Transformant and method for producing 3-hydroxybenzoic acid using same

Abstract

Provided are: a transformant capable of improving the productivity of 3-HBA; and a method for producing 3-HBA using said transformant. In one aspect, the present disclosure pertains to a transformant for producing 3-hydroxybenzoic acid (3-HBA), the transformant being obtained by expressibly introducing, into a microbial host having chorismic acid-producing ability, a gene (A) encoding an enzyme (3-HBA synthase) having activity for producing 3-HBA from chorismic acid, wherein the microbial host is Corynebacterium glutamicum. In another aspect, the present disclosure pertains to a method for producing 3-HBA, the method comprising a step for culturing this transformant to produce 3-HBA.

Description

Transformant and method for producing 3-hydroxybenzoic acid using the same

[0001] The present disclosure relates to a technology for producing 3-hydroxybenzoic acid. In one aspect, the present disclosure relates to a transformant that has been subjected to specific genetic manipulation, and a technology for producing 3-hydroxybenzoic acid using the transformant.

[0002] 3-Hydroxybenzoic acid (CAS number: 99-06-9; synonyms include m-hydroxybenzoic acid and 3-hydroxybenzenecarboxylic acid; hereinafter, sometimes referred to as "3-HBA") crystallizes into a needle shape and is readily soluble in ethanol and ether, but poorly soluble in cold water. Applications of 3-HBA include as a raw material for organic synthesis and as a skin conditioner. It has been reported that 3-HBA can be produced by microbial fermentation. For example, Patent Document 1 proposes the production of 3-HBA by culturing a microorganism belonging to the genus Rhodococcus in a medium containing isophthalic acid as the main carbon source. There is a demand for the creation of microorganisms (transformants) that produce 3-HBA more efficiently.

[0003] Japanese Unexamined Patent Publication No. 7-184671

[0004] The present disclosure provides a new transformant capable of improving 3-HBA productivity, and a method for producing 3-HBA using the transformant.

[0005] In one aspect, the present disclosure relates to a transformant for producing 3-hydroxybenzoic acid (3-HBA), which is obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of producing 3-HBA from chorismate into a microbial host capable of producing chorismate.

[0006] In another aspect, the present disclosure relates to a transformant for producing 3-HBA, which is obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of producing 3-HBA from chorismate into a microbial host capable of producing chorismate, wherein the microbial host is Corynebacterium glutamicum.

[0007] In another aspect, the present disclosure relates to a method for producing 3-HBA, comprising the step of culturing the transformant of the present disclosure to produce 3-HBA.

[0008] In still another aspect, the present disclosure relates to a transformant for producing 3-HBA, obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of producing 3-HBA from chorismate into a microbial host capable of producing chorismate, wherein the gene (A) is derived from at least one selected from the group consisting of Caballeronia calidae, Streptomyces conglobatus, Streptomyces shaanxiensis, Caballeronia cordobensis, Saccharopolyspora halophila, Caballeronia fortuita, Lentzea nigeriaca, Lentzea indica, Actinoplanes bogorensis, and Caballeronia hypogeia, and the microbial host is Corynebacterium glutamicum.

[0009] According to one aspect of the present disclosure, a new transformant capable of improving 3-HBA productivity can be provided, preferably a transformant capable of efficiently producing 3-HBA using sugars etc. as raw materials can be provided. According to one aspect of the present disclosure, a method for producing 3-HBA with improved productivity can be provided.

[0010] Figure 1 is a schematic diagram showing 3-HBA production in the transformant of the present disclosure. Figure 2 is an overall metabolic pathway diagram illustrating the 3-HBA biosynthetic pathway in one embodiment of the transformant of the present disclosure.

[0011] The present disclosure is based on the inventors' discovery that a transformant obtained by introducing a gene (A) encoding an enzyme capable of synthesizing 3-HBA from chorismate into a microbial host capable of producing chorismate can biologically produce 3-HBA using sugars such as glucose as a raw material, and further, can improve 3-HBA productivity. Furthermore, the present disclosure is based on the finding that 3-HBA productivity can be further improved by using Corynebacterium glutamicum capable of producing chorismate as a host.

[0012] In the present disclosure, the term "transformant obtained by introducing a gene" may include a transformant obtained by introducing the gene, and a transformant that can be obtained by introducing the gene.

[0013] In one or more embodiments, the method for introducing the gene may be a method using a general gene recombination technique (for example, the method described in Michael R. Green & Joseph Sambrook, Molecular cloning, Cold Spring Harbor Laboratory Press). In one or more embodiments, the gene may be introduced using a plasmid vector or by integrating the gene into the chromosome of the microbial host.

[0014] In the present disclosure, in one or more embodiments, "introducing a gene in an expressible manner" refers to introducing the introduced gene in a manner that allows the gene to be expressed in a transformant. In one or more embodiments, methods for introducing a gene in an expressible manner include a method of introducing the gene together with an expression regulatory sequence such as a promoter that can induce enhanced expression of the gene. In one or more embodiments, in the present disclosure, the gene introduced together with a promoter that can induce enhanced expression may be a single gene or an operon that can express multiple genes. As a promoter that can induce enhanced expression, a known promoter can be used.

[0015] In the present disclosure, "90% or more identity" with respect to an amino acid sequence or a nucleotide sequence refers to at least 90% identity, and in one or more embodiments, refers to 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity.

[0016] In the present disclosure, "evaluation of identity of amino acid sequences or nucleotide sequences" can be performed using readily available sequence comparison computer programs. In one or more embodiments, computer programs include the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), BLAST (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), and FASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448).

[0017] In the present disclosure, "stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. In one or more embodiments, stringent conditions include conditions under which highly identical base sequences hybridize with each other, but less identical base sequences do not hybridize with each other. In one or more embodiments, high identity between base sequences means an identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% or more. In one or more embodiments, stringent conditions may be those described in "Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol. 2, p. 11.45." Specifically, hybridization may occur at a temperature 5 to 10°C lower than the melting temperature (Tm) of a perfect hybrid.

[0018] In the present disclosure, a "microbial host capable of producing chorismate" refers to a microbial host having at least the ability to biosynthesize chorismate, and in one or more embodiments, may be a microbial host capable of expressing an enzyme having chorismate synthase activity. In one or more embodiments, the microbial host according to the present disclosure may be not only a bacterium having a gene encoding an enzyme having chorismate synthase activity as a wild-type, but also a bacterium that has been artificially transformed so as to be capable of expressing an enzyme having chorismate synthase activity. In one or more embodiments, the microbial host capable of producing chorismate may be a bacterium having a gene encoding an enzyme having chorismate synthase activity as a wild-type, transformed so as to be capable of expressing an enzyme having chorismate synthase activity, or a bacterium transformed so as to enhance expression of the enzyme having chorismate synthase activity. In one or more embodiments, from the viewpoint of further improving 3-HBA productivity, preferred microbial hosts capable of producing chorismate include bacteria into which a gene encoding an enzyme having chorismate synthase activity has been introduced in an expressible manner, bacteria that overexpress the enzyme, and bacteria in which the expression level of the enzyme has been improved. In one or more embodiments, examples of enzymes having chorismate synthase activity include enzymes (EC 4.2.3.5) that catalyze the reaction of synthesizing chorismate from 5-O-(1-carboxyvinyl)-3-phosphoshikimic acid.

[0019] In one or more embodiments, the microbial host according to the present disclosure may, from the viewpoint of further improving 3-HBA productivity, be a microbial host in which the expression level of an enzyme having chorismate synthase activity is improved, or a microbial host in which the expression level of an enzyme having chorismate synthase activity is overexpressed or the expression level can be increased or inducible. The expression level of the enzyme can be adjusted or increased as appropriate by those skilled in the art, for example, by using an appropriate promoter. In one or more embodiments, the transformant according to the present disclosure may further include a microbial host capable of producing chorismate, into which a gene encoding an enzyme having chorismate synthase activity has been introduced.

[0020] In one or more embodiments, a microorganism capable of producing chorismate can be constructed by taking into consideration the description in WO2016 / 027870, the contents of which are incorporated herein by reference.

[0021] The microbial host according to the present disclosure is not particularly limited and may be, for example, a coryneform bacterium, Escherichia coli (bacteria of the genus Escherichia, particularly Escherichia coli), solvent-tolerant bacteria, or yeast. Coryneform bacteria are a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), and are not particularly limited as long as they grow under normal aerobic conditions. In one or more embodiments, examples of coryneform bacteria include bacteria of the genus Corynebacterium, Brevibacterium, Arthrobacter, Mycobacterium, and Micrococcus. In one or a plurality of embodiments, examples of solvent-resistant bacteria include Pseudomonas putida S12, Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas alcaligenes, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas oleovorans, Pseudomonas sp., Rhodococcus erythropolis, Rhodococcus opacus, Burkholderia cepacia, and Paenibacillus illinoisensis.

[0022] The microbial host according to the present disclosure is not particularly limited and may be, for example, a bacterium of the genus Corynebacterium. In one or more embodiments, examples of the Corynebacterium bacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum. The microbial host according to the present disclosure is not particularly limited and may be, for example, Corynebacterium glutamicum from the viewpoint of further improving 3-HBA productivity.

[0023] The microbial host according to the present disclosure is not particularly limited and may be, for example, Corynebacterium glutamicum such as Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), or ATCC13869 (DSM1412), or a transformant thereof.

[0024] [Transformant of the Present Disclosure] In one aspect, the present disclosure relates to a transformant for producing 3-HBA, obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of producing 3-HBA from chorismate into a microbial host capable of producing chorismate. In another aspect, the present disclosure relates to a transformant, wherein the microbial host is Corynebacterium glutamicum capable of producing chorismate. The transformant of the present disclosure is capable of producing 3-HBA. Therefore, in one or more embodiments, the transformant of the present disclosure can also be referred to as a transformant capable of producing 3-HBA. In one or more embodiments, but not particularly limited thereto, the transformant of the present disclosure is capable of producing 3-HBA from chorismate by the gene (A), as shown in FIG. 1 , and preferably is capable of producing chorismate from sugars (e.g., glucose) in vivo in the transformant and then producing 3-HBA from the chorismate.

[0025] [Gene (A)] The gene (A) in the present disclosure is a gene encoding an enzyme having the activity of producing 3-HBA from chorismate. In one or more embodiments, the gene (A) may be a gene encoding a polypeptide having 3-HBA synthase activity. 3-HBA synthase (EC 4.1.3.45) is an enzyme that catalyzes the reaction of producing 3-HBA from chorismate. In one or more embodiments, an example of a gene encoding 3-HBA synthase is the hyg5 gene.

[0026] The origin of gene (A) is not particularly limited, and in one or more embodiments, examples include the genera Actinoplanes, Burkholderia, Caballeronia, Krasilnikovia, Lentzea, Methylibium, Nonomuraea, Rhodococcus, Saccharopolyspora, Streptomyces, Thermobacillus, and Thiobacillus. The names (designations) of the "genus" and "species" of the organisms from which the genes are derived shown in the present disclosure are merely examples, and synonymous alternative names may exist. For example, the genus Lentzea can also be referred to as the genus Lechevalieria.

[0027] In one or more embodiments, the genus Actinoplanes includes Actinoplanes ovalisporus, etc. In one or more embodiments, an example of a hyg5 gene derived from Actinoplanes ovalisporus is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 73. In one or more embodiments, an example of a species of the genus Burkholderia includes Burkholderia contaminans, etc. In one or more embodiments, an example of a hyg5 gene derived from Burkholderia contaminans is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 4 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 74. In one or more embodiments, an example of a species of the genus Caballeronia includes Caballeronia insecticola, etc. In one or more embodiments, an example of a hyg5 gene derived from Caballeronia insecticola is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 7. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 7 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 75. In one or more embodiments, examples of the genus Krasilnikovia include Krasilnikovia cinnamomea, etc. In one or more embodiments, examples of the hyg5 gene derived from Krasilnikovia cinnamomea include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 10. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 10 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 76. In one or more embodiments, examples of the genus Lentzea include Lentzea aerocolonigenes, Lentzea alba, Lentzea flava, Lentzea waywayandensis, and Lentzea xinjiangensis, etc.In one or more embodiments, the hyg5 gene derived from Lentzea aerocolonigenes, the hyg5 gene derived from Lentzea alba, the hyg5 gene derived from Lentzea flava, the hyg5 gene derived from Lentzea waywayandensis, and the hyg5 gene derived from Lentzea xinjiangensis include genes consisting of the nucleotide sequences set forth in SEQ ID NOs: 13, 16, 19, 22, and 25, respectively. The genes consisting of the nucleotide sequences set forth in SEQ ID NOs: 13, 16, 19, 22, and 25 encode polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 77, 78, 79, 80, and 81, respectively. In one or more embodiments, the genus Methylibium includes Methylibium petroleiphilum, etc. In one or more embodiments, the hyg5 gene derived from Methylibium petroleiphilum includes a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 28. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 28 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 82. In one or more embodiments, the genus Nonomuraea includes Nonomuraea africana, etc. In one or more embodiments, an example of a hyg5 gene derived from Nonomuraea africana is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 31. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 31 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 83. In one or more embodiments, an example of a species of the genus Rhodococcus includes Rhodococcus ruber, etc. In one or more embodiments, an example of a hyg5 gene derived from Rhodococcus ruber is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 34. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 34 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 84.In one or more embodiments, the genus Saccharopolyspora includes Saccharopolyspora aridisoli, Saccharopolyspora dendranthemae, Saccharopolyspora flava, etc. In one or more embodiments, the hyg5 gene derived from Saccharopolyspora aridisoli, the hyg5 gene derived from Saccharopolyspora dendranthemae, and the hyg5 gene derived from Saccharopolyspora flava include genes consisting of the nucleotide sequences set forth in SEQ ID NOs: 37, 40, and 43, respectively. The genes consisting of the nucleotide sequences set forth in SEQ ID NOs: 37, 40, and 43 encode polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 85, 86, and 87, respectively. In one or a plurality of embodiments, examples of the genus Streptomyces include Streptomyces chrestomyceticus, Streptomyces eurocidicus, Streptomyces nanshensis, Streptomyces sp. NBRC 114659, Streptomyces rimosus subsp. paromomycinus, Streptomyces uncialis, and Streptomyces zaomyceticus. In one or more embodiments, the hyg5 gene derived from Streptomyces chrestomyceticus, the hyg5 gene derived from Streptomyces eurocidicus, the hyg5 gene derived from Streptomyces nanshensis, the hyg5 gene derived from Streptomyces sp. NBRC 114659, the hyg5 gene derived from Streptomyces rimosus subsp. paromomycinus, the hyg5 gene derived from Streptomyces uncialis, and the hyg5 gene derived from Streptomyces zaomyceticus include genes consisting of the nucleotide sequences shown in SEQ ID NOs: 46, 49, 52, 55, 58, 61, and 64, respectively.In one or more embodiments, genes consisting of the nucleotide sequences set forth in SEQ ID NOs: 46, 49, 52, 55, 58, 61, and 64 encode polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 88, 89, 90, 91, 92, 93, and 94, respectively. In one or more embodiments, the genus Thermobacillus includes Thermobacillus composti, etc. In one or more embodiments, an example of a hyg5 gene derived from Thermobacillus composti is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 67. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 67 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 95. In one or more embodiments, an example of a Thiobacillus includes Thiobacillus denitrificans, etc. In one or more embodiments, an example of a hyg5 gene derived from Thiobacillus denitrificans is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 70. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 70 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 96.

[0028] The gene (A) is not particularly limited and may be, for example, any of the following genes (a), (b), (c), and (d): (a) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96; (b) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein the gene encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate, or a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein the gene encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate;(c) A gene encoding a polypeptide having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids per unit are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, with 100 amino acids as one unit, and having the activity of producing 3-hydroxybenzoic acid from chorismate. or the amino acid sequence represented by SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids per unit, with 100 amino acids considered as one unit, are deleted, substituted, and / or added, and wherein the gene encodes a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate; (D) A gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96. A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate. or a gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, wherein the gene hybridizes under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96, and wherein the gene encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate;

[0029] The gene (A) is not particularly limited and may be, for example, any of the following genes (e), (f), (g), and (h): (e) a gene having the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70; (f) a gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, which encodes a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate, or a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, which encodes a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate;(g) A gene having a base sequence represented by SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, with 100 bases per unit, in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases are deleted, substituted, and / or added, and encoding a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate. or a gene consisting of a nucleotide sequence represented by SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base deletions, substitutions, and / or additions per unit, with 100 bases considered to be one unit, wherein the gene encodes a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate; (h) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, and encodes a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate, or a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, or 70, and encodes a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate;

[0030] In one or more embodiments, the gene (A) is preferably a hyg5 gene derived from any of Actinoplanes ovalisporus, Caballeronia insecticola, Krasilnikovia cinnamomea, Lentzea aerocolonigenes, Lentzea flava, Lentzea waywayandensis, Lentzea xinjiangensis, Methylibium petroleiphilum, Nonomuraea africana, Rhodococcus ruber, Saccharopolyspora aridisoli, Saccharopolyspora flava, Streptomyces chrestomyceticus, Streptomyces eurocidicus, Streptomyces nanshensis, Streptomyces sp. NBRC 114659, Streptomyces uncialis, and Streptomyces zaomyceticus, from the viewpoint of further improving 3-HBA productivity. ovalisporus, Caballeronia insecticola, Krasilnikovia cinnamomea, Lentzea aerocolonigenes, Lentzea flava, Lentzea waywayandensis, Lentzea xinjiangensis, Nonomuraea africana, Rhodococcus ruber, Saccharopolyspora flava, Streptomyces chrestomyceticus, Streptomyces nanshensis, Streptomyces sp.NBRC 114659, and Streptomyces zaomyceticus are more preferred, and hyg5 genes derived from any of Actinoplanes ovalisporus, Caballeronia insecticola, Krasilnikovia cinnamomea, Lentzea aerocolonigenes, Lentzea flava, Rhodococcus ruber, Saccharopolyspora flava, Streptomyces sp. NBRC 114659, and Streptomyces zaomyceticus are even more preferred.

[0031] In one or more embodiments, from the viewpoint of further improving 3-HBA productivity, the gene (A) is preferably a gene having the nucleotide sequence represented by SEQ ID NO: 1, 7, 10, 13, 19, 22, 25, 28, 31, 34, 37, 43, 46, 49, 52, 55, 61, or 64, more preferably a gene having the nucleotide sequence represented by SEQ ID NO: 1, 7, 10, 13, 19, 22, 25, 31, 34, 43, 46, 52, 55, or 64, and even more preferably a gene having the nucleotide sequence represented by SEQ ID NO: 1, 7, 10, 13, 19, 34, 43, 55, or 64. In one or more embodiments, the polypeptide encoded by gene (A) is preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 73, 75, 76, 77, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 93, or 94, more preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 73, 75, 76, 77, 79, 80, 81, 83, 84, 87, 88, 90, 91, or 94, and even more preferably a polypeptide having the amino acid sequence represented by SEQ ID NO: 73, 75, 76, 77, 79, 84, 87, 91, or 94.

[0032] In one or more embodiments, whether a gene is a "gene encoding an enzyme having the activity of producing 3-HBA from chorismate" can be evaluated by carrying out a 3-HBA production reaction using chorismate as a substrate using a transformant prepared by expressibly introducing the gene to be evaluated into Corynebacterium glutamicum. When production of 3-HBA or an increase in the amount of production is confirmed, the gene can be determined to be encoding an enzyme having the activity of producing 3-HBA from chorismate. The 3-HBA production reaction and production measurement can be carried out based on the description in the Examples section of the present specification.

[0033] Although not an essential component, in one embodiment, the transformant of the present disclosure may optionally have reduced or deleted functions of one or more or all of the following enzymes (1) to (5) in order to further improve 3-HBA productivity: (1) 3-dehydroshikimate dehydratase; (2) quinate / shikimate dehydrogenase; (3) 3-hydroxybenzoate 6-monooxygenase; (4) lactate dehydrogenase; (5) phenol 2-monooxygenase.

[0034] In one or more embodiments, in order to further improve 3-HBA productivity, the transformant of the present disclosure may have portions of the genes encoding one or more or all of the enzymes (1) to (5) above disrupted or deleted, or may have portions of all of the five genes encoding 3-dehydroshikimate dehydratase (e.g., the qsuB gene), quinate / shikimate dehydrogenase (e.g., the qsuD gene), 3-hydroxybenzoate 6-monooxygenase (e.g., the genH gene), lactate dehydrogenase (e.g., the ldhA gene), and phenol 2-monooxygenase (e.g., the poxF gene) disrupted or deleted.

[0035] Although not an essential component, in one aspect, the transformant of the present disclosure may optionally have enhanced activity of one or more enzymes contained in at least one pathway selected from the shikimate pathway, the sugar metabolic pathway (iolT1-ppgk), and the nonoxidative pentose phosphate pathway. The enhancement of the enzyme activity is not particularly limited, and may be achieved, for example, by enhancing the expression of a gene encoding the enzyme.

[0036] In one embodiment, the transformant of the present disclosure may have enhanced 3-HBA production from sugars by increasing the production of chorismate, an endogenous precursor, as a reaction substrate for the introduced gene (A).

[0037] Chorismate is the final product of the metabolic pathway from 3-deoxy-D-arabinopeptulosonic acid 7-phosphate (DAHP) to chorismate, known as the shikimate pathway. Therefore, in order to further improve 3-HBA productivity, the metabolic flux of the shikimate pathway may be increased. Figure 2 schematically illustrates an example of an overall sugar metabolic pathway showing the metabolic pathway of 3-HBA using glucose as a raw material. The sugar metabolic pathway in the present disclosure refers to various in vivo reaction systems for breaking down and converting sugars such as glucose into raw materials for the biosynthesis of various compounds, and in one or more embodiments, may include the shikimate pathway, the sugar metabolic pathway (iolT1-ppgk), the nonoxidative pentose phosphate pathway, the TCA cycle, and the like. However, the 3-HBA biosynthetic pathway shown in Figure 2 is merely an example of a sugar metabolic pathway in the present disclosure, and the present disclosure is not limited thereto.

[0038] The metabolic engineering technique for increasing the metabolic flux of the shikimate pathway is not particularly limited, and in one aspect, it may be any of the following pathways (1) to (3), or a combination of two or three of the pathways (1) to (3). (1) High activation by enhancing the expression of each enzyme gene constituting the shikimate pathway or by using heterologous or mutant genes. In one or more embodiments, enzymes constituting the shikimate pathway include DAHP synthase (e.g., aroG), 3-dehydroquinate synthase (e.g., aroB), 3-dehydroquinate dehydratase (e.g., aroD), shikimate dehydrogenase (e.g., aroE), shikimate kinase (e.g., aroK), 5-enolpyruvylshikimate-3-phosphate synthase (e.g., aroA), and chorismate synthase (e.g., aroC). (2) High activation by, for example, enhancing the expression of genes constituting a non-PTS sugar transport pathway, which is an alternative sugar transport / metabolism pathway different from the phosphoenolpyruvate:sugar phosphotransferase system (PTS) typically used as a sugar transporter in wild-type strains. In one or more embodiments, examples of genes constituting the non-PTS sugar transport pathway include the iolT1 and iolT2 genes encoding myo-inositol / glucose transporters, the glk gene encoding glucokinase, and the ppgk gene encoding polyphosphate glucokinase. (3) High activation by, for example, enhancing the expression of genes constituting the nonoxidative pentose phosphate pathway. In one or more embodiments, examples of enzymes constituting the nonoxidative pentose phosphate pathway include transketolase (e.g., tkt) and transaldolase (e.g., tal).

[0039] In one or more embodiments, the metabolic modification may be, but is not limited to, a modification of at least one gene constituting each pathway, or may be a modification of two, three, or four or more genes constituting each pathway, or may be a modification of all of the genes mentioned above in each pathway.

[0040] In one or more embodiments, in order to further improve 3-HBA productivity, the transformant of the present disclosure may have at least one or all of the aroG gene, aroD gene, aroE gene, aroC gene, aroK gene, aroB gene, and aroA gene introduced into the host in order to enhance the activity of at least one or all of the enzymes encoded by these genes, or may have been modified so that the expression of at least one or all of these genes is enhanced.

[0041] In one aspect, the transformant of the present disclosure may be modified to enhance each metabolic pathway by enhancing the expression of the various metabolic pathway genes described above or by highly activating enzyme functions through the use of heterologous (mutated) genes, etc.

[0042] In one aspect of the transformant of the present disclosure, optionally, the expression of a gene encoding pyruvate kinase may not be suppressed, although this is not an essential component. In one aspect of the transformant of the present disclosure, optionally, the function of pyruvate kinase may not be reduced or deleted, although this is not an essential component. Pyruvate kinase is an enzyme that catalyzes the reaction from phosphoenolpyruvate to pyruvate. In one or more embodiments, examples of pyruvate kinase include PykF and PykA.

[0043] In another aspect, the present disclosure relates to a vector for introducing the gene (A) into a microorganism. The vector according to the present disclosure is not particularly limited and may be, for example, a plasmid.

[0044] Construction of Vectors for Transformants The above-described genes can be introduced into a microbial host by amplifying the above-described genes by PCR, cloning them into an appropriate vector that can be amplified in a microbial host such as a coryneform bacterium, and incubating the microbial host in the presence of the vector. In one or more embodiments, examples of promoters include the promoter (PgapA) of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (also referred to as "glyceraldehyde 3-phosphate dehydrogenase") derived from Corynebacterium glutamicum R, the promoter (Pmdh) of the mdh gene encoding malate dehydrogenase, and the promoter (PldhA) of the ldhA gene encoding lactate dehydrogenase, with PgapA being preferred. In one or more embodiments, examples of the terminator include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the trpA terminator of Escherichia coli, and the trp terminator of Brevibacterium lactofermentum, with the rrnB T1T2 terminator being particularly preferred.

[0045] [Preparation of Transformants] Transformation Known methods can be used for transformation without limitation. In one or more embodiments, such known methods include the calcium chloride / rubidium chloride method, the calcium phosphate method, DEAE-dextran-mediated transfection, and electroporation (electric pulse method). When the microbial host is a coryneform bacterium, the electric pulse method is preferred. The electric pulse method can be performed by known methods [e.g., Kurusu, Y. et al., Electroporation-transformation system for coryneform bacteria by auxotrophic complementation. Agric. Biol. Chem. 54:443-447 (1990)] and [Vertes AA et al., Presence of mrr- and mcr-like restriction systems in coryneform bacteria. Res. Microbiol. 144:181-185 (1993)].

[0046] Disruption or Mutation of Host Chromosomal Genes When the microbial host is a coryneform bacterium, genes encoding competing biosynthetic pathways, biosynthetic pathway inhibitors, efflux transporters, etc. may be disrupted or deleted as necessary. The function of an enzyme protein encoded by a specific gene may be improved by introducing mutations into the chromosome. A DNA fragment lacking the entire target gene can be created by ligating DNA fragments before and after the target gene, and then transforming bacteria with the DNA to induce homologous recombination on the chromosome, thereby completely deleting the target gene on the chromosome. Alternatively, a deletion-type gene modified to delete a partial sequence of the target gene so that it does not produce a normally functioning enzyme protein can be created. Bacteria can then be transformed with DNA containing the gene to induce homologous recombination between the deletion-type gene and the gene on the chromosome, thereby replacing the target gene on the chromosome with the deletion-type or disruption-type gene. Even if an enzyme protein encoded by a deletion-type or disruption-type gene is produced, it has a different three-dimensional structure from the wild-type enzyme protein and has reduced or lost function. Furthermore, a mutation can be introduced into a specific location on a chromosome by homologous recombination of a gene fragment containing a specific mutation with the chromosomal region. Gene deletion or disruption by gene replacement using homologous recombination has already been established, and examples include methods using a plasmid containing a temperature-sensitive replication origin, a conjugatively transferable plasmid, and a suicide vector that does not have a replication origin in the host (U.S. Patent No. 6,303,383 and Japanese Patent Laid-Open Publication No. 05-007491). The markerless chromosomal gene introduction vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of a single-crossover strain with the homologous region on the chromosome introduced into the plasmid pCRA725, the strain exhibits kanamycin resistance due to the expression of the kanamycin resistance gene on pCRA725 and lethality on sucrose-containing medium due to the expression of the sacR-sacB genes of Bacillus subtilis, whereas in the case of a double-crossover strain, the strain exhibits kanamycin sensitivity due to the loss of the kanamycin resistance gene on pCRA725 and growth on sucrose-containing medium due to the loss of the sacR-sacB genes.Therefore, the markerless chromosomal gene-introduced strain exhibits sensitivity to kanamycin and the ability to grow in a sucrose-containing medium.

[0047] Growth of Microorganisms In one or more embodiments, the transformant of the present disclosure is preferably cultured and grown under aerobic conditions. In one or more embodiments, the culture conditions for growth include a temperature of about 25°C to about 38°C and a growth period of about 12 to about 48 hours. In one or more embodiments, the growth medium may be a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, other nutrients, and the like. In one or more embodiments, the pH of the medium is about 5 to about 8.

[0048] When the host is a coryneform bacterium, examples of the medium include A medium [Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182-196(2004)] and BT medium [Omumasaba, CA et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103(2004)]. When the host is Escherichia coli, examples of the medium include LB medium, etc.

[0049] In one or more embodiments, examples of carbon sources include carbohydrates or sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol, and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid, and gluconic acid; and alcohols such as ethanol and propanol. Hydrocarbons such as normal paraffin can also be used if desired. One type of carbon source may be used alone, or two or more types may be used in combination. In one or more embodiments, the concentration of these carbon sources in the growth medium is about 0.1 (w / v%) to about 10 (w / v%).

[0050] In one or more embodiments, examples of the nitrogen source include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, urea, aqueous ammonia, sodium nitrate, and potassium nitrate. In one or more embodiments, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolysates, and amino acids can also be used. One nitrogen source may be used alone, or two or more may be used in combination. The concentration of the nitrogen source in the growth medium varies depending on the nitrogen compound used, but in one or more embodiments, it is about 0.1 (w / v%) to about 10 (w / v%).

[0051] In one or more embodiments, examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, ammonium carbonate, and calcium carbonate. These inorganic salts may be used alone or in combination of two or more. The concentration of inorganic salts in the growth medium varies depending on the inorganic salt used, but in one or more embodiments, it is about 0.01 (w / v%) to about 1 (w / v%).

[0052] In one or more embodiments, other nutritional substances include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, hydrolyzed skim soybeans with hydrochloric acid, extracts of animals, plants, or microbial cells, or decomposition products thereof. The concentration of the nutritional substances in the medium varies depending on the nutritional substances used, but in one or more embodiments, it is about 0.1 (w / v%) to about 10 (w / v%). Vitamins may be added as needed. In one or more embodiments, examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0053] [Method for Producing 3-HBA] In another aspect, the present disclosure relates to a method for producing 3-HBA. Specifically, in one aspect, the method for producing 3-HBA of the present disclosure includes a step of culturing the transformant of the present disclosure to produce 3-HBA.

[0054] In one or more embodiments, the production method of the present disclosure may include culturing a transformant of the present disclosure in a medium. In one or more embodiments, culturing a transformant of the present disclosure in a medium may allow the transformant of the present disclosure to metabolize a substrate in the medium, thereby causing the transformant of the present disclosure to produce 3-HBA. In one or more embodiments, the medium preferably contains sugars as a carbon source (substrate). In one or more embodiments, the medium may contain nutrients other than sugars, such as vitamins, yeast extract, and dry yeast.

[0055] In one or more embodiments, the medium may be an inorganic salt medium. In one or more embodiments, the inorganic salt medium may be a medium containing one or more inorganic salts such as monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, ferrous nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, calcium carbonate, urea, ammonium sulfate, and ferrous sulfate. Among these, a medium containing urea, ammonium sulfate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, and ferrous sulfate is preferred. Specific examples of inorganic salt medium include BT medium and A medium. The concentration of inorganic salts in the medium varies depending on the inorganic salt used, but in one or more embodiments, it may be about 0.01 (w / v%) to about 1 (w / v%).

[0056] In one or more embodiments, examples of sugars include glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol. In one or more embodiments, the concentration of sugars in the medium is about 0.1 (w / v%) to 20 (w / v%), and may be 1 (w / v%) to 20 (w / v%), 1 (w / v%) to 10 (w / v%), or 5 (w / v%) to 20 (w / v%). In one or more embodiments, one type of sugar may be used, or two or more types may be used in combination.

[0057] Reaction Conditions In one or more embodiments, reaction conditions include reducing conditions and microaerobic conditions (e.g., conditions under which the dissolved oxygen concentration is controlled). In one or more embodiments, but not limited to, when the microbial host is Corynebacterium glutamicum, the reaction is carried out under both reducing and microaerobic conditions, under which Corynebacterium glutamicum does not substantially grow, thereby enabling more efficient production of 3-HBA. In one or more embodiments, conditions under which the dissolved oxygen concentration is controlled include conditions under which the DO of the medium is 13% or less, 12% or less, 11% or less, 10% or less, or less than 10%. In one or more embodiments, the DO can be measured using a commercially available dissolved oxygen meter.

[0058] In one or more embodiments, the reaction temperature (the survival temperature of the transformant during the reaction) is about 15°C to about 50°C. 3-HBA can be produced efficiently within this temperature range. From the viewpoint of further improving 3-HBA productivity, the reaction temperature is, in one or more embodiments, 16°C or higher, 17°C or higher, 18°C ​​or higher, 19°C or higher, 20°C or higher, 21°C or higher, 22°C or higher, 23°C or higher, 24°C or higher, or 25°C or higher. From the same viewpoint, in one or more embodiments, the reaction temperature is 49°C or lower, 45°C or lower, 40°C or lower, or 35°C or lower.

[0059] In one or more embodiments, the pH of the medium is preferably about 6 to about 8. The pH of the medium during the reaction is preferably controlled to near neutral, particularly about 7.5, using an aqueous ammonia solution, an aqueous sodium hydroxide solution, or the like with a pH controller (for example, Model DT-1023, manufactured by Able Co., Ltd.).

[0060] The reaction time is not particularly limited and can be determined appropriately. In one or more embodiments, the reaction time is about 1 to 7 days, and preferably about 1 to 3 days. In one or more embodiments, the culture may be performed by any of a batch system, a fed-batch system, and a continuous system, with a batch system being preferred.

[0061] In one or more embodiments, the production method of the present disclosure may include recovering 3-HBA from the medium (culture), and may further include purifying the recovered 3-HBA as needed. The method for recovering and purifying 3-HBA from the medium (culture) is not particularly limited. In one or more embodiments, the recovery and / or purification of 3-HBA can be carried out by appropriately combining well-known methods such as ion exchange resin methods, precipitation methods, crystallization methods, recrystallization methods, concentration methods, liquid-liquid extraction methods, and other methods. The 3-HBA accumulated in the culture may be used as is without isolation.

[0062] [Transformant 2 of the Present Disclosure] In yet another aspect, the present disclosure relates to a transformant for producing 3-HBA, obtained by expressibly introducing a gene (A) encoding an enzyme active in producing 3-HBA from chorismate into a microbial host capable of producing chorismate, wherein the gene (A) is derived from at least one selected from the group consisting of Actinoplanes bogorensis, Caballeronia calidae, Caballeronia cordobensis, Caballeronia fortuita, Caballeronia hypogeia, Lentzea indica, Lentzea nigeriaca, Saccharopolyspora halophila, Streptomyces conglobatus, and Streptomyces shaanxiensis, and the microbial host is Corynebacterium glutamicum. The transformant of the present disclosure is capable of producing 3-HBA. Therefore, in one or more embodiments, the transformant of the present disclosure can also be referred to as a transformant capable of producing 3-HBA.

[0063] In one or more embodiments, an example of the hyg5 gene derived from Actinoplanes bogorensis is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 101. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 101 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 131. In one or more embodiments, an example of the hyg5 gene derived from Caballeronia calidae is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 104. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 104 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 132. In one or more embodiments, an example of the hyg5 gene derived from Caballeronia cordobensis is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 107. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 107 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 133. In one or more embodiments, an example of the hyg5 gene derived from Caballeronia fortuita is a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 110. The gene consisting of the nucleotide sequence set forth in SEQ ID NO: 110 encodes a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 134. In one or more embodiments, an example of the hyg5 gene derived from Caballeronia hypogeia is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 113. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 113 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 135. In one or more embodiments, an example of the hyg5 gene derived from Lentzea indica is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 116. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 116 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 136. In one or more embodiments, an example of the hyg5 gene derived from Lentzea nigeriaca is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 119. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 119 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 137.In one or more embodiments, an example of the hyg5 gene derived from Saccharopolyspora halophila is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 122. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 122 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 138. In one or more embodiments, an example of the hyg5 gene derived from Streptomyces conglobatus is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 125. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 125 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 139. In one or more embodiments, an example of the hyg5 gene derived from Streptomyces shaanxiensis is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 128. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 128 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 140.

[0064] In one or more embodiments, the gene (A) in this aspect includes any one of the following genes (i), (j), (k), and (l): (i) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140, or a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. (j) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140, or a gene encoding a polypeptide consisting of an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140. (k) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids in the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140, or a gene encoding a polypeptide consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids in the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140, and which encodes a polypeptide having the activity of producing 3-HBA from chorismate.(l) A gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140, which hybridizes under stringent conditions, and which encodes a polypeptide having the activity of producing 3-HBA from chorismate; or A gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140, which hybridizes under stringent conditions, and which encodes a polypeptide having the activity of producing 3-HBA from chorismate.

[0065] In one or more embodiments, the gene (A) in this aspect includes any one of the following genes (m), (n), (o), and (p): (m) a gene having the nucleotide sequence shown in SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128. (n) A gene having a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, which encodes a polypeptide having the activity of producing 3-HBA from chorismate, or a gene consisting of a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, which encodes a polypeptide having the activity of producing 3-HBA from chorismate. (O) A gene having a base sequence represented by SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, with 100 bases per unit, of 1 to 10 bases deleted, substituted, and / or added per unit, and encoding a polypeptide having the activity of producing 3-HBA from chorismate; or a gene having a base sequence represented by SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, with 100 bases per unit, of 1 to 10 bases deleted, substituted, and / or added per unit, and encoding a polypeptide having the activity of producing 3-HBA from chorismate.(p) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, and encodes a polypeptide having the activity of producing 3-HBA from chorismate; or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 101, 104, 107, 110, 113, 116, 119, 122, 125, or 128, and encodes a polypeptide having the activity of producing 3-HBA from chorismate.

[0066] In one or more embodiments, the gene (A) in this aspect is preferably a hyg5 gene derived from any of Caballeronia calidae, Streptomyces conglobatus, Streptomyces shaanxiensis, Caballeronia cordobensis, Saccharopolyspora halophila, Caballeronia fortuita, and Lentzea nigeriaca, from the viewpoint of further improving 3-HBA productivity.

[0067] In one or more embodiments, the gene (A) in this aspect is preferably a gene having the nucleotide sequence shown in SEQ ID NO: 104, 125, 128, 107, 122, 110, or 119, from the viewpoint of further improving 3-HBA productivity. In one or more embodiments, the polypeptide encoded by the gene (A) in this aspect is preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 132, 139, 140, 133, 138, 134, or 137, from the viewpoint of further improving 3-HBA productivity.

[0068] Although not an essential component, in one or more embodiments, the transformant of this aspect may optionally have reduced or deleted function of one or more or all of the following enzymes (1) to (5) from the viewpoint of further improving 3-HBA productivity: (1) 3-dehydroshikimate dehydratase (qsuB), (2) quinate / shikimate dehydrogenase (qsuD), (3) 3-hydroxybenzoate 6-monooxygenase (genH), (4) lactate dehydrogenase (ldhA), and (5) phenol 2-monooxygenase (poxF).

[0069] In one or more embodiments, in order to further improve 3-HBA productivity, the transformant of this aspect may have a portion of the gene encoding one or more or all of the enzymes (1) to (5) above disrupted or deleted, or may have a portion of all of the five genes encoding 3-dehydroshikimate dehydratase (e.g., the qsuB gene), quinate / shikimate dehydrogenase (e.g., the qsuD gene), 3-hydroxybenzoate 6-monooxygenase (e.g., the genH gene), lactate dehydrogenase (e.g., the ldhA gene), and phenol 2-monooxygenase (e.g., the poxF gene) disrupted or deleted.

[0070] Although not an essential component, in one or more embodiments, the transformant of this aspect may optionally have enhanced activity of one or more enzymes contained in at least one of the shikimate pathway, the sugar metabolic pathway (iolT1-ppgk), and the nonoxidative pentose phosphate pathway. The enhancement of the enzyme activity is not particularly limited, and may be achieved, for example, by enhancing the expression of a gene encoding the enzyme.

[0071] In one or more embodiments, the transformant of the present disclosure may have enhanced 3-HBA production from sugars by increasing production of chorismate, an endogenous precursor that serves as a reaction substrate for the introduced gene (A).

[0072] In one or more embodiments, in order to further improve 3-HBA productivity, the transformant of this aspect may have at least one or all of the aroG gene, aroD gene, aroE gene, aroC gene, aroK gene, aroB gene, and aroA gene introduced into the host in order to enhance the activity of at least one or all of the enzymes encoded by these genes, or may have been modified so that the expression of at least one or all of these genes is enhanced.

[0073] In one or more embodiments, the transformant of this aspect can be used for producing 3-HBA. Thus, in yet another aspect, the present disclosure relates to a method for producing 3-HBA, comprising the step of culturing the transformant of this aspect to produce 3-HBA.

[0074] The contents of each document mentioned in this application are incorporated by reference as part of this disclosure.

[0075] The present disclosure further relates to one or more of the following embodiments: [1] A transformant for producing 3-HBA, obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of producing 3-HBA from chorismic acid into a microbial host capable of producing chorismic acid, wherein the microbial host is Corynebacterium glutamicum. [2] The gene (A) is Lentzea flava, Streptomyces sp. NBRC 114659, Saccharopolyspora flava, Actinoplanes ovalisporus, Caballeronia insecticola, Krasilnikovia cinnamomea, Streptomyces zaomyceticus, Lentzea aerocolonigenes, Rhodococcus ruber, Lentzea xinjiangensis, Lentzea waywayandensis, Streptomyces nanshensis, Streptomyces chrestomyceticus, Nonomuraea africana, Saccharopolyspora aridisoli, Methylibium petroleiphilum, Streptomyces uncialis, Streptomyces eurocidicus, Lentzea alba, Burkholderia contaminans, Saccharopolyspora dendranthemae, Streptomyces rimosus [3] The transformant according to [1] or [2], wherein the gene (A) is derived from at least one selected from the group consisting of the following (a), (b), (c), and (d):(a) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96. (b) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96, wherein the gene encodes a polypeptide having the activity of producing 3-HBA from chorismate. (c) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added per unit, with 100 amino acids being one unit, in the amino acid sequence represented by SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96, and which encodes a polypeptide having the activity of producing 3-HBA from chorismate.(d) A gene that hybridizes under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96, and that encodes a polypeptide having the activity of producing 3-HBA from chorismate. [4] The transformant according to any of [1] to [3], wherein the gene (A) is selected from the group consisting of (e), (f), (g), and (h) below. (e) A gene having a nucleotide sequence represented by SEQ ID NO: 19, SEQ ID NO: 55, SEQ ID NO: 43, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 64, SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 25, SEQ ID NO: 22, SEQ ID NO: 52, SEQ ID NO: 46, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 28, SEQ ID NO: 61, SEQ ID NO: 49, SEQ ID NO: 16, SEQ ID NO: 4, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 67, or SEQ ID NO: 70. (f) A gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence represented by SEQ ID NO: 19, SEQ ID NO: 55, SEQ ID NO: 43, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 64, SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 25, SEQ ID NO: 22, SEQ ID NO: 52, SEQ ID NO: 46, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 28, SEQ ID NO: 61, SEQ ID NO: 49, SEQ ID NO: 16, SEQ ID NO: 4, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 67, or SEQ ID NO: 70, which encodes a polypeptide having the activity of producing 3-HBA from chorismate.(g) A gene having a base sequence represented by SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:43, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:64, SEQ ID NO:13, SEQ ID NO:34, SEQ ID NO:25, SEQ ID NO:22, SEQ ID NO:52, SEQ ID NO:46, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:28, SEQ ID NO:61, SEQ ID NO:49, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:67, or SEQ ID NO:70, with 100 bases per unit, and having 1 to 10 base deletions, substitutions, and / or additions per unit, wherein the gene encodes a polypeptide having the activity of producing 3-HBA from chorismate. (h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 19, SEQ ID NO: 55, SEQ ID NO: 43, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 64, SEQ ID NO: 13, SEQ ID NO: 34, SEQ ID NO: 25, SEQ ID NO: 22, SEQ ID NO: 52, SEQ ID NO: 46, SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 28, SEQ ID NO: 61, SEQ ID NO: 49, SEQ ID NO: 16, SEQ ID NO: 4, SEQ ID NO: 40, SEQ ID NO: 58, SEQ ID NO: 67, or SEQ ID NO: 70, and encodes a polypeptide having the activity of producing 3-HBA from chorismate. [5] The transformant according to any of [1] to [4], wherein at least one enzyme selected from the group consisting of the following (1) to (5) has reduced or deleted function: (1) 3-dehydroshikimate dehydratase (qsuB), (2) quinate / shikimate dehydrogenase (qsuD), (3) 3-hydroxybenzoate 6-monooxygenase (genH), (4) lactate dehydrogenase (ldhA), and (5) phenol 2-monooxygenase (poxF). [6] The transformant according to any one of [1] to [5], wherein the microbial host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof. [7] A method for producing 3-HBA, comprising a step of culturing the transformant according to any one of [1] to [6] to produce 3-HBA.[8] A transformant for producing 3-hydroxybenzoic acid (3-HBA), obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of synthesizing 3-HBA from chorismate into a microbial host capable of producing chorismate, wherein the gene (A) is derived from at least one selected from the group consisting of Caballeronia calidae, Streptomyces conglobatus, Streptomyces shaanxiensis, Caballeronia cordobensis, Saccharopolyspora halophila, Caballeronia fortuita, Lentzea nigeriaca, Lentzea indica, Actinoplanes bogorensis, and Caballeronia hypogeia, and the microbial host is Corynebacterium glutamicum. [9] The transformant according to [8], wherein the gene (A) is selected from the group consisting of the following (i), (j), (k), and (l): (i) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135. (j) A gene encoding a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135, wherein the gene encodes a polypeptide having the activity of producing 3-HBA from chorismate. (k) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids in the amino acid sequence represented by SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135, and the polypeptide has the activity of producing 3-HBA from chorismate.(l) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135, and that encodes a polypeptide having the activity of producing 3-HBA from chorismate.

[10] The transformant according to [8] or [9], wherein the gene (A) is selected from the group consisting of (m), (n), (o), and (p) below. (m) A gene having the nucleotide sequence shown in SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113. (n) A gene having a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113, wherein the nucleotide sequence has deletions, substitutions, and / or additions of 1 to 10 bases per unit, with 100 bases being one unit, and wherein the gene encodes a polypeptide having the activity of producing 3-HBA from chorismate. (o) A gene having a nucleotide sequence having deletions, substitutions, and / or additions of 1 to 10 bases per unit, with 100 bases being one unit, in the nucleotide sequence shown in SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113, wherein the nucleotide sequence has deletions, substitutions, and / or additions of 1 to 10 bases per unit, with the nucleotide sequence being 100 bases being one unit, and wherein the gene encodes a polypeptide having the activity of producing 3-HBA from chorismate. (p) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113, and encodes a polypeptide having the activity of producing 3-HBA from chorismate.

[11] The transformant according to any one of [8] to

[10] , wherein the transformant has a reduced function or is functionally deleted in at least one enzyme selected from the group consisting of the following (1) to (5): (1) 3-dehydroshikimate dehydratase (qsuB), (2) quinate / shikimate dehydrogenase (qsuD), (3) 3-hydroxybenzoate 6-monooxygenase (genH), (4) lactate dehydrogenase (ldhA), and (5) phenol 2-monooxygenase (poxF).

[12] The transformant according to any one of [8] to

[11] , wherein the microbial host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

[13] A method for producing 3-HBA, comprising a step of culturing the transformant according to any one of [8] to

[12] to produce 3-hydroxybenzoic acid.

[0076] The present disclosure will be further described below using examples, but the present disclosure should not be construed as being limited to the following examples.

[0077] Construction of 3-HBA producing strain 1 (1) Preparation and acquisition of chromosomal DNA Actinoplanes ovalisporus JCM 33067, Burkholderia contaminans NBRC 111593, Caballeronia insecticola JCM 31142, Corynebacterium glutamicum R (FERM BP-18976), Krasilnikovia cinnamomea JCM 13252, Lentzea alba JCM 33970, Lentzea flava NBRC 14521, Lentzea waywayandensis NBRC 14970, Lentzea xinjiangensis JCM 15473, Methylibium petroleiphilum ATCC BAA-1232, Nonomuraea africana NBRC 14745, Saccharopolyspora aridisoli JCM 32922, Saccharopolyspora dendranthemae NBRC 108675, Streptomyces chrestomyceticus NBRC 13444, Streptomyces eurocidicus NBRC 13491, Streptomyces sp. NBRC 114659, Streptomyces rimosus subsp. paromomycinus NBRC 15454, Streptomyces zaomyceticus NBRC 13348, and Thiobacillus denitrificans ATCC 25259 were cultured according to the information provided by the strain obtaining institution, and then chromosomal DNA was prepared using a DNA genome extraction kit (illustra bacteria genomicPrep Mini Spin Kit, Cytiva). The 3-hydroxybenzoate synthase genes of Lentzea aerocolonigenes, Rhodococcus ruber, Saccharopolyspora flava, Streptomyces nanshensis, Streptomyces uncialis, and Thermobacillus composti were artificially synthesized.

[0078] (2) Construction of Expression Plasmids for 3-HBA Production-Related Genes The primer sequences used to isolate the 3-hydroxybenzoate synthase gene (gene (A)) are shown in Table 1. PCR was performed using a VeritiPro Thermal Cycler (Thermo Fisher Scientific) and PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) as a reaction reagent.

[0079] The PCR-amplified DNA fragment was introduced into the cloning vector pCRB209 [WO2012 / 033112] containing the PgapA promoter. The cloning vector introduced and the name of the resulting plasmid are shown in Table 2.

[0080] (3) Construction of a Plasmid for Disrupting the Chromosomal Genes of Corynebacterium glutamicum R Strain The DNA region required for markerless disruption of the chromosomal genes of Corynebacterium glutamicum R strain was amplified by PCR. Each PCR fragment could be ligated using a phosphorylated primer. The resulting DNA fragment was introduced into the markerless chromosomal gene modification plasmid pCRA725 [J. Mol. Microbiol. Biotechnol. 8:243-254 (2004), JP 2007-295809]. The primer sequences and the resulting plasmid are shown in Table 3.

[0081] (4) Construction of a chromosome-modified strain (Rmhgk) for 3-HBA production. The markerless chromosome gene modification vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of a single-crossover strain with the homologous region on the chromosome introduced into the pCRA725 plasmid, the strain exhibits kanamycin resistance due to the expression of the kanamycin resistance gene on pCRA725 and lethality on sucrose-containing medium due to the expression of the sacR-sacB gene derived from Bacillus subtilis. In contrast, in the case of a double-crossover strain, the strain exhibits kanamycin sensitivity due to the loss of the kanamycin resistance gene on pCRA725 and growth on sucrose-containing medium due to the loss of the sacR-sacB gene. Therefore, the markerless chromosome gene-modified strain exhibits kanamycin sensitivity and growth on sucrose-containing medium. Using the above-described method, a genetically modified strain was constructed using the aforementioned chromosomal gene disruption plasmid pCRG66. The host strain used was the Corynebacterium glutamicum R ldhA-disrupted strain CRZ1 [Biotechnol Bioeng. Nov;110(11):2938-2948 (2013)]. In addition, to improve the host's ability to produce chorismate, the following plasmids were used: aroG gene chromosomal introduction plasmid pCRB284 [WO2017 / 169399]; aroD gene chromosomal introduction plasmid pCRB291 [WO2017 / 169399]; aroE gene chromosomal introduction plasmid pCRB293 [WO2017 / 169399]; aroCKB gene chromosomal introduction plasmid pCRB286 [WO2017 / 169399]; aroA gene chromosomal introduction plasmid pCRB289 [WO2017 / 169399]; qsuB gene disruption plasmid pCRB229 [WO2017 / 169399]; and qsuD gene disruption plasmid pCRB299 [WO2017 / 169399]. To reduce or eliminate the function of phenol 2-monooxygenase, we also used the poxF gene disruption plasmid pCRG2 [WO2017 / 169399]. The details of this chromosomal gene recombination are summarized in Table 4.

[0082] (5) Construction of strains carrying the 3-HBA production-related gene expression plasmid The above-mentioned 3-HBA production-related gene expression plasmid was introduced into the Corynebacterium glutamicum Rmhgk1 strain to construct gene (A)-introduced strains (MHGK01-MHGK24). An overview of these strains is summarized in Table 5.

[0083] 3-HBA Production Experiment 1 The constructed gene (A)-introduced strains (MHGK01-MHGK24) were cultured in a nutrient medium containing glucose as a carbon source in a 200 ml jar fermenter under controlled conditions of temperature, pH, and dissolved oxygen concentration, and the ability of each strain to produce 3-HBA from glucose was examined. Each strain was pre-cultured overnight in 10 ml of AK-Glc liquid medium (liquid medium A [2 g of (NH2)2CO4, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 1 ml of a mixture of 0.6% (w / v) FeSO4·7H2O and 0.42% (w / v) MnSO4·H2O, 1 ml of 0.02% (w / v) biotin solution, 1 ml of 0.02% (w / v) thiamin solution, 2 g of yeast extract, and 7 g of vitamin A casamino acid dissolved in 1 L of distilled water] supplemented with 4% glucose and 50 μg / ml kanamycin) in a test tube. The strains were then inoculated into 100 ml of fresh AK-Glc liquid medium (8% glucose) to an initial OD610 of 0.5 and cultured in a 200 ml jar fermenter for 72 hours under the following conditions: 33°C, pH 7.5 (automatically adjusted by adding 5 N NaOH), DO > 10% (automatically controlled by agitation speed control between 100 and 2000 rpm), and 1.0 vvm (100 ml air / min). After 72 hours of jar culture, the 3-HBA content in the culture supernatant of each strain was quantified by HPLC analysis. All strains (MHGK01-MHGK24) produced 3-HBA from sugar (glucose). Among them, as shown in the table below, MHGK01, MHGK03, MHGK04, MHGK05, MHGK07, MHGK12, MHGK15, MHGK19 and MHGK22 showed 3-HBA production concentrations exceeding 350 mM, and in particular, MHGK07 had the highest 3-HBA production concentration at 475 mM.

[0084]

[0085] Construction of 3-HBA-producing strains 2 (1) Preparation and acquisition of chromosomal DNA The 3-hydroxybenzoate synthase genes of Actinoplanes bogorensis, Caballeronia calidae, Caballeronia cordobensis, Caballeronia fortuita, Caballeronia hypogeia, Lentzea indica, Lentzea nigeriaca, Saccharopolyspora halophila, Streptomyces conglobatus, and Streptomyces shaanxiensis were artificially synthesized.

[0086] (2) Construction of Expression Plasmids for Genes Associated with 3-HBA Production The primer sequences used to isolate the 3-hydroxybenzoate synthase gene (gene (A)) are shown in Table 7. PCR was performed using a VeritiPro Thermal Cycler (Thermo Fisher Scientific) and PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) as a reaction reagent.

[0087] The PCR-amplified DNA fragment was introduced into the cloning vector pCRB209 [WO2012 / 033112] containing the PgapA promoter. The cloning vector introduced and the name of the resulting plasmid are shown in Table 8.

[0088] (3) Construction of a Plasmid for Disrupting Chromosomal Genes in Corynebacterium glutamicum R Strain The DNA region required for markerless disruption of chromosomal genes in Corynebacterium glutamicum R strain was amplified by PCR. Each PCR fragment could be ligated using a phosphorylated primer. The resulting DNA fragment was introduced into the markerless chromosomal gene modification plasmid pCRA725 [J. Mol. Microbiol. Biotechnol. 8:243-254 (2004), JP 2007-295809]. The primer sequences and the resulting plasmid are shown in Table 9.

[0089] (4) Construction of a chromosome-modified strain (Rmhgk) for 3-HBA production. The markerless chromosome gene modification vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of a single-crossover strain with the homologous region on the chromosome introduced into the pCRA725 plasmid, the strain exhibits kanamycin resistance due to the expression of the kanamycin resistance gene on pCRA725 and lethality on sucrose-containing medium due to the expression of the sacR-sacB gene derived from Bacillus subtilis. In contrast, in the case of a double-crossover strain, the strain exhibits kanamycin sensitivity due to the loss of the kanamycin resistance gene on pCRA725 and growth on sucrose-containing medium due to the loss of the sacR-sacB gene. Therefore, the markerless chromosome gene-modified strain exhibits kanamycin sensitivity and growth on sucrose-containing medium. Using the above-described method, a genetically modified strain was constructed using the aforementioned chromosomal gene disruption plasmid pCRG66. The host strain used was the Corynebacterium glutamicum R ldhA-disrupted strain CRZ1 [Biotechnol Bioeng. Nov;110(11):2938-2948 (2013)]. In addition, to improve the host's ability to produce chorismate, the following plasmids were used: aroG gene chromosomal introduction plasmid pCRB284 [WO2017 / 169399]; aroD gene chromosomal introduction plasmid pCRB291 [WO2017 / 169399]; aroE gene chromosomal introduction plasmid pCRB293 [WO2017 / 169399]; aroCKB gene chromosomal introduction plasmid pCRB286 [WO2017 / 169399]; aroA gene chromosomal introduction plasmid pCRB289 [WO2017 / 169399]; qsuB gene disruption plasmid pCRB229 [WO2017 / 169399]; and qsuD gene disruption plasmid pCRB299 [WO2017 / 169399]. To reduce or eliminate the function of phenol 2-monooxygenase, we also used the poxF gene disruption plasmid pCRG2 [WO2017 / 169399]. The details of this chromosomal gene recombination are summarized in Table 10.

[0090] (5) Construction of strains carrying the 3-HBA production-related gene expression plasmid The above-mentioned 3-HBA production-related gene expression plasmid was introduced into the Corynebacterium glutamicum Rmhgk1 strain to construct gene (A)-introduced strains (MHGK25-MHGK34). An overview of these strains is summarized in Table 11.

[0091] 3-HBA Production Experiment 2 The constructed gene (A)-introduced strains (MHGK25-MHGK34) were cultured in a nutrient medium containing glucose as a carbon source in a 200 ml jar fermenter under controlled conditions of temperature, pH, and dissolved oxygen concentration, and the ability of each strain to produce 3-HBA from glucose was examined. Each strain was pre-cultured overnight in 10 ml of AK-Glc liquid medium (liquid medium A [2 g of (NH2)2CO4, 7 g of (NH4)2SO4, 0.5 g of KH2PO4, 0.5 g of K2HPO4, 0.5 g of MgSO4·7H2O, 1 ml of a mixture of 0.6% (w / v) FeSO4·7H2O and 0.42% (w / v) MnSO4·H2O, 1 ml of 0.02% (w / v) biotin solution, 1 ml of 0.02% (w / v) thiamin solution, 2 g of yeast extract, and 7 g of vitamin A casamino acid dissolved in 1 L of distilled water] supplemented with 4% glucose and 50 μg / ml kanamycin) in a test tube. The strains were then inoculated into 100 ml of fresh AK-Glc liquid medium (8% glucose) to an initial OD610 of 0.5 and cultured in a 200 ml jar fermenter at 33°C, pH 7.5 (automatically adjusted by adding 5 N NaOH), DO > 10% (automatically controlled by agitation speed control between 100 and 2000 rpm), and 1.0 vvm (100 ml air / min) for 72 hours. After 72 hours of jar culture, the 3-HBA content in the culture supernatant of each strain was quantified by HPLC analysis. All strains (MHGK25-MHGK34) produced 3-HBA from glucose. As shown in the table below, MHGK26, MHGK27, MHGK33, and MHGK34 produced 3-HBA at concentrations exceeding 350 mM, with MHGK26 producing the highest concentration of 3-HBA at 491 mM.

[0092]

[0093] The abbreviated compound names and their CAS numbers shown in Figure 2 (an overall metabolic pathway diagram for explaining the biosynthetic pathway of 3-HBA) are shown below. It goes without saying that the "compound names" shown below are only examples, and that synonymous alternative names may exist. It also goes without saying that the "CAS numbers" shown below are not necessarily a comprehensive list.

[0094] The enzymes encoded by the genes shown in Figure 2 (an overall metabolic pathway diagram for explaining the biosynthetic pathway of 3-HBA) and their EC numbers are shown below. The "enzyme names" shown below are examples, and it goes without saying that there may be synonymous alternative names. Furthermore, the "enzymes" encoded by the "genes" shown below are examples, and it goes without saying that there may be "genes" that encode "enzymes" with multiple functions.

[0095]

Claims

1. A transformant for producing 3-hydroxybenzoic acid (3-HBA), obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of producing 3-HBA from chorismic acid into a microbial host capable of producing chorismic acid, wherein the microbial host is Corynebacterium glutamicum.

2. The gene (A) is Lentzea flava, Streptomyces sp. NBRC 114659, Saccharopolyspora flava, Actinoplanes ovalisporus, Caballer insectoniaicola, Krasilnikovia cinnamomea, Streptomyces zaomyceticus, Lentzea aerocolonigenes, Rhodococcus ruber, Lentzea xinjiangensis, Lentzea waywayandensis, Streptomyces nanshensis, Streptomyces chrestomyceticus, Nonomuraea africana, Saccharopolyspora aridisoli, Methylibium petroleiphilum, Streptomyces uncialis, Streptomyces eurocidicus, Lentzea alba, Burkholderia contaminans, Saccharopolyspora dendranthemae, Streptomyces rimosus The transformant according to claim 1, which is derived from at least one selected from the group consisting of subsp. paromomycinus, Thermobacillus composti, and Thiobacillus denitrificans.

3. The transformant according to claim 1, wherein the gene (A) is selected from the group consisting of the following: (a) a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:

96. (b) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96, wherein the gene encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate. (c) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids in the amino acid sequence represented by SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96, wherein the amino acid sequence is a unit of 100 amino acids, and the gene encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate.(d) A gene that hybridizes under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:87, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:94, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:81, SEQ ID NO:80, SEQ ID NO:90, SEQ ID NO:88, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:82, SEQ ID NO:93, SEQ ID NO:89, SEQ ID NO:78, SEQ ID NO:74, SEQ ID NO:86, SEQ ID NO:92, SEQ ID NO:95, or SEQ ID NO:96, and that encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate.

4. The transformant according to claim 1, wherein the gene (A) is selected from the group consisting of the following (e), (f), (g), and (h): (e) a gene having the nucleotide sequence set forth in SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:43, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:64, SEQ ID NO:13, SEQ ID NO:34, SEQ ID NO:25, SEQ ID NO:22, SEQ ID NO:52, SEQ ID NO:46, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:28, SEQ ID NO:61, SEQ ID NO:49, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:67, or SEQ ID NO:

70. (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:43, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:64, SEQ ID NO:13, SEQ ID NO:34, SEQ ID NO:25, SEQ ID NO:22, SEQ ID NO:52, SEQ ID NO:46, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:28, SEQ ID NO:61, SEQ ID NO:49, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:67, or SEQ ID NO:70, which encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate. (g) A gene having a base sequence represented by SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:43, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:64, SEQ ID NO:13, SEQ ID NO:34, SEQ ID NO:25, SEQ ID NO:22, SEQ ID NO:52, SEQ ID NO:46, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:28, SEQ ID NO:61, SEQ ID NO:49, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:67, or SEQ ID NO:70, with 1 to 10 base deletions, substitutions, and / or additions per unit, where 100 bases constitute one unit, and which encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate.(h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO:19, SEQ ID NO:55, SEQ ID NO:43, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:64, SEQ ID NO:13, SEQ ID NO:34, SEQ ID NO:25, SEQ ID NO:22, SEQ ID NO:52, SEQ ID NO:46, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:28, SEQ ID NO:61, SEQ ID NO:49, SEQ ID NO:16, SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:58, SEQ ID NO:67, or SEQ ID NO:70, and encodes a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate.

5. The transformant according to claim 1, wherein the transformant has reduced or no function of at least one enzyme selected from the group consisting of the following (1) to (5): (1) 3-dehydroshikimate dehydratase (qsuB), (2) quinate / shikimate dehydrogenase (qsuD), (3) 3-hydroxybenzoate 6-monooxygenase (genH), (4) lactate dehydrogenase (ldhA), and (5) phenol 2-monooxygenase (poxF).

6. The transformant according to claim 1, wherein the microbial host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

7. A method for producing 3-hydroxybenzoic acid, comprising the step of culturing the transformant according to any one of claims 1 to 6 to produce 3-hydroxybenzoic acid.

8. A transformant for producing 3-hydroxybenzoic acid (3-HBA), obtained by expressibly introducing a gene (A) encoding an enzyme having the activity of synthesizing 3-HBA from chorismic acid into a microbial host capable of producing chorismic acid, wherein the gene (A) is derived from at least one selected from the group consisting of Caballeronia calidae, Streptomyces conglobatus, Streptomyces shaanxiensis, Caballeronia cordobensis, Saccharopolyspora halophila, Caballeronia fortuita, Lentzea nigeriaca, Lentzea indica, Actinoplanes bogorensis, and Caballeronia hypogeia, and the microbial host is Corynebacterium glutamicum.

9. The transformant according to claim 8, wherein the gene (A) is selected from the group consisting of the following (i), (j), (k), and (l): (i) a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO:

135. (j) a gene encoding a polypeptide having an amino acid sequence that is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135, and encoding a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate. (k) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, and / or added per unit of 100 amino acids in the amino acid sequence shown in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135, and the gene encoding the polypeptide has the activity of producing 3-hydroxybenzoic acid from chorismate. (l) A gene that hybridizes under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 132, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 133, SEQ ID NO: 138, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 136, SEQ ID NO: 131, or SEQ ID NO: 135, and the gene encoding the polypeptide has the activity of producing 3-hydroxybenzoic acid from chorismate.

10. The transformant according to claim 8, wherein the gene (A) is selected from the group consisting of the following (m), (n), (o), and (p): (m) a gene having the nucleotide sequence shown in SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO:

113. (n) a gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113, and encoding a polypeptide having the activity of synthesizing 3-hydroxybenzoic acid from chorismate. (o) A gene having a nucleotide sequence represented by SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113, with 1 to 10 base deletions, substitutions, and / or additions per unit of 100 bases, and encoding a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate. (p) A gene that hybridizes under stringent conditions to a gene having a nucleotide sequence complementary to the nucleotide sequence represented by SEQ ID NO: 104, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 107, SEQ ID NO: 122, SEQ ID NO: 110, SEQ ID NO: 119, SEQ ID NO: 116, SEQ ID NO: 101, or SEQ ID NO: 113, and encoding a polypeptide having the activity of producing 3-hydroxybenzoic acid from chorismate.

11. The transformant according to claim 8, wherein the transformant has reduced or no function of at least one enzyme selected from the group consisting of the following (1) to (5): (1) 3-dehydroshikimate dehydratase (qsuB), (2) quinate / shikimate dehydrogenase (qsuD), (3) 3-hydroxybenzoate 6-monooxygenase (genH), (4) lactate dehydrogenase (ldhA), and (5) phenol 2-monooxygenase (poxF).

12. The transformant according to claim 8, wherein the microbial host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof.

13. A method for producing 3-hydroxybenzoic acid, comprising the step of culturing the transformant according to any one of claims 8 to 12 to produce 3-hydroxybenzoic acid.

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