutant proteins, and mutant nucleotide sequences can be applied mutatis mutandis to any of the proteins and genes described herein (including, but not limited to, TyrB, LeuA, LeuB, LeuC, LeuD, KdcA, and AldH proteins, and the genes encoding them, such as the tyrB, leuA, leuB, leuC, leuD, kdcA, and aldH genes, and their homologs).

[0087] The term "a bacterium modified to attenuate expression of a gene" can mean that the bacterium has been modified such that expression of a gene is attenuated in the modified bacterium. Expression of a gene can be attenuated, for example, by inactivation of the gene.

[0088] The term "gene is inactivated" can mean that the modified gene encodes a protein that is completely inactive or non-functional compared to the wild-type or unmodified gene. The modified DNA region can be a deletion of part of a gene or a deletion of the entire gene, or a deletion of the gene from which the gene is encoded. Substitution of one or more bases (missense mutation) resulting in amino acid substitution of the protein Codon introduction (nonsense mutation), resulting in a reading frame shift of the gene deletion of one or two bases, insertion of a drug resistance gene and / or a transcription termination signal, or The gene may not be naturally expressed by modifying the adjacent region of the gene (which includes sequences that control gene expression, such as promoters, enhancers, attenuators, and ribosome binding sites). Gene inactivation can be achieved, for example, by UV irradiation or mutagenesis using nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), site-directed mutagenesis, or other methods. insertion, gene disruption using homologous recombination, and / or "Red / ET-driven integration" or insertion-deletion mutagenesis based on "λRed / ET-mediated integration" (Yu D. et al., Recombination can be carried out by conventional methods such as those described in "An efficient recombination system for chromosome engineering in Escherichia coli," Proc. Natl. Acad. Sci. USA, 2000, 97(11):5978-5983; Datsenko KA and Wanner BL, "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products," Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645; Zhang Y. et al., "A new logic for DNA engineering using recombination in Escherichia coli," Nature Genet., 1998, 20:123-128).

[0089] The term "bacteria modified to attenuate expression of a gene" refers to the modified bacterium being modified to attenuate expression of a gene by attenuating the expression of the gene from the control regions (which may include promoters, enhancers, operators, attenuators and termination signals, ribosome binding sites, and other expression control elements) operably linked to the gene. and other examples (e.g., WO9534672 A1; Carrier TA and Keasling JD, Library of synthetic 5' secondary structures to (See, e.g., manipulate mRNA stability in Escherichia coli, Biotechnol. Prog., 1999, 15:58-64).

[0090] The term "operably linked to the gene" when referring to a regulatory region means that the regulatory region controls (e.g., enhances, increases, constitutively regulates) the expression of the nucleotide sequence. "Conservative" can mean linked to the base sequence of a nucleic acid molecule or gene so as to achieve expression of a gene product encoded by the base sequence, specifically, normal, basal, anti-terminating, attenuated, deregulated, reduced, or suppressed expression, specifically, expression of a gene product encoded by the base sequence.

[0091] The term "a bacterium modified to attenuate gene expression" can also mean that the bacterium has been modified so that the expression level (i.e., amount of expression) of the gene in the modified bacterium is reduced compared to an unmodified strain (e.g., a wild-type strain or a parent strain). A reduction in the expression level of a gene can be measured, for example, as a reduction in the expression level of the gene per cell (which may be the average expression level of the gene per cell). The term "expression level of a gene" or "expression amount of a gene" can mean, for example, the amount of the expression product of the gene (e.g., the amount of mRNA of the gene or the amount of the protein encoded by the gene). The bacterium may have an expression level of the gene per cell that is, for example, 50% or less, 20% or less, 10% or less, 5% or less, or less than that of an unmodified bacterium. or may be modified to reduce it to 0%.

[0092] The term "bacteria modified to attenuate expression of the metJ gene" can mean that the bacterium has been modified such that the total amount and / or total activity of the corresponding gene product (i.e., the encoded protein) is reduced in the modified bacterium compared to the unmodified bacterium. A reduction in the total amount or activity of the protein can be measured, for example, as a reduction in the amount or activity of the protein per cell (which can be the average amount or activity of the protein per cell). The bacterium can be modified such that the activity of the protein per cell is, for example, 50% of that of the unmodified bacterium. It may be modified to reduce it to 20% or less, 10% or less, 5% or less, or 0%.

[0093] Examples of unmodified bacteria used for the comparison include wild-type strains of bacteria belonging to the genus Escherichia, such as E. coli MG1655 (ATCC 47076) and E. coli W3110 (ATCC 27325), and wild-type strains of bacteria belonging to the genus Pantoea, such as P. ananatis AJ13355 (FERM BP-6614). The unmodified bacteria to be subjected to the method also include parent strains that have not been modified to attenuate gene expression or bacteria in which gene expression has not been attenuated.

[0094] Gene expression is controlled by weaker promoters and other gene expression control sequences on chromosomal DNA. The promoter strength can be weakened by substituting It is defined by the frequency of initiation. An example of a method for assessing promoter strength is given in Goldstein MA. (Goldstein MA and Doi RH, Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)) and the like. As disclosed in

[1999] , promoters can also be weakened by introducing one or more base substitutions into the promoter region of a gene. Furthermore, the number of base substitutions in the Shine-Dalgarno (SD) sequence, and / or the spacer between the SD sequence and the start codon, and / or the sequence immediately upstream or downstream of the start codon in the ribosome binding site (RBS) can be reduced. It is known that substitution of a single nucleotide significantly affects the translation efficiency of mRNA, and modification of this RBS may result in a decrease in gene transcription.

[0095] The expression of a gene can be specifically expressed by, for example, the coding region of the gene (US Pat. No. 5,175,107) or the gene itself. By inserting a transposon or insertion sequence (IS) into the region that controls gene expression or ultraviolet irradiation or nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine It can also be weakened by conventional methods such as mutagenesis using NTG. The incorporation of specific mutations can be achieved by known dye transfer methods, for example, based on λRed / ET-mediated recombination (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645). This can be done by color body editing.

[0096] The copy number of a gene or the presence or absence of a gene can be determined, for example, by restriction digestion of chromosomal DNA. Then, Southern blotting using probes based on the gene sequence or fluorescent in It can be measured by performing fluorescence in situ hybridization (FISH) or other methods. The level of gene expression can be determined by measuring the amount of mRNA transcribed from the gene using a variety of well-known methods, such as Northern blotting or quantitative RT-PCR. The amount of protein encoded by the gene can be measured by well-known methods, such as SDS-PAGE followed by immunoblotting (Western blotting) or mass spectrometry of the protein sample.

[0097] Preparation of plasmid DNA, DNA cleavage, DNA ligation, DNA transformation, selection of oligonucleotides as primers, introduction of mutations, and other DNA recombinant molecular manipulation and molecular cloning. The method for cloning may be a conventional method well known to those skilled in the art. Such a method is described, for example, in Sambrook J., Fritsch EF and Maniatis T., "Molecular Cloning: A Laboratory Manual”, 2 nded., Cold Spring Harbor Laboratory Press (1989) or Green MR and Sambrook JR, “Molecular Cloning: A Laboratory Manual”, 4 th ed., Cold Spring Harbor Laboratory Press (2012); Bernard R. Glick, Jack J. Pasternak and Cheryl L. Patten, “Molecular Biotechnology: principles and applications of recombinant DNA”, 4 th ed., Washington, DC, ASM Press (2009).

[0098] Any method can be used for manipulation using recombinant DNA, including conventional methods such as transformation, transfection, infection, conjugation, and mobilization. Transformation, transfection, infection, conjugation, or mobilization of bacteria with DNA encoding a protein can confer the bacteria the ability to synthesize the protein encoded by the DNA. Methods for transformation, transfection, infection, conjugation, and mobilization include any method. For example, for efficient DNA transformation and transfection, E. coli K-12 cells can be transformed to be more permeable to DNA. A method of treating cells with calcium chloride has been reported (Mandel M. and Higa A., Calcium-dependent bacteriophage DNA infection, J. Mol. Biol., 1970, 53:159-162). Specialized and / or generalized transformation methods have been described (Morse ML et al., Transduction in Escherichia coli K-12, Genetics, 1956, 41(1):142-156; Miller JH, Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor La. Press, 1972). Random and / or targeted transfer of DNA into the host microorganism. Other methods for integration can be applied, such as "Mu-driven integration / amplification" (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871), "Red / ET-driven integration" or "λRed / ET-mediated integration" (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA 2000, 97(12):6640-45; Zhang Y., et al., Nature Genet., 1998, 20:123-128). Furthermore, for multiple insertion of desired genes, Mu-driven replicative transposition (Akhverdyan et al., Appl. Microbiol. Biotechnol., 2011, 91:857-871) and chemically induced recA-dependent homologous recombination leading to amplification of the desired gene. Inducible chromosome evolution (Tyo KEJ et al., Nature Biotechnol., 2009, 27:760-765) Additionally, other methods utilizing various combinations of transposition, site-specific and / or homologous Red / ET-mediated recombination, and / or P1-mediated generalized transduction (see, e.g., Minaeva N. et al., BMC Biotechnology, 2008, 8:63; Koma D. et al., Appl. Microbiol. Biotechnol., 2012, 93(2):815-829) can be used.

[0099] There may be differences in DNA sequences between families, genera, species, or strains within the order Enterobacteriaceae. Thus, the tyrB, leuA, leuB, leuC, and leuD genes are not limited to the genes having the nucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7, and 9, but may also include variant nucleotide sequences relative to SEQ ID NOS: 1, 3, 5, 7, and 9. The present invention may also include genes having or homologous thereto that encode variants of the TyrB, LeuA, LeuB, LeuC, and LeuD proteins. Similarly, the TyrB, LeuA, LeuB, LeuC, and LeuD proteins may be genes having the amino acid sequences shown in SEQ ID NOs: 4, 6, 8, and 10. Proteins having, but not limited to, variant amino acid sequences of SEQ ID NOs: 4, 6, 8, and 10, or or proteins homologous thereto.

[0100] The term "variant protein" may refer to a protein having a variant amino acid sequence.

[0101] The term "mutant protein" refers to a protein that has one or more amino acid sequences that differ from the wild-type amino acid sequence of the protein. is a single amino acid sequence, whether it is a substitution, deletion, insertion, and / or addition of several amino acid residues. The term "mutant" may refer to a protein having 1 to 150 or more mutations in its amino acid sequence, which maintains the same activity or function as the wild-type protein, or whose three-dimensional structure is not significantly altered compared to the wild-type or unmodified protein. The number of mutations in a mutant protein depends on the position of the amino acid residue in the three-dimensional structure of the protein or the type of amino acid residue. The number of changes in a mutant protein is not strictly limited, but may be 1 to 150, in another example, 1 to 100, in another example, 1 to 50, in another example, 1 to 20, in another example, 1 to 20, in another example, 1 to 30, in another example, 1 to 30, in another example, 1 to 40, in another example, 1 to 50, in another example, 1 to 150, in another example, 1 to 100, in another example, 1 to 50, in another example, In some cases, it is 1 to 30, in other cases it is 1 to 15, in other cases it is 1 to 10, or in other cases it is 1 to 5. This is because some amino acids have a high degree of homology to one another, and such changes do not affect activity or function or significantly alter the three-dimensional structure of the protein relative to the wild-type or unmodified protein. Thus, a mutant protein may have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of the entire wild-type amino acid sequence of a protein, as long as the activity or function is maintained or the three-dimensional structure of the protein is not significantly altered relative to the wild-type or unmodified protein. Above, or above 99%, the parameter " The homology may be a protein having an amino acid sequence that has homology defined as "identity." As used herein, the term "homology" may mean "identity" (which is identity between amino acid residues). Sequence identity between two sequences is calculated as the percentage of matching residues between the two sequences when the two sequences are aligned for maximum correspondence.

[0102] Examples of substitution, deletion, insertion, and / or addition of one or several amino acid residues include conservative mutations so that the activity and characteristics of the mutant protein are maintained or the three-dimensional structure of the protein does not change significantly compared to the unmodified protein (e.g., wild-type protein, etc.). A typical conservative mutation is a conservative substitution. Conservative substitutions are not limited to, but include, when the substitution site is an aromatic amino acid, those between Phe, Trp, and Tyr. If the amino acid is a hydrophobic amino acid, it is between Ala, Leu, Ile, and Val. If the substitution site is a hydrophilic amino acid, it is between Glu, Asp, Gln, Asn, Ser, His, and Thr. If the substitution site is a polar amino acid, it is between Ala, Leu, Ile, and Val. between Gln and Asn in the case of a basic amino acid substitution, and between Lys, Arg, and His in the case of a basic amino acid substitution. When the substitution site is an acidic amino acid, Asp and Glu are substituted with each other, and when the substitution site is an amino acid having a hydroxyl group, Ser and Thr are substituted with each other. Examples of conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His or Lys, substitution of Asn with Glu, Gln, Lys, His or Asp, substitution of Asp with Asn, Glu or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp or Arg, substitution of Glu with Asn, Gln, Lys or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg or Asp. or Tyr substitution; Ile to Leu, Met, Val or Phe substitution; Leu to Ile, Met, Val or Phe substitution; Lys to Asn, Glu, Gln, His or Arg substitution; Met to Ile, Leu, Val or substitution of Phe; substitution of Phe with Trp, Tyr, Met, Ile, or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe, or Trp; and substitution of Val with Met, Ile, or Leu.

[0103] Examples of substitution, deletion, insertion, and / or addition of one or several amino acid residues also include non-conservative mutations, provided that the mutations are at one or more different positions in the amino acid sequence. Further secondary mutations may maintain the activity or function of the mutant protein or may result in a The modification is performed so that the three-dimensional structure of the protein is not significantly altered relative to the unmodified protein (eg, wild-type protein).

[0104] The percentage of identity of amino acid sequences can be calculated using the blastp algorithm. More specifically, the percentage of identity of amino acid sequences can be calculated using the blastp algorithm provided by the National Center for Biotechnology Information (NCBI) with default settings. The percentage of identity between base sequences can be calculated using the scoring parameters (Matrix: BLOSUM62; Gap Costs: Existence = 11, Extension = 1; Compositional Adjustments: Conditional compositional score matrix adjustment). The percentage of identity between base sequences can be calculated using the blastn algorithm. More specifically, the percentage of identity between base sequences can be calculated using the blastn algorithm provided by NCBI with default scoring parameters (Match / Mismatch Scores = 1, -2; Gap Costs = Linear).

[0105] As mentioned above, tyrosine aminotransferase activity specific to various bacteria belonging to the order Enterobacteriaceae Protein homologs of TyrB having the following structure are known. Examples of such homolog proteins specific to bacteria belonging to the order Enterobacteriaceae are listed in the NCBI database (National Center for Biotechnology Information, ncbi.nlm.nih.gov / protein / ) under the accession numbers of their amino acid sequences: , phylogenetic data, along with an indication of homology values ​​(as "identity," which is amino acid identity) are shown in Table 1 .

[0106] [Table 1]

[0107] The term "variant sequence" refers to any variant sequence according to the standard genetic code (see, e.g., Lewin B., "Genes VIII", 2004, Pearson Education, Inc., Upper Saddle River, NJ 07458). Therefore, a gene encoding a protein having a wild-type amino acid sequence can be used to refer to a base sequence encoding a protein having a wild-type amino acid sequence using synonymous amino acid codons. The gene may be a gene having a variant base sequence due to the degeneracy of the genetic code.

[0108] The term "mutant nucleotide sequence" includes, but is not limited to, a mutant nucleotide sequence having tyrosine aminotransferase activity. As long as the term encodes a protein having the desired sequence, it can also refer to a nucleotide sequence that can hybridize under stringent conditions to a nucleotide sequence complementary to the wild-type nucleotide sequence or a probe that can be prepared from the nucleotide sequence. The term "stringent conditions" can encompass conditions under which specific hybrids are formed, for example, hybrids with a homology defined as the parameter "identity" when using the computer program blastn, of 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, and non-specific hybrids, for example, hybrids with lower homology than the above, are not formed. Stringent conditions include, for example, salt concentrations of 1x SSC (standard sodium citrate or standard sodium chloride) and 0.1% SDS (sodium dodecyl sulfate), and in another example, 0.1 × SSC, 0.1% SDS, at 60°C or 65°C, for one or more times, or for two or more times. For example, wash three times. The wash time depends on the type of membrane used for blotting, but generally follows the manufacturer's recommendation. For example, Amersham Hybond TM -N+ positively charged nylon membrane (GE Healthcare) stringent conditions The recommended washing time is 15 minutes. The washing step can be performed two or three times. A portion of the sequence complementary to the wild-type nucleotide sequence may be used as a probe. Such a probe can be prepared by PCR using oligonucleotides prepared based on the wild-type nucleotide sequence as primers and a DNA fragment containing the nucleotide sequence as a template. It is recommended that the probe length be more than 50 bp, but the length may vary depending on the hybridization conditions. The length can be selected within the range of 100 bp to 1 kbp, and is usually 100 bp to 1 kbp. For example, when a DNA fragment having a length of about 300 bp is used as a probe, washing conditions after hybridization can be, for example, 2×SSC, 0.1% SDS at 50°C, 60°C, or 65°C.

[0109] The term "mutant nucleotide sequence" may also refer to a nucleotide sequence that encodes a mutant protein.

[0110] The nucleotide sequence of the gene encoding the wild-type protein specific to the E. coli species has already been determined. Therefore, the mutant nucleotide sequence encoding the mutant protein of the wild-type protein was obtained by using the DNA of the same bacterial species and primers prepared based on the nucleotide sequence of the wild-type gene. The wild-type gene can be isolated by PCR (polymerase chain reaction; refer to White TJ et al., The polymerase chain reaction, Trends Genet., 1989, 5(6):185-189) or by site-directed mutagenesis, in which DNA containing the wild-type gene is treated in vitro with, for example, hydroxylamine. The microorganism carrying the gene, e.g., E. coli species, is exposed to ultraviolet (UV) irradiation or similar treatment. These proteins can be obtained by treatment with mutagens commonly used in bacteria, such as N-methyl-N'-nitro-nitrosoguanidine (NTG) or nitrous acid, or can be chemically synthesized as full-length gene constructs. Genes encoding proteins or mutant proteins from other bacteria in the order Enterobacteriaceae can also be obtained in a similar manner.

[0111] The term "wild-type" (which means "native") when referring to proteins (e.g., "wild-type proteins") and genes (e.g., "wild-type genes") is used. " and "natural" respectively refer to wild-type bacteria (e.g. For example, E. coli MG1655 strain (ATCC 47076), E. coli W3110 strain (ATCC 27325), P. ananatis A "wild-type protein" may refer to native proteins and genes that are naturally present and / or expressed and / or naturally produced in a wild-type strain of bacteria belonging to the order Enterobacteriaceae, such as Enterobacteriaceae or Erwiniaceae, such as strain AJ13355 (FERM BP-6614). Because proteins are encoded by genes, a "wild-type protein" may be encoded by a "wild-type gene" that occurs naturally in the genome of a wild-type bacterium.

[0112] The term "wild-type protein" refers to a protein that is expressed by a wild-type or parent strain belonging to the order Enterobacteriaceae, e.g. It is naturally produced by wild-type strains E. coli MG1655 and P. ananatis AJ13355. It may refer to a native protein. A wild-type protein may be encoded by a "wild-type gene" or by an "unmodified gene" that occurs naturally in the genome of a wild-type bacterium. Wild-type proteins and genes may therefore have a "wild-type amino acid sequence" and a "wild-type nucleotide sequence" as the primary structure of the protein and gene.

[0113] The term "native to" when referring to a protein or nucleic acid that is native to a particular organism (e.g., a bacterial species, etc.) can mean a protein or nucleic acid that is native to that organism. That is, a protein or nucleic acid unique to a particular organism may refer to a protein or nucleic acid, respectively, that naturally occurs in that organism and can be isolated and sequenced from that organism by methods known to those skilled in the art. Furthermore, since the amino acid sequence or nucleotide sequence of a protein or nucleic acid, respectively, isolated from an organism in which the protein or nucleic acid exists can be easily determined, the term "unique" when referring to a protein or nucleic acid may also refer to a protein or nucleic acid obtained, for example, by genetic engineering techniques, including recombinant DNA technology, or chemical synthesis, as long as the resulting amino acid sequence or nucleotide sequence of the protein or nucleic acid is identical to the amino acid sequence or nucleotide sequence of a protein or nucleic acid naturally occurring in the organism. Amino acid sequences unique to a particular organism include, but are not limited to, peptides, oligopeptides, polypeptides (which include proteins, specifically enzymes), and the like. Examples of nucleotide sequences specific to a particular organism include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including, but not limited to, expression regulatory sequences (including promoters, attenuators, terminators, etc.), genes, intergenic sequences, and nucleotide sequences encoding signal peptides, protein pro-regions, and artificial amino acid sequences. Specific examples of amino acid sequences and nucleotide sequences and their homologs specific to various organisms are described herein, including, for example, proteins having the amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, and 10, respectively (these are expressed in E. coli species). The salts shown in SEQ ID NOS: 1, 3, 5, 7, and 9 may be specific to bacteria. (It may be encoded by a gene having the base sequence).

[0114] In addition to the above-mentioned properties, the bacteria may have specific properties such as various nutritional requirements, drug resistance, drug sensitivity, drug dependence, etc., without departing from the scope of the present invention.

[0115] 2. Method The methods described herein include methods for producing 2-methylbutyric acid using the bacteria described herein. The method for producing 2-methylbutyric acid includes cultivating the bacteria in a culture medium (also referred to as "culturing") to produce, excrete, and / or accumulate 2-methylbutyric acid in the culture medium, the bacterial cells, or both, and recovering the 2-methylbutyric acid from the culture medium and / or the bacterial cells. The method may further optionally include purifying the 2-methylbutyric acid from the culture medium and / or the bacterial cells. 2-Methylbutyric acid may be produced in the free form, a salt thereof, or a mixture thereof. Thus, the term "2-methylbutyric acid" may refer to, for example, 2-methylbutyric acid in the free form, a salt thereof, or a mixture thereof. For example, salts of 2-methylbutyric acid, such as sodium salt, potassium salt, or ammonium salt, may be produced by the method. This is possible because carboxylic acids (to which 2-methylbutyric acid belongs) can react under fermentation conditions with neutralizing agents such as inorganic or organic substances to form salts in a typical acid-base neutralization reaction, which is a chemical characteristic of carboxylic acids that is clear to those skilled in the art.

[0116] The cultivation of bacteria, and the recovery and optional purification of 2-methylbutyric acid from the medium, etc. can be carried out in the same manner as in conventional fermentation methods for producing L-amino acids using microorganisms. That is, the cultivation of bacteria, and the recovery and purification of 2-methylbutyric acid from the medium, etc. can be carried out under conditions suitable for the cultivation of bacteria and conditions suitable for the recovery and purification of L-amino acids, which are well known to those skilled in the art. This may be done by using

[0117] The medium used is not particularly limited as long as it contains at least a carbon source and allows the bacteria described herein to grow and produce 2-methylbutyric acid. The medium may be a synthetic or natural medium, such as a typical medium containing a carbon source, a nitrogen source, a sulfur source, a phosphorus source, inorganic ions, and other organic and inorganic components as needed. Carbon sources include sugars such as glucose, sucrose, lactose, galactose, fructose, arabinose, maltose, xylose, trehalose, ribose, and starch hydrolysates; alcohols such as ethanol, glycerol, mannitol, and sorbitol; organic acids such as gluconic acid, fumaric acid, citric acid, malic acid, and succinic acid; and fatty acids. Nitrogen sources include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen sources such as soybean hydrolysates; ammonia gas; and aqueous ammonia. Peptone, yeast extract, meat extract, malt extract, and corn steep liquor can also be used. The medium may contain one or more of these nitrogen sources. Examples of sulfur sources include ammonium sulfate, magnesium sulfate, iron sulfate, and manganese sulfate. The medium may contain a phosphorus source in addition to a carbon source, a nitrogen source, and a sulfur source. Phosphorus sources that can be used include potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and phosphate polymers such as pyrophosphate. Vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12, as well as other necessary substances, such as adenine, nucleic acids such as RNA, amino acids, peptone, casamino acids, and organic nutrients such as yeast extract, can be present in appropriate amounts (even trace amounts). In addition to these, small amounts of calcium phosphate, iron ions, manganese ions, and the like may be added, if necessary.

[0118] The culturing can be carried out under conditions suitable for culturing the bacteria used in the method for producing 2-methylbutyric acid. For example, the culturing can be carried out under aerobic conditions for 16 to 72 hours or 16 to 65 hours. The culture temperature during the culturing can be controlled within the range of 30 to 45°C or 30 to 37°C. The pH can be adjusted between 5 and 8 or between 6.0 and 7.5. The pH can be adjusted using inorganic or organic acidic or alkaline substances, such as urea, calcium carbonate, or ammonia gas.

[0119] After cultivation, 2-methylbutyric acid can be recovered from the medium. Specifically, 2-methylbutyric acid present outside the bacterial cells can be recovered from the medium. Furthermore, after cultivation, 2-methylbutyric acid can be recovered from the bacterial cells. Specifically, the bacterial cells are disrupted, and solids such as the bacterial cells and the disrupted bacterial cell suspension (also known as cell debris) are removed to obtain a supernatant, from which 2-methylbutyric acid can be recovered. Disruption of the bacterial cells can be carried out by well-known methods, such as ultrasonic disruption using high-frequency sound waves. Solids can be removed by centrifugation or membrane filtration. 2-Methylbutyric acid can be recovered from the medium, supernatant, etc., by conventional techniques, such as concentration, crystallization, membrane treatment, ion exchange chromatography, flash chromatography, thin-layer chromatography, and medium- or high-pressure liquid chromatography. These methods may be used alone or in appropriate combinations. [Example]

[0120] The invention will now be more precisely described with reference to the following non-limiting examples.

[0121] Example 1 Construction of E. coli strain L1190-1 A 2-methylbutyrate-producing strain was constructed from the L-isoleucine-producing E. coli strain NS1547. NS1547 was cloned as MG1655 Δtdh rhtA* mini-Mu::P lac -laсI-ilvA* P L -SD1-ilvG*MEDA genotype.

[0122] The NS1547 strain has a mutation in the rhtA gene (rhtA23) (US20010049126 A1) that confers tolerance to high concentrations of threonine (>40 mg / mL) or homoserine (>5 mg / mL) and improves threonine production, and a deletion of the tdh gene (SEQ ID NO: 11) inhibits threonine degradation (Shakalis IO and Gusyatiner MM, Participation of threonine dehydrogenase, threonine desaminase, and serine transhydroxymethylase in threonine degradation). Escherichia coli cells K-12, Biotekhnologiya, 1990, (2):16-17). The NS1547 strain was engineered with the expression cassette mini-Mu::P lac -laсI-ilvA*-kan (SEQ ID NO: 12) (Sycheva EV et al., Aerobic catabolism of threonine in Escherichia coli strain with feedback resistant biosynthetic threonine deaminase, Biotekhnologiya, 2003, (4):22-34), The set contains a feedback-resistant threonine deaminase. The mutant ilvA gene and the lacI repressor gene were inserted into P lac The cassette contains the gene for threonine deaminase (which converts threonine to 2-ketobutyrate) under the control of the promoter. The NS1547 strain contains the expression cassette P L -SD1-ilvG*MEDA (SEQ ID NO: 13), the cassette containing SD1 and P of λ phage operably linked to the designated modified Shine-Dalgarno sequence (SD sequence from pET22b(+) plasmid (Novagen)). L of the ilvG*MEDA operon under the control of the promoter This artificial operon (ilvG*MEDA) contains a mutant ilvG gene (ilvG*). The gene has two base pairs (aa) inserted between bases at positions 981 and 982 from the start codon of the gene, i.e., upstream of the sequence TGActggca (EP1627884 B1). This restores the frameshift in the wild-type ilvG gene, resulting in the expression of feedback-resistant acetolactate synthase II.

[0123] Cassette dacA::mini-Mu::cat-P thrThe NS1547 strain was transduced with the threonine operon-attB-thrA*BC (SEQ ID NO: 14), which provides expression of the threonine operon with the region required for attenuation deleted. The threonine operon contains a mutation in the thrA gene (thrA442) (Akhverdyan VZ et al., Development of the mini-Mu system providing effective integration and amplification of the genetic material into the Escherichia coli chromosome, Biotekhnologiya, 2007, (3):3-20), which renders aspartokinase I-homoserine dehydrogenase I insensitive to feedback inhibition by threonine. This cassette was placed into the dacA gene of E. coli and flanked by the phage mini-Mu L / R ends for mini-Mu-mediated transfer (Akhverdyan VZ et al., Application of the bacteriophage Mu-driven system for the integration / amplification of target genes in the chromosomes of engineered Gram-negative bacteria). - mini review, Appl. Microbiol. Biotechnol., 2011, 91(4):857-871), and chloramphenicol resistance (Cm R ) marker is inserted.

[0124] Cm RThe transductants were cultured in LB medium (Sambrook, J. and Russell, DW "Molecular Cloning: A Laboratory Manual”, 3 rd ed., Cold Spring Harbor Laboratory Press (2001) The strains were selected on plates containing Luria-Bertani medium (also known as Luria-Bertani medium, as described previously), agar (1.5%), and Cm (20 mg / L) and confirmed by PCR using primers P1 (SEQ ID NO: 15) and P2 (SEQ ID NO: 16). The PCR validation conditions were as follows: a 5-minute denaturation step at 94°C; a 25-cycle profile of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. The length of DNA fragment 1 (SEQ ID NO: 17) obtained by the reaction using the DNA of the parent strain NS1547 as a template was 1256 bp. thr The length of DNA fragment 2 (SEQ ID NO: 18) obtained by the reaction using the NS1547 dacA::mini-Mu::cat-P thr -attB-thrA*BC strain Cm R Marker excision via λ-Int / Xis As a result, strain L1178-1 was obtained. The excision was confirmed by PCR as described above. Strain NS1547 dacA::mini-Mu::cat-P thr The length of DNA fragment 2 (SEQ ID NO: 18) obtained by the reaction using the cell DNA of the L1178-1 strain as a template was 2452 bp. The length of DNA fragment 3 (SEQ ID NO: 19) obtained in this reaction was 842 bp.

[0125] Expression cassette ΔcynX::cat-P L-ilvA* (SEQ ID NO: 20) was introduced into strain L1178-1 by P1 transduction. This cassette was placed in the cynX gene of E. coli, and the mutant ilvA* gene Child (ilvA 1237 ) (This is due to the substitution of g with a at position 1237 from the start codon of the gene, resulting in Glu 412 The L-isoleucine overproducing strain of Escherichia coli K-12, which was replaced by Lys (Hashiguchi K. et al., Construction of an L-isoleucine overproducing strain of Escherichia coli K-12, Biosci. Biotechnol. Biochem., 1999, 63(4):672-679), introduced a feedback-resistant threonine deaminase into the P L (encoding a gene under the control of a promoter).

[0126] Cm R Transductants were selected and amplified using primers P3 (SEQ ID NO: 21) and P4 (SEQ ID NO: 22). The PCR conditions were as follows: a 5-minute denaturation step at 94°C; The profile was 25 cycles of 94°C for 30 seconds, 59°C for 30 seconds, and 72°C for 1 minute; with a final extension of 7 minutes at 72°C. No DNA fragments were observed in the reaction using the DNA of the parent strain L1178-1 as a template. ΔcynX::cat-P L The length of DNA fragment 4 (SEQ ID NO: 23) obtained by the reaction using the DNA of the -ilvA* strain cells as a template was 2131 bp. L -ilvA* strain Cm R The marker was excised via λ-Int / Xis, resulting in the L1190-1 strain. The parent strain L1178-1 ΔcynX::cat-P was confirmed by PCR. LThe length of DNA fragment 4 (SEQ ID NO: 23) obtained by the reaction using the -ilvA* bacterial cell DNA as a template was 2131 bp. The length of DNA fragment 5 (SEQ ID NO: 24) obtained by the reaction using the L1190-1 bacterial cell DNA as a template was 534 bp.

[0127] Example 2 Construction of E. coli strain L1194-2 For the biosynthesis of 2-methylbutyric acid, the 2-methylbutyric acid biosynthesis gene was introduced into strain L1190-1 (Example 1 The plasmid pAH162-tetA-tetR-kdcA-aldH was used, which contains the heterologous kdcA gene native to Lactococcus lactis, codon-optimized for expression in E. coli. (Savrasova EA et al., Use of the valine biosynthetic pathway to convert glucose into isobutanol, J. Ind. Microbiol. Biotechnol., 2011, 38(9):1287-1294) and the E. coli specific aldH gene. A 3195 bp promoterless DNA fragment of kdcA-aldH (SEQ ID NO: 11) was inserted into the valine biosynthetic pathway (Savrasova EA et al., Use of the valine biosynthetic pathway to convert glucose into isobutanol, J. Ind. Microbiol. Biotechnol., 2011, 38(9):1287-1294) and the E. coli specific aldH gene. No. 25) was digested with MlsI / SalI to prepare the integrative vector pAH162-λattL-Tc R -λattR (Minaeva NI et al., Dual-In / Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure, BMC Biotechnol., 2008, 8:63). +Transformants of the strain (Herrero M. et al., Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria, J. Bacteriol., 1990, 172:6557-6567) were selected on medium supplemented with tetracycline (Tc). Correct insertion was confirmed by restriction analysis and PCR using primers P5 (SEQ ID NO: 26) and P6 (SEQ ID NO: 27) and P7 (SEQ ID NO: 28) and P8 (SEQ ID NO: 29). The PCR verification conditions were as follows: a 5-minute denaturation step at 94°C; a 25-cycle profile of 94°C for 30 seconds, 61°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. The parent strain CC118 λpir + No DNA fragments were observed in the reaction using the DNA of the CC118 λpir strain as a template. + Reaction using the DNA of the pAH162-tetA-tetR-kdcA-aldH strain as a template and primers P5 (SEQ ID NO: 26) and P6 (SEQ ID NO: 27) The length of DNA fragment 6 (SEQ ID NO: 30) obtained in step 1 was 1045 bp. + The length of DNA fragment 7 (SEQ ID NO: 31) obtained by a reaction using the DNA of the pAH162-tetA-tetR-kdcA-aldH strain cells as a template and primers P7 (SEQ ID NO: 28) and P8 (SEQ ID NO: 29) was 865 bp.

[0128] The plasmid pAH162-tetA-tetR-kdcA-aldH was then transferred to E. coli containing the plasmid pAH123. MG1655 Δ(φ80-attB) trs5-10::φ80-attB strain (Minaeva NI et al., Dual-In / Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with The trs5-10::pAH162-tetA-tetR-kdcA-aldH cassette was integrated into the artificial locus trs5-10::φ80-attB by φ80-driven integration. MG1655 Δ(φ80-attB) strain cells carrying the trs5-10::pAH162-tetA-tetR-kdcA-aldH cassette were selected on plates containing LB medium, agar (1.5%), and Tc (40 mg / L). The integration was confirmed to be successful. The sequence of the clone was confirmed by PCR using primers P9 (SEQ ID NO: 32) and P10 (SEQ ID NO: 33). The PCR conditions were as follows: denaturation at 94°C for 5 minutes; 25 cycles of 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. No DNA fragments were observed in the reaction using the DNA of the parent strain MG1655 Δ(φ80-attB) trs5-10::φ80-attB as a template. DNA fragment 8 (SEQ ID NO: 34) obtained in the reaction using the DNA of the MG1655 Δ(φ80-attB) trs5-10::pAH162-tetA-tetR-kdcA-aldH strain as a template was 1629 bp long. The vector portion was excised via λ-Int / Xis to obtain the MG1655 Δ(φ80-attB) trs5-10::kdcA-aldH strain. R The marker was excised using primers P9 (SEQ ID NO: 32) and P11 (SEQ ID NO: 33). The PCR conditions were as follows: 94°C for 5 minutes. denaturation of the DNA from the MG1655 Δ(φ80-attB) trs5-10::pAH162-tetA-tetR-kdcA-aldH strain; 25 cycles of denaturation at 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. The length of DNA fragment 9 (SEQ ID NO: 36) obtained by the reaction using the above as a template was 4603 bp. The length of DNA fragment 10 (SEQ ID NO: 37) obtained by the reaction using the MG1655 Δ(φ80-attB) trs5-10::kdcA-aldH strain cell DNA as a template was 1312 bp.

[0129] Then, to ensure high-level constitutive expression, lac repressor binding sites (Figure 2) were inserted. P tagged with the cat gene, containing half of tac The regulatory region of the promoter (De Boer HA et al., The tac promoter: a functional hybrid derived from the trp and lac promoters, Proc. Natl. Acad. Sci. USA, 1983, 80(1): 21-25) was inserted onto the kdcA-aldH operon by a method called "λRed-dependent integration" developed by Datsenko and Wanner (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97(12): 6640-6645). Following this procedure, the flanking regions of the kdcA gene and the cat gene in the template chromosome were Child and P tac PCR primers P12 (SEQ ID NO: 38) and P13 (SEQ ID NO: 39) were constructed that are homologous to both flanking regions of the promoter. tac -lacZ strain (Katashkina JI (Tuning of expression level of the genes of interest located in the bacterial chromosome, Mol. Biol. (Mosk.), 2005, 39(5):719-726) was used as a template for PCR. The PCR conditions were as follows: denaturation at 95°C for 3 minutes; denaturation at 95°C for 1 minute, 34°C for 30 seconds, and 72°C for 1 minute. The profile of the first two cycles was 80 s at 95°C; the final cycle was 30 s at 95°C, 30 s at 50°C, and 80 s at 72°C. The profile was 28 cycles followed by a final step at 72°C for 5 minutes. The resulting 1768 bp DNA fragment 11 (SEQ ID NO: 40) was purified using a Silica Bead DNA Gel Extraction Kit (Thermo Scientific) and used to electroporate the MG1655 Δ(φ80-attB) trs5-10::kdcA-aldH strain containing the plasmid pKD46. Plasmid pKD46 (Datsenko KA and Wanner BL, Proc. Natl. Acad. Sci. USA, 2000, 97(12):6640-6645) contains a 2154 bp DNA fragment (positions 31088 to 33241) of phage λ (GenBank, accession No. J02459) and encodes the genes for the λRed homologous recombination system (gamma, beta, and exo genes) in an arabinose-inducible P araB Promoter Restrictions The plasmid pKD46 is required for integration of the DNA fragment into the bacterial chromosome.

[0130] Electrocompetent cells were prepared as follows. The MG1655 Δ(φ80-attB) trs5-10::kdcA-aldH strain containing the pKD46 plasmid was cultured overnight at 30°C in LB medium containing ampicillin (100 mg / L), and then transferred to 5 mL of SOB medium (Sambrook J. et al., "Molecular Cloning: A Laboratory Manual," 2012) supplemented with ampicillin (100 mg / L) and L-arabinose (1 mM). nd The culture was diluted 100-fold with PBS (Cold Spring Harbor Laboratory Press (1989)). The cells were cultured at 30°C with aeration (250 rpm) until the OD600 reached approximately 0.6, then concentrated 100-fold and stored on ice. The cells were washed three times with cold deionized water to make them electrocompetent. Electroporation was carried out using 200 mkL of the cells and approximately 100 ng of DNA fragment 11 (SEQ ID NO: 40). The cells were then cultured in 1 mL of SOC medium (Sambrook J. et al., "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press (1989)) at 37°C for 2.5 hours, and then transferred to LB Transfer onto plates containing medium, agar (1.5%), and chloramphenicol (20 μg / mL). The plasmid pKD46 was then cultured at 37°C to select for chloramphenicol-resistant recombinants. To remove the plasmid, the strain was subcultured once on L-agar medium supplemented with Cm (20 μg / mL) at 42°C, and the resulting individual colonies were tested for sensitivity to ampicillin. In this way, the MG1655 Δ(φ80-attB) trs5-10::cat-Ptac-kdcA-aldH strain was selected. tacIntroduction of the promoter was confirmed by PCR using primers P14 (SEQ ID NO: 41) and P6 (SEQ ID NO: 27). The PCR conditions were as follows: denaturation at 94°C for 5 minutes; 25 cycles of 94°C for 30 seconds, 59°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. Parent strain MG1655 Δ(φ80-attB) No DNA fragments were observed in the reaction using trs5-10::kdcA-aldH bacterial DNA as a template. tac The length of DNA fragment 12 (SEQ ID NO: 42) obtained in the reaction using the -kdcA-aldH strain cell DNA as a template was 1030 bp.

[0131] Finally, the expression cassette trs5-10::cat-P tac The trs5-10::cat-Ptac-kdcA-aldH cassette was introduced into the chromosome of the L1190-1 strain (Example 1) by P1 transduction. The L1190-1 strain was selected on plates containing LB medium, agar (1.5%), and Cm (20 mg / L). tac Introduction of the -kdcA-aldH cassette was verified by PCR as described above. R The marker was excised via λ-Int / Xis, resulting in strain L1194-2, which was confirmed by PCR using primers P15 (SEQ ID NO: 43) and P11 (SEQ ID NO: 35). Parent strain L1190-1 trs5-10::cat-P tac DNA obtained by reaction using -kdcA-aldH bacterial DNA as a template The length of fragment 13 (SEQ ID NO: 44) was 2777 bp. The length of DNA fragment 14 (SEQ ID NO: 45) obtained by the reaction was 1167 bp.

[0132] Example 3 Construction of 2-methylbutyric acid producing E. coli strain L1201-1 Expression cassette P L -ilvG*M-ΔilvE::cat-DA was introduced into the L1194-2 strain (Example 2). L The MG1655 strain carrying the -ilvG*M-ΔilvE::cat-DA modification was constructed by the Red-dependent integration method described above. Following this procedure, PCR primers P16 (SEQ ID NO: 46) and P17 (SEQ ID NO: 47) were constructed, which are homologous to both the flanking regions of the ilvE gene in the template plasmid and the gene conferring chloramphenicol resistance. Plasmid pMW118-attL-cat-attR (Katashkina JI) et al., Tuning of expression level of the genes of interest located in the bacterial chromosome, Mol. Biol. (Mosk), 2005, 39(5):719-726) was used as a template for PCR reaction. The PCR conditions were as follows: denaturation at 95°C for 3 minutes; denaturation at 95°C for 1 minute, 34°C for 30 seconds, and 72°C for 1 minute. The profile of the first two cycles was 80 s at 95°C; the final cycle was 30 s at 95°C, 30 s at 50°C, and 80 s at 72°C. Profile after 28 cycles; final extension at 72°C for 5 min. Resulting DNA fragment 15 (1713 bp) (SEQ ID NO: 48) was purified using a Silica Bead DNA Gel Extraction Kit (Thermo Scientific). and MG1655 P containing the plasmid pKD46. L -SD1-ilvG*MEDA strain was electroporated. R Recombinants were selected, and the deletion of the ilvE gene was verified by PCR using primers P18 (SEQ ID NO: 49) and P19 (SEQ ID NO: 50). The PCR verification conditions were as follows: denaturation at 95°C for 3 minutes; 25 cycles of denaturation at 95°C for 30 seconds, 59°C for 30 seconds, and 72°C for 1 minute; Final extension at 7°C for 7 min. Parent strain MG1655 P L-SD1-ilvG*MEDA bacterial DNA was used as a template. The length of the DNA fragment 16 (SEQ ID NO: 51) obtained was 1354 bp. L The length of DNA fragment 17 (SEQ ID NO: 52) obtained in the reaction using the DNA of the -ilvG*M-ΔilvE::cat-DA strain cells as a template was 2015 bp.

[0133] Then, the expression cassette P L The ΔilvG*M-ΔilvE::cat-DA was introduced into the L1194-2 strain by P1 transduction. L Cm of strain L1194-2 carrying -ilvG*M-ΔilvE::cat-DA R Select the bacteria The cassette P L -SD1-ilvG*MEDA is a cassette P L -ilvG*M-ΔilvE::cat The substitution with -DA was confirmed by PCR as described above. R The marker was excised via λ-Int / Xis, resulting in the IlvE deletion strain L1201-1. Excision was confirmed by PCR using primers P18 (SEQ ID NO: 49) and P19 (SEQ ID NO: 50) as described above. P L DNA fragment 17 (SEQ ID NO: 1) obtained by the reaction using the DNA of the -ilvG*M-ΔilvE::cat-DA cells as a template The length of DNA fragment 18 (SEQ ID NO: 53) obtained by the reaction using the DNA of the L1201-1 strain as a template was 405 bp.

[0134] Example 4 Construction of E. coli L1201-1 ΔtyrB::cat strain A deletion of the tyrB gene in the chromosome of E. coli strain MG1655 (ATCC 47076) was constructed using the Red-dependent integration method described above. Following this procedure, PCR primers P20 (SEQ ID NO: 54) and P21 (SEQ ID NO: 55) were constructed, which are homologous to both the flanking regions of the tyrB gene and the gene conferring chloramphenicol resistance in the template plasmid. The plasmid pMW118-attL-cat-attR was used as the template for the PCR reaction. The PCR conditions were as follows: 95°C for 3 minutes, followed by denaturation. activity; a profile of 2 initial cycles at 95°C for 1 min, 34°C for 30 s, and 72°C for 80 s; a profile of 28 final cycles at 95°C for 30 s, 50°C for 30 s, and 72°C for 80 s; and a final extension at 72°C for 5 min. The resulting DNA fragment 19 (1713 bp) (SEQ ID NO: 56) was purified using a Silica Bead DNA Gel Extraction Kit (Thermo Scientific) and used to electroporate E. coli MG1655 strain containing the plasmid pKD46. R Select the recombinants and identify Cm in the selected mutants. R The deletion of the genetically tagged tyrB gene was verified by PCR using primers P22 (SEQ ID NO: 57) and P23 (SEQ ID NO: 58). The PCR verification conditions were as follows: denaturation at 95°C for 3 minutes; The PCR reaction profile was 25 cycles of 55°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; with a final extension of 7 minutes at 72°C. DNA fragment 20 (SEQ ID NO: 59) obtained in the reaction using the parent strain MG1655 cell DNA as a template was 1,430 bp in length. DNA fragment 21 (SEQ ID NO: 60) obtained in the reaction using the MG1655 ΔtyrB::cat cell DNA as a template was 1,894 bp in length. In this way, the MG1655 ΔtyrB::cat strain was obtained.

[0135] The MG1655 ΔtyrB::cat strain was transformed into the L1201-1 strain (Example 3) by P1 transfection of the tyrB gene deletion. CmR The recombinants were selected, and the deletion of the tyrB gene in the selected mutants was verified by PCR as described above. was obtained.

[0136] Example 5 Production of 2-methylbutyric acid and by-products using E. coli L1201-1 ΔtyrB::cat strain Production The modified strain L1201-1 ΔtyrB::cat and the control strain L1201-1 were cultured in LB medium at 37°C for 6 hours. Next, 0.1 mL of the resulting culture was inoculated into 2 mL of fermentation medium (supplemented with Ile, Val, and Leu (200 mg / L each) for the L1201-1 strain; supplemented with Ile, Val, Leu, Tyr, and Phe (200 mg / L each) for the L1201-1 ΔtyrB::cat strain) in a 20 × 200-mm test tube and cultured at 30°C for 66 hours on a rotary shaker at 238 rpm. After the culture, the accumulated 2 The accumulated 3-methylbutyric acid and isobutyric acid were measured by GC (gas chromatography) analysis (Preparatory Example 1). The measurement was carried out by the analysis (Preparatory Example 2).

[0137] The results of three independent test tube fermentations are shown in Table 2. As can be seen from Table 2, the modified strain L1201-1 ΔtyrB::cat was able to accumulate a reduced amount of 3-methylbutyric acid (3-MB) compared to the control strain L1201-1. As can be seen from Table 2, the modified strain L1201-1 ΔtyrB::cat was able to accumulate a reduced amount of isobutyric acid (IBA) compared to the control strain L1201-1.

[0138] [Table 2]

[0139] Example 6 Construction of E. coli L1201-1 ΔtyrB ΔleuABCD::cat strain A deletion of the leuABCD operon in the chromosome of E. coli MG1655 strain was constructed by the Red-dependent integration method described above. PCR primers homologous to both the flanking regions of the nucleotide sequence and the gene conferring chloramphenicol resistance were identified. The primers P24 (SEQ ID NO: 61) and P25 (SEQ ID NO: 62) were constructed. The plasmid pMW118-attL-cat-attR was used as a template for the PCR reaction. The PCR conditions were as follows: denaturation at 95°C for 3 minutes; The first two cycles were profiled at 95°C for 1 min, 34°C for 30 s, and 72°C for 80 s; the last 28 cycles were profiled at 95°C for 30 s, 50°C for 30 s, and 72°C for 80 s; and a final extension at 72°C for 5 min. The isolated DNA fragment 22 (1713 bp) (SEQ ID NO: 63) was purified using a Silica Bead DNA Gel Extraction Kit (Thermo Scientific) and used to electroporate E. coli MG1655 strain containing the plasmid pKD46. R Select the recombinants and identify Cm in the selected mutants. R The deletion of the gene-tagged leuABCD operon was verified by PCR using primers P26 (SEQ ID NO: 64) and P27 (SEQ ID NO: 65). The PCR verification conditions were as follows: denaturation at 95°C for 3 minutes; 25 cycles of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. DNA fragment 23 (SEQ ID NO: 66) obtained using the parent strain MG1655 cell DNA as a template was 5172 bp in length. DNA fragment 24 (SEQ ID NO: 67) obtained using the MG1655 ΔleuABCD::cat cell DNA as a template was 1868 bp in length. Thus, the MG1655 ΔleuABCD::cat strain was obtained.

[0140] The MG1655 ΔleuABCD::cat strain was used as a donor for P1 transduction of the leuABCD operon deletion into the L1201-1 ΔtyrB strain. R By cutting out the marker The L1201-1 ΔtyrB strain was obtained from the L1201-1 ΔtyrB::cat strain (Example 4). The PCR was confirmed by PCR using the primers P22 (SEQ ID NO: 57) and P23 (SEQ ID NO: 58). The PCR conditions were as follows: denaturation at 95°C for 3 minutes; a 25-cycle profile of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 7 minutes. The DNA of the parent strain L1201-1 ΔtyrB::cat was used as a template. The length of DNA fragment 21 (SEQ ID NO: 60) obtained in the reaction using the DNA of the L1201-1 ΔtyrB strain as a template was 1894 bp. The length of DNA fragment 25 (SEQ ID NO: 68) obtained in the reaction using the DNA of the L1201-1 ΔtyrB strain as a template was 284 bp. R Recombinants were obtained, and the deletion of the leuABCD operon in the selected mutants was verified by PCR as described above, thus obtaining the strain L1201-1 ΔtyrB ΔleuABCD::cat.

[0141] Example 7: Production of 2-methylbutyric acid and its derivatives using E. coli L1201-1 ΔtyrB ΔleuABCD::cat strain Biomaterial production The modified strain L1201-1 ΔtyrB ΔleuABCD::cat and the control strain L1201-1 ΔtyrB::cat were cultured in LB The cells were cultured in the medium at 37°C for 6 hours. Then, 0.1 mL of the resulting culture medium was placed in a 20 × 200 mm test tube. 2 mL of fermentation medium (supplemented with Ile, Val, Leu, Tyr, and Phe (200 mg / L each)) in The bacteria were inoculated into the spores and cultured at 30°C for 66 hours on a rotary shaker at 238 rpm. The accumulated 2-methylbutyric acid was measured by GC analysis (Preparatory Example 1). The accumulated 3-methylbutyric acid was measured by GC-MS analysis (Preparatory Example 2).

[0142] The results of three independent test tube fermentations are shown in Table 3. As can be seen from Table 3, the modified strain L1201-1 ΔtyrB ΔleuABCD::cat was able to accumulate a smaller amount of 3-methylbutyric acid compared to the control strain L1201-1.

[0143] [Table 3]

[0144] Preparatory Example 1 GC Analysis of 2-Methylbutyric Acid and Isobutyric Acid A Shimadzu GC-2014 gas chromatography instrument equipped with a flame ionization detector (FID) was used. This assay was used to analyze 2-methylbutyric acid and isobutyric acid in fermentation media. The samples were resuspended in a 1% (v / v) formic acid solution in ethanol and mixed. The samples were then vortexed for 3 minutes and centrifuged at 13,000 rpm for 5 minutes. The supernatant was used directly for the analysis of 2-methylbutyric acid and isobutyric acid. Standards were directly dissolved in a mixture of ethanol and 1% (v / v) formic acid. The calibration range was 5-160 mg / L. Other parameters were as follows: Column: InertCap Pure-WAX (GL science Inc.) Column dimensions: ID 0.25 mm, length 30 m, film thickness 0.5 μm Column gradient: 65°C (5 min hold) - 5°C / min - 200°C - 10°C / min - 240 °C Injector temperature: 240°C Injection volume: 1.0 μL Injection split mode: 1:10 Carrier gas: He Control mode: Linear velocity Pressure: 89.0 kPa Total flow rate: 14.0 mL / min Column flow rate: 1.0 mL / min Linear velocity: 26.1 cm / sec Purge flow rate: 3.0 mL / min Detector: FID 260°C Before injection: Wash with sample, 8 μL (3 times) After injection: Wash with ethanol, 8 μL (3 times)

[0145] Preparatory Example 2 GC-MS Analysis of 3-Methylbutyric Acid The supernatant of the fermentation medium was collected from both tubes and combined to a volume of 2 mL. The combined volume was filtered through a 0.2 μm pore size filter to remove the bacterial cells. Then, 1 mL of the diluted sample was used for GC-MS analysis. GC-FID analysis was performed on an Agilent 7890A chromatograph equipped with a SUPELCO β-DEX 120 column. The column dimensions were as follows: ID 0.25 mm, length 30 m, film thickness 0.25 mm. μm; limit of detection (LOD) 0.05 mg / L.

[0146] While the present invention has been described in detail with reference to illustrative embodiments, it will be apparent to those skilled in the art that various modifications and equivalents can be made without departing from the scope of the invention. [Industrial Applicability]

[0147] The method of the present invention is useful for producing highly purified 2-methylbutyric acid by bacterial fermentation.

Claims

1. 1. A method for producing 2-methylbutyric acid, comprising: (i) 2-methyl- ... Cultivating 2-methylbutyric acid-producing bacteria in a medium to produce and accumulate 2-methylbutyric acid in the medium, the bacterial cells, or both; (ii) recovering 2-methylbutyric acid from the medium or the bacterial cells, or both; Including, the bacterium has been modified to attenuate expression of a gene encoding a protein having tyrosine aminotransferase activity; The method, wherein the gene is selected from the group consisting of: (a) a gene comprising the nucleotide sequence shown in SEQ ID NO: 1; (b) a gene comprising a nucleotide sequence capable of hybridizing under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, encoding a protein having tyrosine aminotransferase activity, wherein the stringent conditions are such that a homology of 90% or more is obtained; A condition where hybrids are formed and hybrids with less than 90% homology are not formed. Transmission; (c) Substitution, deletion, or insertion of 1 to 30 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2; and / or a gene encoding a protein comprising an amino acid sequence containing an addition, wherein the protein has tyrosine aminotransferase activity; and (d) A gene comprising a variant base sequence of SEQ ID NO: 1, wherein the variant base sequence is due to the degeneracy of the genetic code.

2. The method of claim 1, wherein the gene encoding a protein having tyrosine aminotransferase activity is the tyrB gene.

3. 3. The method of claim 1 or 2, wherein the protein having tyrosine aminotransferase activity is selected from the group consisting of: (A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2; (B) Substitution, deletion, or insertion of 1 to 30 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2, and / or an addition, wherein the protein has tyrosine aminotransferase activity; and (C) an amino acid sequence having 90% or more identity to the entire amino acid sequence shown in SEQ ID NO: 2 1. A protein comprising the formula:

4. The method according to any one of claims 1 to 3, wherein the expression of a gene encoding a protein having tyrosine aminotransferase activity is attenuated by inactivating the gene.

5. The method of claim 4, wherein the gene encoding the protein having tyrosine aminotransferase activity is deleted.

6. The method according to any one of claims 1 to 5, wherein the bacterium is Escherichia coli or Pantoea ananatis.

7. The method according to any one of claims 1 to 6, wherein the bacterium has been further modified to attenuate expression of one or more genes selected from the group consisting of leuA, leuB, leuC, and leuD genes.

8. The method according to any one of claims 1 to 7, wherein the amount of 2-methylbutyric acid by-product is reduced compared to an unmodified bacterium.

9. 9. The method of claim 8, wherein the by-product is selected from the group consisting of 3-methylbutyric acid, isobutyric acid, L-alloisoleucine, D-alloisoleucine, and combinations thereof.

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