Microorganism, and method for producing salicylic acid or salt thereof using same

Abstract

The present disclosure provides: a novel microorganism capable of improving the productivity of salicylic acid or a salt thereof; and a method for producing salicylic acid or a salt thereof using the microorganism.　In one embodiment, the present disclosure pertains to a microorganism having: a gene that encodes an enzyme having the activity to produce isochorismic acid from chorismic acid; and a gene (A) that encodes an enzyme having the activity to produce salicylic acid from isochorismic acid and / or a gene (B) that encodes an enzyme having both the activity to produce isochorismic acid from chorismic acid and the activity to produce salicylic acid from isochorismic acid.

Description

Microorganisms and methods for producing salicylic acid or its salts using the same

[0001] The present disclosure relates to a technique for producing salicylic acid or a salt thereof. In one aspect, the present disclosure relates to a microorganism that has been subjected to specific genetic manipulation, and a technique for producing salicylic acid or a salt thereof using the microorganism.

[0002] Salicylic acid (CAS number: 69-72-7; synonyms include 2-hydroxybenzoic acid, o-hydroxybenzoic acid, and 2-hydroxybenzenecarboxylic acid) crystallizes in a needle shape and is easily soluble in ethanol and ether, but poorly soluble in cold water. Applications of salicylic acid include keratin solvents and antiseptics. Salicylic acid can also be produced by microbial fermentation (see, for example, Patent Document 1), and there is a demand for the creation of microorganisms that produce salicylic acid more efficiently.

[0003] Japanese Patent Application Laid-Open No. 2020-184993

[0004] The present disclosure provides a novel microorganism capable of improving the productivity of salicylic acid or a salt thereof, and a method for producing salicylic acid or a salt thereof using the microorganism.

[0005] In one aspect, the present disclosure relates to a microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid, and a gene (A) encoding an enzyme having the activity of producing salicylic acid from isochorismic acid and / or a gene (B) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid, wherein gene (A) is at least one gene selected from the group consisting of (A1) to (A9) below, and gene (B) is at least one gene selected from the group consisting of (B1) to (B9) below. (A1) A gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98; (A2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98; (A3) 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: 94, 95, 96, 97, or 98; (A4) A gene that hybridizes under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98; (A5) A gene having a nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13; (A6) A gene having a nucleotide sequence having 50% or more identity to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13; (A7) A gene having a nucleotide sequence represented by SEQ ID NO: 1, 4, 7, 10, or 13, in which 1 to 10 bases have been deleted, substituted, and / or added per unit of 100 bases; (A8) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence represented by SEQ ID NO: 1, 4, 7, 10, or 13;(A9) A gene derived from at least one selected from the group consisting of the genus Zymobacter, the genus Microbulbifer, the genus Paracoccus, and Pseudomonas. (B1) A gene encoding a polypeptide having an amino acid sequence set forth in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113; (B2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113; (B3) 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: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113; (B4) A gene hybridizing 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: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113; (B5) A gene having a nucleotide sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58; (B6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58; (B7) A gene having a nucleotide sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58, where 100 bases are considered as one unit, and having 1 to 10 base deletions, substitutions, and / or additions per unit;(B8) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58; (B9) A gene derived from at least one selected from the group consisting of the genera Streptomyces, Actinoplanes, Citrobacter, Mycolicibacterium, Gordonia, Saccharopolyspora, Aspergillus, Sorangium, and Mycobacteroides;

[0006] In another aspect, the present disclosure relates to a method for producing salicylic acid or a salt thereof, comprising culturing a microorganism of the present disclosure to produce salicylic acid or a salt thereof.

[0007] According to one aspect of the present disclosure, a novel microorganism capable of improving productivity of salicylic acid or a salt thereof can be provided, preferably a microorganism capable of efficiently producing salicylic acid or a salt thereof using sugars or the like as raw materials. According to one aspect of the present disclosure, a method for producing salicylic acid or a salt thereof with improved productivity can be provided.

[0008] Figure 1 is a schematic diagram showing salicylic acid production in one embodiment of the microorganism of the present disclosure. Figure 2 is a schematic diagram showing salicylic acid production in another embodiment of the microorganism of the present disclosure. Figure 3 is a schematic diagram showing salicylic acid production in yet another embodiment of the microorganism of the present disclosure. Figure 4 is a schematic diagram showing salicylic acid production in yet another embodiment of the microorganism of the present disclosure. Figure 5 is a schematic diagram showing salicylic acid production in yet another embodiment of the microorganism of the present disclosure. Figure 6 is a schematic diagram showing salicylic acid production in yet another embodiment of the microorganism of the present disclosure. Figure 7 is an overall metabolic pathway diagram illustrating the biosynthetic pathway for salicylic acid production in one embodiment of the microorganism of the present disclosure.

[0009] The present disclosure is based on the discovery by the present inventors that, in one or more embodiments, a microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and a gene (A) encoding an enzyme having the activity of producing salicylic acid from isochorismic acid, the gene (A) being selected from the group consisting of (A1) to (A9) above, and a gene (B) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid, the gene (B) being at least one selected from the group consisting of (B1) to (B9) above, can biologically produce salicylic acid using sugars such as glucose as a raw material, and can further improve salicylic acid productivity compared to conventional methods (e.g., the method disclosed in Patent Document 1).

[0010] In the present disclosure, "50% or more identity" with respect to an amino acid sequence or a nucleotide sequence refers to at least 50% identity, and in one or more embodiments, refers to 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 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, 99.9% or more, or 100% identity.

[0011] 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).

[0012] 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 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°C to 10°C lower than the melting temperature (Tm) of a perfect hybrid.

[0013] In this disclosure, the term "native" used in reference to an enzyme, gene, etc. possessed by a microorganism is used to indicate that the enzyme or gene is present in the wild-type of the microorganism, while the term "exogenous" is used to indicate that the enzyme or gene is inherent to the microorganism, i.e., is not originally present in the microorganism, but is introduced from outside.

[0014] [Microorganism of the Present Disclosure] In one or more embodiments, the microorganism of the present disclosure relates to a microorganism having: a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid; and at least one of a gene (A) encoding an enzyme having the activity of producing salicylic acid from isochorismic acid, selected from the group consisting of the following (A1) to (A9), and a gene (B) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid, selected from the group consisting of the following (B1) to (B9). In one or more embodiments, the microorganism of the present disclosure is capable of producing salicylic acid from chorismic acid, the final product of the shikimic acid pathway (e.g., Figures 1 to 6).

[0015] In one or more embodiments, the present disclosure relates to a microorganism capable of producing salicylic acid or having improved salicylic acid production ability, the microorganism having a gene encoding an enzyme active in producing isochorismic acid from chorismic acid and at least one of the gene (A) and the gene (B). In one or more embodiments, the microorganism of the present disclosure may have one or more of the three types of genes (the gene encoding the enzyme active in producing isochorismic acid from chorismic acid, the gene (A), and the gene (B)) introduced in an expressible manner, or may have enhanced expression of one or more genes, or may have one or more genes introduced in an expressible manner and enhanced expression of the remaining genes. Thus, in one or more embodiments, the microorganism of the present disclosure may also be referred to as a transformant. In one or more embodiments, the microorganism of the present disclosure may also be referred to as a transformant having salicylic acid production ability or improved salicylic acid production ability by introducing and / or enhancing expression of the above-mentioned genes.

[0016] In the present disclosure, the "transformant obtained by introducing a gene" may include a transformant obtained by introducing the gene in an expressible manner, and a transformant that can be obtained by introducing the gene in an expressible manner.

[0017] 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, examples of the gene introduction include gene introduction using a plasmid vector and integration into the chromosome of the microbial host.

[0018] 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.

[0019] In the present disclosure, "enhanced gene expression" may, in one or more embodiments, include an increase in the expression level of the polypeptide encoded by the target gene, an increase in the activity of the polypeptide encoded by the target gene, and / or an increase in the transcription level of the target gene, compared to a strain in which expression of the gene (target gene) is not enhanced (or before the expression enhancement). In one or more embodiments, the gene may be exogenous, or may be one that the microorganism (host) itself inherently possesses.

[0020] In one or more embodiments, the microorganism of the present disclosure includes the following transformants (1) to (8).(1) A transformant obtained by expressibly introducing a gene (C) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and a gene (A) selected from the group consisting of the following (A1) to (A9) into a microbial host (FIG. 1); (2) A transformant obtained by expressibly introducing a gene (C) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid, a gene (A) selected from the group consisting of the following (A1) to (A9), and a gene (B) selected from the group consisting of the following (B1) to (B9) into a microbial host (FIG. 2); (3) A transformant obtained by expressibly introducing a gene (A) selected from the group consisting of the following (A1) to (A9) and a gene (B) selected from the group consisting of the following (B1) to (B9) into a microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid (FIG. 3); (4) A transformant obtained by expressibly introducing a gene (C) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and a gene (B) selected from the group consisting of the following (B1) to (B9) into a microbial host (FIG. 4); (5) A transformant obtained by expressibly introducing a gene (B) selected from the group consisting of the following (B1) to (B9) into a microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid (FIG. 5); (6) A transformant obtained by expressibly introducing a gene (A) selected from the group consisting of the following (A1) to (A9) into a microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid (FIG. 6); (7) A transformant obtained by expressibly introducing a gene (C) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid into a microorganism having at least one of a gene (A) selected from the group consisting of the following (A1) to (A9) and a gene (B) selected from the group consisting of the following (B1) to (B9); (8) A transformant in which expression of a gene (C) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid, a gene (A) selected from the group consisting of the following (A1) to (A9), and a gene (B) selected from the group consisting of the following (B1) to (B9) is enhanced.

[0021] [Gene (A)] The microorganism of the present disclosure has a gene (A) encoding an enzyme having the activity of producing salicylic acid from isochorismate, the gene (A) being selected from the group consisting of (A1) to (A9) below. In one or more embodiments, the gene (A) encodes a polypeptide having the activity of isochorismate pyruvate lyase (ipl, EC number: 4.2.99.21). In one or more embodiments, an example of a gene encoding isochorismate pyruvate lyase is the pchB gene. In one or more embodiments, the gene (A) in the microorganism of the present disclosure may be a foreign gene, a gene native to the microorganism, or a gene in which expression of the gene native to the microorganism has been enhanced.

[0022] In the present disclosure, a "gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: XX" refers to a gene encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO: XX. In the present disclosure, a "gene having the nucleotide sequence shown in SEQ ID NO: XX" refers to a gene comprising the nucleotide sequence shown in SEQ ID NO: XX.

[0023] In one or more embodiments, the genera Zymobacter, Microbulbifer, Paracoccus, and Pseudomonas are examples of the origin of the gene (A). 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 there may be synonymous alternative names.

[0024] In one or more embodiments, examples of the Zymobacter genus include Zymobacter palmae. In one or more embodiments, examples of the pchB gene derived from Zymobacter palmae include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94.

[0025] In one or more embodiments, the genus Microbulbifer includes Microbulbifer sp. In one or more embodiments, the pchB gene derived from Microbulbifer sp. includes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 4. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 4 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 95.

[0026] In one or more embodiments, an example of the genus Paracoccus is Paracoccus denitrificans. In one or more embodiments, an example of the pchB gene derived from Paracoccus denitrificans is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 7. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 7 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 96.

[0027] In one or more embodiments, examples of the genus Pseudomonas include Pseudomonas fluorescens and Pseudomonas putida. In one or more embodiments, an example of the pchB gene derived from Pseudomonas fluorescens is 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: 97. In one or more embodiments, an example of the pchB gene derived from Pseudomonas putida is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 13. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 13 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 98.

[0028] In one or more embodiments, gene (A) is a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98, or a gene having the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13, and is preferably a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13.

[0029] In one or more embodiments, the gene (A) may be a gene selected from the group consisting of the following genes (A2) to (A4) and (A6) to (A9): The genes (A2) to (A4) and (A6) to (A9) encode enzymes having the activity of producing salicylic acid from isochorismic acid. (A2) a gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98, or a gene encoding a polypeptide consisting of an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98; (A3) a gene encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98, in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids considered as one unit, or a gene encoding a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98, in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids considered as one unit; (A4) 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: 94, 95, 96, 97, or 98, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94, 95, 96, 97, or 98; (A6) A gene that has a nucleotide sequence that is 50% or more identical to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13, or a gene consisting of a nucleotide sequence that is 50% or more identical to the nucleotide sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13;(A7) A gene having a base sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13, with 1 to 10 base deletions, substitutions, and / or additions per unit of 100 bases, or a gene consisting of a base sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13, with 1 to 10 base deletions, substitutions, and / or additions per unit of 100 bases; (A8) A gene that hybridizes under stringent conditions with a gene having a base sequence complementary to a gene having the base sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13, or a gene that hybridizes under stringent conditions with a gene consisting of a base sequence complementary to a gene consisting of the base sequence shown in SEQ ID NO: 1, 4, 7, 10, or 13; (A9) A gene derived from at least one gene selected from the group consisting of the genera Zymobacter, Microbulbifer, Paracoccus, and Pseudomonas. ;

[0030] [Gene (B)] The microorganism of the present disclosure has a gene (B) encoding an enzyme having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid, the gene (B) being selected from the group consisting of (B1) to (B9) below. In one or more embodiments, the gene (B) encodes a polypeptide having salicylate synthase (irp, EC number: 5.4.4.2, 4.2.99.21) activity. In one or more embodiments, an example of a gene encoding salicylate synthase is an irp gene. In one or more embodiments, the gene (B) in the microorganism of the present disclosure may be a foreign gene, a gene native to the microorganism, or a gene in which expression of the gene native to the microorganism has been enhanced.

[0031] In one or more embodiments, examples of the origin of the genera (B) include the genera Streptomyces, Actinoplanes, Citrobacter, Mycolicibacterium, Gordonia, Saccharopolyspora, Aspergillus, Sorangium, and Mycobacteroides.

[0032] In one or more embodiments, examples of the genus Streptomyces include Streptomyces glaucescens, Streptomyces pharetrae, Streptomyces rapamycinicus, Streptomyces violaceoruber, Streptomyces lividans, and Streptomyces venezuelae. Synonymous names for Streptomyces violaceoruber include Streptomyces coelicolor. In one or more embodiments, examples of irp genes derived from Streptomyces glaucescens, Streptomyces pharetrae, Streptomyces rapamycinicus, Streptomyces violaceoruber, Streptomyces lividans, and Streptomyces venezuelae include genes consisting of the nucleotide sequences set forth in SEQ ID NOs: 16, 19, 22, 25, 28, and 58, respectively. The genes consisting of the base sequences shown in SEQ ID NOs: 16, 19, 22, 25, 28, and 58 encode polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 99, 100, 101, 102, 103, and 113, respectively.

[0033] In one or more embodiments, the genus Actinoplanes includes Actinoplanes sp. In one or more embodiments, an irp gene derived from Actinoplanes sp. includes 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: 104.

[0034] In one or more embodiments, the genus Citrobacter includes Citrobacter koseri. In one or more embodiments, an IRP gene derived from Citrobacter koseri includes genes consisting of the nucleotide sequences shown in SEQ ID NOs: 34 and 37. 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: 105. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 37 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 106.

[0035] In one or more embodiments, the genus Mycolicibacterium includes Mycolicibacterium smegmatis. In one or more embodiments, an irp gene derived from Mycolicibacterium smegmatis includes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 40. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 40 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 107.

[0036] In one or more embodiments, the genus Gordonia includes Gordonia terrae. In one or more embodiments, an irp gene derived from Gordonia terrae includes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 43. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 43 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 108.

[0037] In one or more embodiments, examples of the genus Saccharopolyspora include Saccharopolyspora erythraea. In one or more embodiments, examples of the irp gene derived from Saccharopolyspora erythraea include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 46. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 46 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 109.

[0038] In one or more embodiments, the genus Aspergillus includes Aspergillus clavatus. In one or more embodiments, an irp gene derived from Aspergillus clavatus includes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 49. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 49 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 110.

[0039] In one or more embodiments, examples of the genus Sorangium include Sorangium cellulosum. In one or more embodiments, examples of the irp gene derived from Sorangium cellulosum include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 52. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 52 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 111.

[0040] In one or more embodiments, examples of the genus Mycobacteroides include Mycobacteroides immunogenum. In one or more embodiments, examples of the irp gene derived from Mycobacteroides immunogenum include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 55. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 55 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 112.

[0041] In one or more embodiments, the gene (B) is a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113, or a gene encoding a polypeptide having a nucleotide sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58. The gene is preferably a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58.

[0042] In one or more embodiments, the gene (B) may be a gene selected from the group consisting of the following genes (B2) to (B4) and (B6) to (B9): The genes (B2) to (B4) and (B6) to (B9) encode enzymes having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid. (B2) a gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113, or a gene encoding a polypeptide consisting of an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113; (B3) a gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113, in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids being considered as one unit; a gene encoding a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113, in which 1 to 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids being considered as one unit;(B4) 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 represented by SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113, or a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene encoding a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113; (B6) a gene having a nucleotide sequence having 50% or more identity to the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58, or a gene consisting of a nucleotide sequence having 50% or more identity to the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58; (B7) A gene having a base sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58, with 100 bases considered as one unit, in which 1 to 10 bases have been deleted, substituted, and / or added per unit; or a gene consisting of a base sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58, with 100 bases considered as one unit, in which 1 to 10 bases have been deleted, substituted, and / or added per unit; (B8) a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to a gene consisting of the nucleotide sequence represented by SEQ ID NO: 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, or 58;(B9) A gene derived from at least one selected from the group consisting of the genera Streptomyces, Actinoplanes, Citrobacter, Mycolicibacterium, Gordonia, Saccharopolyspora, Aspergillus, Sorangium, and Mycobacteroides;

[0043] In one or more embodiments, the gene (B) is preferably a gene derived from the genus Streptomyces in order to further improve salicylic acid productivity. In one or more embodiments, the gene (B) is preferably a gene derived from Streptomyces glaucescens, Streptomyces pharetrae, Streptomyces rapamycinicus, Streptomyces violaceoruber, Streptomyces lividans, Actinoplanes sp., or Streptomyces venezuelae in order to further improve salicylic acid productivity.

[0044] In one or more embodiments, the gene (B) may be a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 99, 100, 101, 102, 103, 104, or 113, 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: 99, 100, 101, 102, 103, 104, or 113, a gene having the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, or 58, or a gene encoding a polypeptide having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, or 58, from the viewpoint of further improving salicylic acid productivity. A gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 99, 100, 101, 102, 103, 104, or 113, 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: 99, 100, 101, 102, 103, 104, or 113, a gene consisting of the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, or 58, or a gene consisting of a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 16, 19, 22, 25, 28, 31, or 58 is more preferred.

[0045] [Gene (C)] The microorganism of the present disclosure has a gene (C) encoding an enzyme having the activity of producing isochorismate from chorismate. In one or more embodiments, the gene (C) encodes a polypeptide having isochorismate synthase (ics, EC number: 5.4.4.2) activity. In one or more embodiments, examples of genes encoding isochorismate synthase include the entC gene, the dhbC gene, and the menF gene. In one or more embodiments, the gene (C) in the microorganism of the present disclosure may be one that the microorganism naturally has, may be a gene in which expression of a gene that the microorganism naturally has is enhanced, or may be an exogenous gene.

[0046] In one or more embodiments, examples of the origin of the genera (C) include Enterobacter, Escherichia, Klebsiella, Bacillus, Citrobacter, Cronobacter, Marinomonas, Zymobacter, Photobacterium, Xenorhabdus, and Corynebacterium.

[0047] In one or more embodiments, the genus Enterobacter includes Enterobacter cloacae, etc. In one or more embodiments, the entC gene derived from Enterobacter cloacae includes a gene consisting of the nucleotide sequence shown in SEQ ID NO: 61. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 61 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 114.

[0048] In one or more embodiments, examples of the genus Escherichia include Escherichia coli and Escherichia fergusonii. In one or more embodiments, an example of the entC gene derived from Escherichia coli is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 64. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 64 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 115. In one or more embodiments, an example of the entC gene of Escherichia fergusonii is a gene consisting of the nucleotide sequence shown in SEQ ID NO: 70. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 70 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 117.

[0049] In one or more embodiments, examples of the genus Klebsiella include Klebsiella variicola. In one or more embodiments, examples of the entC gene derived from Klebsiella variicola include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 67. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 67 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 116.

[0050] In one or more embodiments, examples of the genus Bacillus include Bacillus amyloliquefaciens. In one or more embodiments, examples of the dhbC gene derived from Bacillus amyloliquefaciens include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 73. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 73 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 118.

[0051] In one or more embodiments, examples of the genus Citrobacter include Citrobacter koseri. In one or more embodiments, examples of the entC gene derived from Citrobacter koseri include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 76. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 76 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 119.

[0052] In one or more embodiments, examples of the genus Cronobacter include Cronobacter sakazakii. In one or more embodiments, examples of the entC gene derived from Cronobacter sakazakii include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 79. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 79 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 120.

[0053] In one or more embodiments, examples of the genus Marinomonas include Marinomonas mediterranea. In one or more embodiments, examples of the entC gene derived from Marinomonas mediterranea include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 82. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 82 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 121.

[0054] In one or more embodiments, examples of the Zymobacter genus include Zymobacter palmae. In one or more embodiments, examples of the entC gene derived from Zymobacter palmae include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 85. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 85 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 122.

[0055] In one or more embodiments, examples of the genus Photobacterium include Photobacterium profundum. In one or more embodiments, examples of the entC gene derived from Photobacterium profundum include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 88. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 88 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 123.

[0056] In one or more embodiments, examples of the genus Xenorhabdus include Xenorhabdus bovienii. In one or more embodiments, examples of the entC gene derived from Xenorhabdus bovienii include a gene consisting of the nucleotide sequence shown in SEQ ID NO: 91. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 91 encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 124.

[0057] In one or more embodiments, the genus Corynebacterium includes bacteria exemplified as the genus Corynebacterium described below, with Corynebacterium glutamicum being preferred. In one or more embodiments, an example of the menF gene derived from Corynebacterium glutamicum 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: 126.

[0058] In one or more embodiments, the gene (C) is a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, or a gene having the nucleotide sequence shown in SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125, and is preferably a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125.

[0059] In one or more embodiments, the gene (C) may be a gene selected from the group consisting of the following genes (C2) to (C4) and (C6) to (C9). The genes (C2) to (C4) and (C6) to (C9) encode enzymes having the activity of producing isochorismic acid from chorismate. (C2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, or a gene encoding a polypeptide consisting of an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126; (C3) 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: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, 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: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, (C4) a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, 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: 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 126, and a gene that hybridizes under stringent conditions;(C6) a gene having a nucleotide sequence having 50% or more identity to the nucleotide sequence represented by SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125, or a gene consisting of a nucleotide sequence having 50% or more identity to the nucleotide sequence represented by SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125; (C7) A gene having a base sequence represented by SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125, with 100 bases per unit, in which 1 to 10 bases have been deleted, substituted, and / or added per unit; or a gene consisting of a base sequence represented by SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125, with 100 bases per unit, in which 1 to 10 bases have been deleted, substituted, and / or added per unit; (C8) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence shown in SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125, or a gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene consisting of the nucleotide sequence shown in SEQ ID NO: 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, or 125; (C9) A gene derived from at least one gene selected from the group consisting of the genera Enterobacter, Escherichia, Klebsiella, Bacillus, Citrobacter, Cronobacter, Marinomonas, Zymobacter, Photobacterium, Xenorhabdus, and Corynebacterium;

[0060] [Microbial Host] In one or more embodiments, the host for the microorganism or transformant of the present disclosure may include coryneform bacteria, Escherichia coli (bacteria of the genus Escherichia, particularly Escherichia coli), solvent-tolerant bacteria, yeast, and the like. 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, coryneform bacteria may 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.

[0061] In one or more embodiments, the microbial host may be a coryneform bacterium or a transformant thereof. In one or more embodiments, the transformant of a coryneform bacterium may be a transformant modified to improve chorismate production. Wild-type coryneform bacteria are known to lack a gene corresponding to gene (A) or a gene corresponding to gene (B), but possess a gene corresponding to gene (C). Therefore, when the microbial host is a coryneform bacterium, in one or more embodiments, the microorganism of the present disclosure preferably has at least one of the genes (A) and (B) introduced in an expressible manner, and more preferably has at least one of the genes (A) and (B) introduced in an expressible manner and gene (C) introduced in an expressible manner, or the expression of gene (C) is enhanced.

[0062] The microbial host is not particularly limited, and from the viewpoint of improving salicylic acid productivity, it may be, for example, a Corynebacterium genus bacterium or a transformant thereof. In one or more embodiments, examples of Corynebacterium genus bacterium include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum. The microbial host in the present disclosure is not particularly limited, and from the viewpoint of improving salicylic acid productivity, it may be, for example, Corynebacterium glutamicum or a transformant thereof.

[0063] The microbial host is not particularly limited, and from the viewpoint of improving salicylic acid productivity, it may be, for example, Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or the like, or a transformant thereof.

[0064] In one or more embodiments, the microbial host may be a microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid. In one or more embodiments, examples of the microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid include Escherichia coli and Corynebacterium sp., with Corynebacterium glutamicum being preferred.

[0065] In one or more embodiments, the microorganism of the present disclosure, from the viewpoint of further improving salicylic acid productivity, comprises at least one selected from the group consisting of the pchB gene derived from Zymobacter palmae, the entC gene derived from Enterobacter cloacae, the entC gene derived from Escherichia coli, the entC gene derived from Klebsiella variicola, the entC gene derived from Escherichia fergusonii, the dhbC gene derived from Bacillus amyloliquefaciens, the entC gene derived from Citrobacter koseri, the entC gene derived from Cronobacter sakazakii, the entC gene derived from Marinomonas mediterranea, the entC gene derived from Zymobacter palmae, and the entC gene derived from Photobacterium profundum, preferably the entC gene derived from Enterobacter cloacae, the entC gene derived from Escherichia coli, the entC gene derived from Klebsiella variicola, the entC gene derived from Escherichia fergusonii, the entC gene derived from Bacillus amyloliquefaciens, the entC gene derived from Citrobacter koseri, the entC gene derived from Cronobacter sakazakii, the entC gene derived from Marinomonas mediterranea, the entC gene derived from Zymobacter palmae, and the entC gene derived from Photobacterium profundum. Examples of such transformants include those obtained by expressibly introducing into Corynebacterium glutamicum at least one gene selected from the group consisting of the dhbC gene derived from Citrobacter koseri, the entC gene derived from Cronobacter sakazakii, the entC gene derived from Marinomonas mediterranea, and the entC gene derived from Zymobacter palmae, more preferably the pchB gene derived from Zymobacter palmae and at least one gene selected from the group consisting of the entC gene derived from Enterobacter cloacae, the entC gene derived from Escherichia coli, the entC gene derived from Klebsiella variicola, and the entC gene derived from Escherichia fergusonii.In one or more embodiments, the microorganism of the present disclosure, from the viewpoint of further improving salicylic acid productivity, includes a transformant obtained by expressibly introducing into Corynebacterium glutamicum a pchB gene derived from Paracoccus denitrificans and at least one gene selected from the group consisting of an entC gene derived from Marinomonas mediterranea and an entC gene derived from Escherichia coli.In one or more embodiments, the microorganism of the present disclosure, from the viewpoint of further improving salicylic acid productivity, includes a transformant obtained by expressibly introducing into Corynebacterium glutamicum a pchB gene derived from Microbulbifer sp. and at least one gene selected from the group consisting of an entC gene derived from Marinomonas mediterranea, an entC gene derived from Enterobacter cloacae, and an entC gene derived from Escherichia coli.

[0066] In one or more embodiments, from the viewpoint of further improving salicylic acid productivity, the microorganism of the present disclosure includes a transformant obtained by expressibly introducing into Corynebacterium glutamicum the entC gene derived from Enterobacter cloacae and at least one gene selected from the group consisting of the pchB gene derived from Zymobacter palmae and the pchB gene derived from Microbulbifer sp., preferably the pchB gene derived from Zymobacter palmae. In one or more embodiments, from the viewpoint of further improving salicylic acid productivity, the microorganism of the present disclosure includes a transformant obtained by expressibly introducing into Corynebacterium glutamicum the entC gene derived from Escherichia coli and at least one gene selected from the group consisting of the pchB gene derived from Zymobacter palmae and the pchB gene derived from Microbulbifer sp., preferably the pchB gene derived from Zymobacter palmae.

[0067] In order to further improve salicylic acid productivity, one or more embodiments of the microorganism of the present disclosure include a transformant obtained by expressibly introducing the pchB gene derived from Zymobacter palmae and the irp gene derived from Streptomyces venezuelae into Corynebacterium glutamicum.

[0068] In one or more embodiments, the microorganism of the present disclosure, from the viewpoint of further improving salicylic acid productivity, includes a transformant obtained by expressibly introducing at least one of an irp gene derived from Streptomyces venezuelae and an irp gene derived from Streptomyces glaucescens and at least one of an entC gene derived from Enterobacter cloacae and an entC gene derived from Escherichia coli into Corynebacterium glutamicum.In one or more embodiments, the microorganism of the present disclosure, from the viewpoint of further improving salicylic acid productivity, includes a transformant obtained by expressibly introducing an irp gene derived from Streptomyces venezuelae and at least one of an entC gene derived from Escherichia coli and an entC gene derived from Enterobacter cloacae, preferably the entC gene derived from Escherichia coli, into Corynebacterium glutamicum. Examples of the microorganism of the present disclosure include a transformant obtained by expressibly introducing the irp gene derived from Streptomyces glaucescens and the entC gene derived from Escherichia coli into Corynebacterium glutamicum.

[0069] In one or more embodiments, from the viewpoint of further improving salicylic acid productivity, the microorganism of the present disclosure includes a transformant obtained by expressibly introducing into Corynebacterium glutamicum at least one gene selected from the group consisting of an irp gene derived from Streptomyces venezuelae, an irp gene derived from Streptomyces glaucescens, an irp gene derived from Streptomyces pharetrae, and an irp gene derived from Streptomyces rapamycinicus, preferably at least one gene selected from the group consisting of an irp gene derived from Streptomyces venezuelae, an irp gene derived from Streptomyces glaucescens, and an irp gene derived from Streptomyces pharetrae, more preferably an irp gene derived from Streptomyces venezuelae.

[0070] In order to further improve salicylic acid productivity, one or more embodiments of the microorganism of the present disclosure include a transformant obtained by expressibly introducing the pchB gene derived from Zymobacter palmae, the irp gene derived from Streptomyces venezuelae, and the entC gene derived from Escherichia coli into Corynebacterium glutamicum.

[0071] Although not an essential component, in one aspect, the microorganism of the present disclosure may optionally have reduced or absent functions of one or more or all of the following enzymes (1) to (3) in order to further improve salicylic acid productivity: (1) 3-dehydroshikimate dehydratase; (2) quinate / shikimate dehydrogenase; (3) lactate dehydrogenase.

[0072] In one or more embodiments, in order to further improve salicylic acid productivity, the microorganism of the present disclosure may have portions of the genes encoding one or more or all of the enzymes (1) to (3) above disrupted or deleted, and it is preferable that portions of all three genes, namely, the gene encoding 3-dehydroshikimate dehydratase (e.g., the qsuB gene), the gene encoding quinate / shikimate dehydrogenase (e.g., the qsuD gene), and the gene encoding lactate dehydrogenase (e.g., the ldhA gene), be disrupted or deleted.

[0073] Although not an essential component, in one aspect, the microorganism 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.

[0074] In one aspect, the microorganisms of the present disclosure may have enhanced salicylic acid production from sugars by increasing chorismate production.

[0075] Chorismate is the final product of the shikimate pathway, a metabolic pathway from 3-deoxy-D-arabinopeptulosonic acid 7-phosphate (DAHP) to chorismate. Therefore, in order to further improve salicylic acid productivity, the metabolic flux of the shikimate pathway may be increased. Figure 7 schematically illustrates an example of an overall sugar metabolic pathway, showing the metabolic pathway of salicylic acid using glucose as a raw material. The sugar metabolic pathway in the present disclosure refers to various in vivo reaction systems for decomposing 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 salicylic acid biosynthetic pathway shown in Figure 7 is merely an example of a sugar metabolic pathway in the present disclosure, and the present disclosure is not limited thereto.

[0076] 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).

[0077] 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.

[0078] In order to further improve salicylic acid productivity, the microorganism 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, or may have been modified so that the expression of at least one or all of these genes is enhanced, in order to enhance the activity of at least one or all of the enzymes encoded by each of these genes. In order to further improve salicylic acid productivity, the microorganism of the present disclosure preferably has the aroG gene, aroD gene, aroE gene, aroC gene, aroK gene, aroB gene, and aroA gene introduced into the host in an expressible manner, and the activity of the enzymes encoded by each gene enhanced.

[0079] In one aspect, the microorganism of the present disclosure may be modified such that each metabolic pathway is strengthened 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.

[0080] In another aspect, the present disclosure relates to a vector for introducing at least one of the above genes (A), (B), and (C) into a microorganism (host). The vector according to the present disclosure is not particularly limited and may be, for example, a plasmid.

[0081] Vector Construction: 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.

[0082] [Preparation of Microorganisms of the Present Disclosure] In one or more embodiments, the microorganisms of the present disclosure can be obtained using known transformation methods. Any known transformation method can be used 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)].

[0083] Disruption or Mutation of Host Chromosomal Genes: When the microbial host is a coryneform bacterium or a transformant thereof, 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 bacteria can be transformed with the DNA to induce homologous recombination on the chromosome, thereby completely deleting the target gene on the chromosome. Alternatively, a deletion-type gene can be created by deleting a partial sequence of the target gene and modifying it so that it does not produce a normally functioning enzyme protein. Bacteria can then be transformed with DNA containing the gene to induce homologous recombination between the deletion-type gene and the chromosomal gene, thereby replacing the target gene on the chromosome with the deletion-type or disrupted gene. Even if an enzyme protein encoded by a deletion-type or disrupted gene is produced, it has a different three-dimensional structure from the wild-type enzyme protein and exhibits 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, etc.). 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.

[0084] Growth of Transformants In one or more embodiments, the microorganisms and transformants of the present disclosure are 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 time 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.

[0085] 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.

[0086] 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%).

[0087] 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%).

[0088] 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%).

[0089] 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.

[0090] [Method for Producing Salicylic Acid] In another aspect, the present disclosure relates to a method for producing salicylic acid. Specifically, in one aspect, the method for producing salicylic acid of the present disclosure includes a step of culturing the microorganism of the present disclosure to produce salicylic acid.

[0091] In one or more embodiments, the production method of the present disclosure may include culturing the microorganism of the present disclosure in a culture medium. In one or more embodiments, the culture medium includes a raw material. In one or more embodiments, examples of the raw material include chorismic acid and a carbon source such as sugars. In one or more embodiments, the culture medium preferably includes sugars as a carbon source. In one or more embodiments, the culture medium may include nutrients other than sugars, such as vitamins, yeast extract, and dry yeast.

[0092] 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%).

[0093] 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.

[0094] In one or more embodiments, the reaction conditions include reducing conditions and microaerobic conditions (for example, conditions under which the dissolved oxygen concentration is controlled), etc. In one or more embodiments, which are not particularly limited, when the microbial host is Corynebacterium glutamicum, the reaction is carried out under both reducing conditions and microaerobic conditions in which Corynebacterium glutamicum does not substantially grow, thereby enabling more efficient production of salicylic acid.

[0095] In one or more embodiments, the reaction temperature (the temperature at which the transformant survives during the reaction) is about 15°C to about 50°C. Salicylic acid can be produced efficiently within this temperature range. From the viewpoint of further improving salicylic acid productivity, the reaction temperature is 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, the reaction temperature is 49°C or lower, 45°C or lower, 40°C or lower, or 35°C or lower.

[0096] In one or more embodiments, the pH of the medium is preferably about 6 to about 8. During the reaction, it is preferable to control the pH 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.).

[0097] 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.

[0098] In one or more embodiments, the production method of the present disclosure may include recovering salicylic acid from the medium (culture), and may further include purifying the recovered salicylic acid as needed. The method for recovering and purifying salicylic acid from the medium (culture) is not particularly limited. In one or more embodiments, recovery and purification of salicylic acid from the medium (culture) 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 salicylic acid accumulated in the culture may be used as is without isolation.

[0099] The contents of each document mentioned in this application are incorporated by reference as part of this disclosure.

[0100] The present disclosure further relates to one or more of the following embodiments: [1] A microorganism having a gene encoding an enzyme having an activity of producing isochorismic acid from chorismic acid, and the following gene (A) and / or the following gene (B): Gene (A): At least one gene selected from the group consisting of the following (A1) to (A9), which is a gene encoding an enzyme having an activity of producing salicylic acid from isochorismic acid. (A1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A3) 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:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A4) 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:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A5) A gene having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:13; (A6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13; (A7) A gene having a nucleotide sequence having 1 to 10 base deletions, substitutions, and / or additions per unit of 100 bases in the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13; (A8) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13;(A9) A gene derived from at least one selected from the group consisting of the genera Zymobacter, Microbulbifer, Paracoccus, and Pseudomonas. Gene (B): At least one gene selected from the group consisting of the following (B1) to (B9), which encodes an enzyme having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid: (B1) A gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113; (B2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113; (B3) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids per unit of 100 amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, or SEQ ID NO:113; (B4) A gene hybridizing under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, or SEQ ID NO:113;(B5) A gene having a nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58; (B6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58; (B7) A gene having a nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58, with 100 nucleotides per unit; (B8) A gene hybridizing under stringent conditions to a gene having a nucleotide sequence complementary to a gene having a nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58; (B9) A gene derived from at least one selected from the group consisting of the genera Streptomyces, Actinoplanes, Citrobacter, Mycolicibacterium, Gordonia, Saccharopolyspora, Aspergillus, Sorangium, and Mycobacteroides. [2] The microorganism according to [1], wherein the gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid has at least one gene (C) selected from the group consisting of the following (C1) to (C9):(C1) A gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 126; (C2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, or SEQ ID NO: 126; (C3) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids per unit of 100 amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, or SEQ ID NO:126; (C4) A gene hybridizing 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:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, or SEQ ID NO:126; (C5) A gene having a nucleotide sequence represented by SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, or SEQ ID NO: 125; (C6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence represented by SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, or SEQ ID NO: 125;(C7) A gene having a nucleotide sequence represented by SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, or SEQ ID NO:125, with 100 nucleotides per unit, having 1 to 10 nucleotide deletions, substitutions, and / or additions; (C8) A gene hybridizing under stringent conditions to a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence represented by SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, or SEQ ID NO:125; (C9) A gene derived from at least one selected from the group consisting of the genera Enterobacter, Escherichia, Klebsiella, Bacillus, Citrobacter, Cronobacter, Marinomonas, Zymobacter, Photobacterium, Xenorhabdus, and Corynebacterium. [3] The microorganism according to [1] or [2], into which at least one of (1) to (11) is introduced in an expressible manner. (1) A gene encoding isochorismate pyruvate lyase having an amino acid sequence having 50% or more identity with the pchB gene from Zymobacter palmae or the amino acid sequence of the enzyme encoded by the gene, and the entC gene from Enterobacter cloacae, the entC gene from Escherichia coli, the entC gene from Klebsiella variicola, the entC gene from Escherichia fergusonii, the dhbC gene from Bacillus amyloliquefaciens, the entC gene from Citrobacter koseri, the entC gene from Cronobacter sakazakii, the entC gene from Marinomonas mediterranea, the entC gene from Zymobacter palmae, and the entC gene from Photobacteriumprofundum, and a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by any of the above genes, preferably at least one selected from the group consisting of the entC gene from Enterobacter cloacae, the entC gene from Escherichia coli, the entC gene from Klebsiella variicola, the entC gene from Escherichia fergusonii, the dhbC gene from Bacillus amyloliquefaciens, the entC gene from Citrobacter koseri, the entC gene from Cronobacter sakazakii, the entC gene from Marinomonas mediterranea, and the entC gene from Zymobacter palmae, and a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by any of the above genes, more preferably the entC gene from Enterobacter cloacae, the entC gene from Escherichia coli, the entC gene from Klebsiella (2) at least one selected from the group consisting of the entC gene from Paracoccus variicola, the entC gene from Escherichia fergusonii, and a gene encoding an isochorismate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by any of the above genes; (3) the pchB gene from Paracoccus denitrificans, or a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by the above genes, and the entC gene from Marinomonas mediterranea, and the entC gene from Escherichia(3) at least one selected from the group consisting of the pchB gene from Microbulbifer sp. or a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by said gene, and at least one selected from the group consisting of the entC gene from Marinomonas mediterranea, the entC gene from Enterobacter cloacae, and the entC gene from Escherichia coli, and a gene encoding an isochorismate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by any of said genes; (4) the entC gene from Enterobacter cloacae or a gene encoding an isochorismate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by said gene, and the pchB gene from Zymobacter palmae, and Microbulbifer (4) At least one selected from the group consisting of the pchB gene derived from Escherichia coli or a gene encoding an isochorismate pyruvate lyase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by any of the above genes, preferably the pchB gene derived from Zymobacter palmae or a gene encoding an isochorismate pyruvate lyase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by the above gene; (5) The entC gene derived from Escherichia coli or a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by the above gene, and the pchB gene derived from Zymobacter palmae, and Microbulbiferand a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by any of the genes, preferably a pchB gene from Zymobacter palmae or a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by the gene; (6) a pchB gene from Zymobacter palmae or a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by the gene, and a gene encoding a salicylate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the irp gene from Streptomyces venezuelae or an irp gene from Streptomyces venezuelae; (7) an irp gene from Streptomyces venezuelae and a gene encoding a salicylate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by the gene; (7) at least one selected from the group consisting of the irp gene from Streptomyces venezuelae or a gene encoding a salicylate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by any of the genes, and at least one selected from the group consisting of the entC gene from Enterobacter cloacae, the entC gene from Escherichia coli, and a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by any of the genes; (8) at least one selected from the group consisting of the irp gene from Streptomyces venezuelae or a gene encoding a salicylate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by the gene, and the entC gene from Escherichia coli and Enterobacter(9) at least one selected from the group consisting of the entC gene from Escherichia coli, the entC gene from Escherichia cloacae, and a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by any of the genes, preferably the entC gene from Escherichia coli or a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by the gene; (10) a gene encoding a salicylate synthase having an amino acid sequence having 50% or more identity with the irp gene from Streptomyces glaucescens or an amino acid sequence of the enzyme encoded by the gene, and a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the entC gene from Escherichia coli or an amino acid sequence of the enzyme encoded by the gene; (11) an irp gene from Streptomyces venezuelae, an irp gene from Streptomyces glaucescens, an irp gene from Streptomyces pharetrae, and a gene encoding an isochorismate synthase having an amino acid sequence having 50% or more identity with the amino acid sequence of the enzyme encoded by the gene; At least one selected from the group consisting of an irp gene derived from Streptomyces rapamycinicus, and at least one selected from the group consisting of a gene encoding a salicylate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by any of the above genes, preferably at least one selected from the group consisting of an irp gene derived from Streptomyces venezuelae, an irp gene derived from Streptomyces glaucescens, and an irp gene derived from Streptomyces pharetrae, and at least one selected from the group consisting of a gene encoding a salicylate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the enzyme encoded by any of the above genes, more ...(11) a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the irp gene derived from Zymobacter palmae or the enzyme encoded by said gene; (12) a gene encoding an isochorismate pyruvate lyase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the pchB gene derived from Zymobacter palmae or the enzyme encoded by said gene, a gene encoding a salicylate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the irp gene derived from Streptomyces venezuelae or the enzyme encoded by said gene, and a gene encoding an isochorismate synthase having an amino acid sequence that is 50% or more identical to the amino acid sequence of the entC gene derived from Escherichia coli or the enzyme encoded by said gene. [4] The microorganism according to any of [1] to [3], which has been genetically modified so that one or more or all of the enzymes selected from the following (1) to (3) are functionally reduced or functionally deleted: (1) 3-dehydroshikimate dehydratase, (2) quinate / shikimate dehydrogenase, (3) lactate dehydrogenase. [5] The microorganism according to any one of [1] to [4], wherein all of three genes encoding 3-dehydroshikimate dehydratase (e.g., the qsuB gene), quinate / shikimate dehydrogenase (e.g., the qsuD gene), and lactate dehydrogenase (e.g., the ldhA gene) are partially disrupted or deleted. [6] The microorganism according to any one of [1] to [5], wherein at least one gene selected from the group consisting of the aroG gene, the aroD gene, the aroE gene, the aroC gene, the aroK gene, the aroB gene, and the aroA gene, preferably the aroG gene, the aroD gene, the aroE gene, the aroC gene, the aroK gene, the aroB gene, and the aroA gene, is introduced into a host in an expressible manner, and the activity of the enzyme encoded by each gene is enhanced. [7] The microorganism according to any one of [1] to [6], wherein the microorganism is a coryneform bacterium or a transformant of a coryneform bacterium.[8] The microorganism according to any one of [1] to [7], wherein the microorganism is Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum. [9] The microorganism according to any one of [1] to [8], wherein the microorganism is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), or ATCC13869 (DSM1412), or a transformant thereof.

[10] The microorganism according to [8] or [9], wherein a gene encoding an enzyme having an activity of producing isochorismic acid from chorismic acid derived from the host (Corynebacterium glutamicum) is expressed in the microorganism.

[11] The microorganism according to [8] or [9], wherein the microorganism expresses the menF gene derived from the host (Corynebacterium glutamicum).

[12] A method for producing salicylic acid or a salt thereof, comprising a step of culturing the microorganism according to any one of [1] to

[11] to produce salicylic acid or a salt thereof.

[13] The production method according to

[12] , which comprises culturing the microorganism in a medium containing sugars.

[14] The production method according to

[12] or

[13] , which comprises culturing under conditions in which the dissolved oxygen concentration is controlled.

[15] The production method according to

[14] , wherein the conditions are oxygen-deficient conditions.

[16] The production method according to any one of

[12] to

[15] , which comprises recovering salicylic acid or a salt thereof from the medium.

[0101] 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.

[0102] [Table 1] Scheme (1) of solvent-free DNA synthesis of Aspergillus clavatus JCM 1718, Bacillus amyloliquefaciens NBRC 15535、Citrobacter koseri ATCC BAA-895D-5、Cronobacter sakazakii JCM 1233、Enterobacter cloacae subsp. cloacae NBRC 13535、Escherichia coli K-12 MG1655、Escherichia fergusonii NBRC 102419、Gordonia terrae ATCC 25594、Klebsiella varicola JCM 12419、Marinomonas mediterranea NBRC 103028. Microbulbifer sp.NBRC 101765, Mycobacteroides immunogenum JCM 12691, Mycolicibacterium smegmatis ATCC 700084, Paracoccus denitrificans NBRC 102528, Photobacterium profundum JCM 10084, Pseudomonas fluorescens NBRC 14160, Pseudomonas putida ATCC 700007, Saccharopolyspora erythraea NBRC 13426, Streptomyces violaceoruber ATCC BAA-471, Streptomyces glaucescens JCM 4377, Streptomyces rapamycinicus ATCC 29253, Streptomyces venezuelae NBRC 13096, Streptomyces lividans NBRC Chromosomal DNA of Bacillus subtilis ATCC 35675, Xenorhabdus bovienii ATCC 35271, and Zymobacter palmae NBRC 102412 was prepared using a DNA genome extraction kit (Illustra bacteria genomicPrep Mini Spin Kit, Cytiva) after culturing according to the information provided by the strain provider. The salicylate synthase genes of Actinoplanes sp., Sorangium cellulosum, and Streptomyces pharetrae were synthesized.

[0103] (2) Construction of Salicylic Acid Production-Related Gene Expression Plasmids. The primer sequences used to isolate the isochorismate pyruvate lyase (pchB) gene (gene (A)), salicylate synthase (irp) gene (gene (B)), and isochorismate synthase (entC, dhbC) genes (gene (C)) are shown in Table 1. PCR was performed using a VeritiPro thermal cycler (Thermo Fisher Scientific) with PrimeSTAR HS DNA Polymerase (Takara Bio Inc.) as the reaction reagent. The PCR-amplified DNA fragment was introduced into the cloning vector pCRB209 [WO2012 / 033112] containing the PgapA promoter. The cloning vectors used and the resulting plasmid names are shown in Table 1.

[0104] Each gene sequence, including the promoter and terminator, was transferred from the above-mentioned plasmids to the cloning vector pCRB22. The names and outlines of the resulting plasmids are shown in Tables 2 to 5.

[0105] (3) Construction of a chromosomally modified strain for salicylic acid production. The markerless chromosomal 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 chromosomal gene modified strain exhibits kanamycin sensitivity and growth on sucrose-containing medium. Using the above method, the genetically modified strain Rsali1 was constructed using a chromosomal gene disruption plasmid. The host strain used was the Corynebacterium glutamicum R ldhA-disrupted strain CRZ1 [Biotechnol Bioeng. Nov;110(11):2938-2948(2013)]. In addition, the following plasmids were used for chromosomal introduction of the aroG gene (pCRB284 [WO2017 / 169399]), aroD gene (pCRB291 [WO2017 / 169399]), aroE gene (pCRB293 [WO2017 / 169399]), aroCKB gene (pCRB286 [WO2017 / 169399]), aroA gene (pCRB289 [WO2017 / 169399]), qsuB gene (pCRB229 [WO2017 / 169399]), and qsuD gene (pCRB299 [WO2017 / 169399]). An overview of the chromosomal gene recombination is shown in Table 6.

[0106] (4) Construction of a strain carrying a salicylic acid production-related gene expression plasmid The above-mentioned salicylic acid production-related gene expression plasmid was introduced into the Corynebacterium glutamicum Rsali1 strain. An overview of this strain is summarized in Table 7.

[0107] Example 2 Salicylic Acid Production Experiment (1) The gene (A)-introduced strains SALI-38 to SALI-42 constructed in Example 1 were cultured in a nutrient medium (liquid medium A + 8% glucose) containing glucose as a carbon source in a 200 ml-scale jar fermenter under controlled conditions of temperature, pH, and oxygen supply, and the ability of each strain to produce salicylic acid from glucose was examined. Each strain was precultured 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) MnSO4·2H2O, 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 fresh AK-Glc liquid medium (liquid medium prepared by adding 8% glucose to the above liquid medium A) to an initial OD610 of approximately 1, and cultured in a 200 ml jar fermenter at 33°C and pH 7-8 (automatically adjusted by adding an aqueous NaOH solution) for 72 hours. After 72 hours of jar culture, the salicylic acid content in the culture supernatant of each strain was quantified by HPLC analysis. The results confirmed that salicylic acid was produced by all strains.

[0108] [Example 3] "Salicylic acid" production experiment (2) Using the strains SALI-23 to SALI-36 into which gene (B) constructed in Example 1 had been introduced, fed-batch culture was carried out in a 200 ml-scale jar fermenter using a nutrient medium containing glucose as a carbon source under controlled conditions of temperature, pH, and oxygen supply, and the ability of each strain to produce salicylic acid from glucose was examined. The detailed experimental conditions were the same as in "salicylic acid" production experiment (1). The resulting salicylic acid production concentrations are shown in Table 8.

[0109] As in Example 1, test tube culture was performed using the strain SALI-37, into which the gene (B) derived from Streptomyces venezuelae had been introduced. As in the "salicylic acid" production experiment (1), the strain SALI-37 was pre-cultured overnight in a test tube, and then inoculated into 10 ml of fresh AK-Glc liquid medium so that the initial OD610 became 1. Shaking culture was performed in the test tube at 33°C for 72 hours. As a result, the concentration of salicylic acid produced was 8.2 mM.

[0110] [Example 4] "Salicylic acid" production experiment (3) Using the SALI-19 strain into which gene (A) + gene (B) constructed in Example 1 had been introduced, fed-batch culture was carried out in a 200 ml-scale jar fermenter using a nutrient medium containing glucose as a carbon source under controlled conditions of temperature, pH, and oxygen supply, and the ability of each strain to produce salicylic acid from glucose was examined. The detailed experimental conditions were the same as in "salicylic acid" production experiment (1). As a result, the salicylic acid production concentration was 235.4 mM after 48 hours and 343.8 mM after 72 hours.

[0111] [Example 5] "Salicylic acid" production experiment (4) Using the strains SALI-1 to SALI-17 into which gene (A) + gene (C) constructed in Example 1 had been introduced, the ability of each strain to produce salicylic acid from glucose was examined when they were cultured in a nutrient medium containing glucose as a carbon source in a 200 ml-scale jar fermenter under controlled conditions of temperature, pH, and oxygen supply. The detailed experimental conditions were the same as in "salicylic acid" production experiment (1). As a result, the salicylic acid production concentration of each strain was as shown in Table 9.

[0112] [Example 6] "Salicylic acid" production experiment (5) Using the strains SALI-20 and SALI-21 into which gene (B) + gene (C) constructed in Example 1 had been introduced, fed-batch culture was carried out in a 200 ml-scale jar fermenter using a nutrient medium containing glucose as a carbon source under controlled conditions of temperature, pH, and oxygen supply, and the ability of each strain to produce salicylic acid from glucose was examined. The detailed experimental conditions were the same as in "salicylic acid" production experiment (1). The resulting salicylic acid production concentrations are shown in Table 10.

[0113] As a reference example, the strain SALI-22, into which the gene (B) derived from Streptomyces venezuelae and the gene (C) derived from Escherichia coli had been introduced, was cultured in a test tube in AK-Glc liquid medium at 33°C for 72 hours in the same manner as in Example 1. As a result, the concentration of salicylic acid produced was 12.1 mM.

[0114] Example 7: Salicylic Acid Production Experiment (2) Using the strain SALI-18, into which gene (A), gene (B), and gene (C) constructed in Example 1 were introduced, fed-batch culture was carried out in a 200 ml-scale jar fermenter using a nutrient medium containing glucose as a carbon source under controlled conditions of temperature, pH, and oxygen supply, and the ability of each strain to produce salicylic acid from glucose was examined. Detailed experimental conditions were the same as in Salicylic Acid Production Experiment (1). As a result, the salicylic acid production concentration was 302.9 mM after 48 hours and 413.1 mM after 72 hours.

[0115] The abbreviated compound names and their CAS numbers shown in Figure 7 (an overall metabolic pathway diagram for explaining the biosynthetic pathway of salicylic acid) are listed below. It goes without saying that the "compound names" shown below are merely examples, and that synonymous alternative names may exist. It also goes without saying that the "CAS numbers" shown below are not necessarily comprehensive.

[0116] The enzymes encoded by the genes shown in Figure 7 (an overall metabolic pathway diagram for explaining the biosynthetic pathway of salicylic acid) 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.

[0117]

Claims

1. A microorganism having a gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid, and the following gene (A) and / or the following gene (B): Gene (A): At least one gene selected from the group consisting of the following (A1) to (A9), which is a gene encoding an enzyme having the activity of producing salicylic acid from isochorismic acid. (A1) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence shown in SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A3) 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:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A4) 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:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, or SEQ ID NO:98; (A5) A gene having the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, or SEQ ID NO:13; (A6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13; (A7) A gene having a nucleotide sequence having 1 to 10 base deletions, substitutions, and / or additions per unit of 100 bases in the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13; (A8) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13;(A9) A gene derived from at least one selected from the group consisting of the genera Zymobacter, Microbulbifer, Paracoccus, and Pseudomonas. Gene (B): At least one gene selected from the group consisting of the following (B1) to (B9), which encodes an enzyme having the activity of producing isochorismic acid from chorismic acid and the activity of producing salicylic acid from isochorismic acid: (B1) A gene encoding a polypeptide having an amino acid sequence represented by SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113; (B2) A gene encoding a polypeptide having an amino acid sequence having 50% or more identity with the amino acid sequence represented by SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, or SEQ ID NO: 113; (B3) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids per unit of 100 amino acids are deleted, substituted, and / or added in the amino acid sequence represented by SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, or SEQ ID NO:113; (B4) A gene hybridizing under stringent conditions to a gene having a nucleotide sequence complementary to a gene encoding a polypeptide having the amino acid sequence represented by SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, or SEQ ID NO:113;(B5) A gene having a nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58; (B6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58; (B7) A gene having a nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58, with 100 nucleotides per unit; (B8) A gene hybridizing under stringent conditions to a gene having a nucleotide sequence complementary to a gene having a nucleotide sequence represented by SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NO: 43, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 52, SEQ ID NO: 55, or SEQ ID NO: 58; (B9) A gene derived from at least one selected from the group consisting of the genera Streptomyces, Actinoplanes, Citrobacter, Mycolicibacterium, Gordonia, Saccharopolyspora, Aspergillus, Sorangium, and Mycobacteroides; 2. The microorganism according to claim 1, wherein the gene encoding an enzyme having the activity of producing isochorismic acid from chorismic acid has at least one gene (C) selected from the group consisting of (C1) to (C9) below: (C1) a gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, or SEQ ID NO:126; (C2) a gene encoding a polypeptide having an amino acid sequence having 50% or more identity to the amino acid sequence set forth in SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, or SEQ ID NO:126; (C3) A gene encoding a polypeptide having an amino acid sequence in which 1 to 10 amino acids per unit of 100 amino acids are deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, or SEQ ID NO:126; (C4) A gene hybridizing 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:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, or SEQ ID NO:126; (C5) A gene having a nucleotide sequence represented by SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, or SEQ ID NO: 125; (C6) A gene having a nucleotide sequence having 50% or more identity with the nucleotide sequence represented by SEQ ID NO: 61, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 79, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 91, or SEQ ID NO: 125;(C7) A gene having a nucleotide sequence represented by SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, or SEQ ID NO:125, with 100 nucleotides per unit, having 1 to 10 nucleotide deletions, substitutions, and / or additions; (C8) A gene hybridizing under stringent conditions to a gene having a nucleotide sequence complementary to a gene having the nucleotide sequence represented by SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, or SEQ ID NO:125; (C9) A gene derived from at least one selected from the group consisting of the genera Enterobacter, Escherichia, Klebsiella, Bacillus, Citrobacter, Cronobacter, Marinomonas, Zymobacter, Photobacterium, Xenorhabdus, and Corynebacterium; 3. The microorganism according to claim 1, wherein the microorganism is a coryneform bacterium or a transformant of a coryneform bacterium.

4. The microorganism according to claim 1, wherein the microorganism is Corynebacterium glutamicum or a transformant of Corynebacterium glutamicum.

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

6. A method for producing salicylic acid or a salt thereof, comprising the step of culturing the microorganism according to any one of claims 1 to 5 to produce salicylic acid or a salt thereof.

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