Transformant, and method for producing 6-hydroxyhexanoic acid or adipic acid using same

Abstract

Provided are: a novel transformant with which the productivity of 6-hydroxyhexanoic acid (6HH) and / or adipic acid can be improved; and a method for producing 6HH or adipic acid using said transformant.　In one aspect, the present disclosure pertains to a transformant for producing 6HH, the transformant being obtained by expressibly introducing, into a microbial host having the ability to produce succinyl-CoA and malonyl-CoA, a gene (A) encoding an enzyme having the activity of producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA and a gene (B) encoding an enzyme having the activity of producing adipic acid semialdehyde from adipyl-CoA, wherein the microbial host is Corynebacterium glutamicum. In another aspect, the present disclosure pertains to a method for producing 6HH, the method comprising a step for culturing this transformant to produce 6HH. In another aspect, the present disclosure pertains to a method for producing adipic acid, the method comprising a step for culturing this transformant to produce adipic acid.

Description

Transformed organism and method for producing 6-hydroxyhexanoic acid or adipic acid using the same

[0001] This disclosure relates to technologies for the production of 6-hydroxyhexanoic acid and adipic acid. In one aspect, this disclosure relates to a genetically modified transformant and a technology for producing 6-hydroxyhexanoic acid or adipic acid using the transformant.

[0002] 6-hydroxyhexanoic acid (CAS number: 1191-25-9; 6-hydroxycaproic acid, ε-hydroxycaproic acid, etc. are synonymous; hereafter, it may be referred to as "6HH" or "6-HH") can be converted to ε-caprolactone by dehydration and cyclization. ε-caprolactone is used as a monomer for polycaprolactone, a biodegradable plastic. Furthermore, ε-caprolactone can be converted to ε-caprolactam by reaction with ammonia, which is used as a monomer for nylon-6. Methods for producing 6-HH by microorganisms include those using cyclohexane, cyclohexanol, or 1,6-hexanediol as raw materials. For example, Non-Patent Literature 1 shows the production of 6HH using the oxidative activity of 1,6-hexanediol possessed by Gluconobacter oxydans. Non-Patent Literature 2 also shows the production of 6-HH by the oxidation reaction of cyclohexane using a recombinant strain of Pseudomonas taiwanensis. However, all of these are manufacturing methods that use chemically synthesized substances produced from petroleum as raw materials. Patent Document 1 discloses a recombinant microorganism having a pathway for producing adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol, or 6-amino-1-hexanol, and discloses a method for simultaneously producing these multiple compounds from biomass-derived raw materials such as glucose by a fermentation method using the microorganism, and that 6-HH is included in the pathway. Furthermore, adipic acid can be used as a raw material for 6,6-nylon, and as a raw material for 1,6-hexanediol, which is used as a raw material for polyester and polyurethane.

[0003] International Publication No. 2022 / 210708

[0004] Sang-Hyun Pyo, Ji Hoon Park, Vanessa Srebny and Rajni Hatti-Kaul. 2020. A sustainable synthetic route for biobased 6-hydroxyhexanoic acid, adipic acid and ε-caprolactone by integrating bio- and chemical catalysis. Green Chem. 22:4450-4455.Lisa Bretschneider, Ingebong Heuschkel, Martin Wegner, Martin Lindmeyer, Katja Buhler, Rohan Karande and Bruno Buhler. 2021. Front. Catal. 1:683248.

[0005] There is a demand for the creation of microorganisms (transformants) that produce 6HH and adipic acid from biomass-derived raw materials such as glucose. The present disclosure provides a novel transformant capable of improving the productivity of 6HH and / or adipic acid, and a method for producing 6HH or adipic acid using the transformant.

[0006] In one aspect, the present disclosure relates to a transformant for producing 6HH, which is obtained by expressibly introducing, into a microbial host capable of producing succinyl-CoA and malonyl-CoA, the following: a gene (A) encoding an enzyme having an activity of generating 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme having an activity of generating adipic acid semialdehyde from adipyl-CoA.

[0007] In other embodiments, this disclosure relates to a transformant for producing 6HH obtained by introducing into a microbial host capable of producing succinyl-CoA and malonyl-CoA a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme active in producing adipic acid semialdehyde from adipyl-CoA, wherein the microbial host is Corynebacterium glutamicum.

[0008] In other embodiments, this disclosure relates to a method for producing 6HH, which includes the step of culturing the transformants of this disclosure to produce 6HH.

[0009] In further embodiments, this disclosure relates to a transformant for producing adipic acid, obtained by introducing into a microbial host capable of producing succinyl-CoA and malonyl-CoA a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme active in producing adipic acid semialdehyde from adipyl-CoA.

[0010] In further embodiments, this disclosure relates to transformants for producing adipic acid, obtained by introducing a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA into a microbial host capable of producing succinyl-CoA and malonyl-CoA.

[0011] In further embodiments, this disclosure relates to a method for producing adipic acid, which includes the step of culturing the transformants of this disclosure to produce adipic acid.

[0012] According to this disclosure, in one embodiment, a novel transformant capable of improving the productivity of 6HH and / or adipic acid can be provided, preferably a transformant capable of efficiently producing 6HH and / or adipic acid using sugars or the like as raw materials. According to this disclosure, in one embodiment, a method for producing 6HH or adipic acid with improved productivity can be provided.

[0013] Figure 1 schematically shows 6HH production in the transformant of the present disclosure. Figure 2 is an overall metabolic pathway diagram illustrating the biosynthetic pathway of 6HH in one form of the transformant of the present disclosure. Figure 3 is a graph showing an example of the results of an adipic acid production experiment by test tube culture in the examples. Figure 4 is a graph showing an example of the results of a 6HH production experiment by test tube culture in the examples. Figure 5 is a graph showing an example of the results of gene (A) (PamA homolog) search by test tube culture in the examples.

[0014] This disclosure is based on the inventors' discovery that a transformant obtained by introducing a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme active in producing adipic acid semialdehyde from adipyl-CoA, into a microbial host capable of producing succinyl-CoA and malonyl-CoA, can, for example, biologically produce 6HH and / or adipic acid using sugars such as glucose (biomass-derived raw materials) as raw materials, and furthermore, can improve the productivity of 6HH and / or adipic acid. Furthermore, this disclosure is based on the finding that the productivity of 6HH and / or adipic acid can be further improved by using Corynebacterium glutamicum, which is capable of producing succinyl-CoA and malonyl-CoA, as the host. More specifically, conventional methods (for example, Patent Document 1) use an enzyme that produces 3-oxoadipyl-CoA from succinyl-CoA and acetyl-CoA. This enzyme is energetically dominant in the reaction that decomposes 3-oxoadipyl-CoA into succinyl-CoA and acetyl-CoA. On the other hand, the enzyme encoded by gene (A) used in this disclosure (an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA) substantially irreversibly produces 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, accompanied by the decarboxylation of malonyl-CoA. In other words, this disclosure is based on the finding that the enzyme predominantly produces 3-oxoadipyl-CoA in the forward reaction, and the reverse reaction that decomposes 3-oxoadipyl-CoA hardly occurs, thus improving the production efficiency of 3-oxoadipyl-CoA compared to conventional methods, and thereby improving the productivity of 6HH and adipic acid, which will be described later.

[0015] In this disclosure, “transformed organism obtained by introducing a gene” may include a transformed organism obtained by introducing the gene, and a transformed organism that may be obtained by introducing the gene.

[0016] Methods for introducing genes include, in one or more embodiments, methods using general genetic engineering techniques (for example, the method described in Michael R. Green & Joseph Sambrook, Molecular Cloning, Cold Spring Harbor Laboratory Press). Gene introduction methods include, in one or more embodiments, gene introduction using plasmid vectors or integration into the chromosomes of a microbial host.

[0017] In this disclosure, "introducing a gene in an expressible manner" means, in one or more embodiments, introducing the introduced gene in a manner that allows it to be expressed in the transformant. Methods for introducing a gene in an expressible manner include, in one or more embodiments, introducing the gene together with an expression regulatory sequence such as a promoter capable of inducing increased gene expression. In this disclosure, the gene introduced together with a promoter capable of inducing increased expression may, in one or more embodiments, be a single gene or an operon capable of expressing multiple genes. In one or more embodiments, a known promoter can be used as the promoter capable of inducing increased expression.

[0018] In this disclosure, "90% or more identity" with respect to an amino acid sequence or base sequence means at least 90% identity, and in one or more embodiments, means 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 99.9% or more identity.

[0019] In this disclosure, "identity of amino acid sequence or nucleotide sequence" can be performed using readily available sequence comparison computer programs. Examples of such computer programs in one or more embodiments include the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387-395), BLAST (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), and FASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448).

[0020] In this disclosure, “stringent conditions” means conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. In one or more embodiments, stringent conditions include conditions under which highly identical base sequences hybridize, but less identical base sequences do not hybridize. In one or more embodiments, high identity between base sequences includes cases where the identity is 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 also be the conditions described in Molecular Cloning, A Laboratory Manual, Second Edition, 1989, Vol2, p11.45. Specifically, hybridization may occur at a temperature 5 to 10°C lower than the melting temperature (Tm) of the complete hybrid.

[0021] In this disclosure, "a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: XX" means a gene encoding a polypeptide containing the amino acid sequence shown in SEQ ID NO: XX. In this disclosure, "a gene having the nucleotide sequence shown in SEQ ID NO: XX" means a gene containing the nucleotide sequence shown in SEQ ID NO: XX.

[0022] In this disclosure, "microbial host having the ability to produce succinyl-CoA and malonyl-CoA" refers to a microbial host having the ability to biosynthesize succinyl-CoA and malonyl-CoA, and in one or more embodiments, a microbial host capable of expressing an enzyme having the activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity. The enzyme having acetyl-CoA carboxylase activity can produce malonyl-CoA from acetyl-CoA. In one or more embodiments, the microbial host according to this disclosure may be not only a bacterium having genes encoding an enzyme having the activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity in the wild type, but also a bacterium that has been artificially transformed to be able to express an enzyme having the activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity. In one or more embodiments, a microbial host capable of producing succinyl-CoA and malonyl-CoA may be a bacterium that has been transformed in a state in which it can express the enzyme that has the activity to produce succinyl-CoA from 2-oxoglutaric acid and the enzyme that has acetyl-CoA carboxylase activity, in which the wild type has genes encoding an enzyme that has the activity to produce succinyl-CoA from 2-oxoglutaric acid and the enzyme that has acetyl-CoA carboxylase activity.As a microbial host capable of producing succinyl-CoA and malonyl-CoA, in one or more embodiments, from the viewpoint of further improving the productivity of 6HH, it is preferable to have a bacterium into which genes encoding an enzyme having the activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity have been introduced so that they can be expressed, a bacterium in which the enzyme is overexpressed, and a bacterium in which the expression level of the enzyme is improved. Enzymes having the activity to produce succinyl-CoA from 2-oxoglutaric acid include, in one or more embodiments, enzyme complexes consisting of SucA, SucB, and Lpd (EC: 1.2.4.2, EC: 2.3.1.61, EC: 1.8.1.4), enzyme complexes consisting of Kgd, AceF, and Lpd (EC: 1.2.4.2, EC: 2.3.1.12, EC: 1.8.1.4), and KorAB (EC: 1.2.7.3) complexes. Enzymes having acetyl-CoA carboxylase activity include, in one or more embodiments, enzymes having the activity to catalyze the reaction for the synthesis of malonyl-CoA from acetyl-CoA. Enzymes having activity to catalyze the reaction for the synthesis of malonyl-CoA from acetyl-CoA include, in one or more embodiments, an enzyme complex consisting of AccBC and AccD1 derived from Corynebacterium, and an enzyme complex consisting of AccA, AccB, AccC, and AccD derived from Escherichia coli. In one or more embodiments, the microbial host according to this disclosure is preferably modified so that a gene encoding an enzyme having acetyl-CoA carboxylase activity can be expressed, and the gene encoding an enzyme having acetyl-CoA carboxylase activity is incorporated into the chromosome in an expressible manner.Examples of genes encoding enzymes having acetyl-CoA carboxylase activity include, in one or more embodiments, the accBC and accD1 genes derived from Corynebacterium, and the accA, accB, accC, and accD genes derived from Escherichia coli. The accBC and accD1 genes derived from Corynebacterium glutamicum R strain, and the accA, accB, accC, and accD genes derived from Escherichia coli can be used. In one or more embodiments, the microbial host according to this disclosure has been modified so that the accBC and accD1 genes derived from Corynebacterium glutamicum R strain, or the accA, accB, accC, and accD genes derived from Escherichia coli, can be incorporated into the chromosome in an expressible manner. The amino acid sequence of AccBC derived from Corynebacterium glutamicum R strain is described in SEQ ID NO: 97, and the nucleotide sequence of the accBC gene encoding AccBC is described in SEQ ID NO: 98. The amino acid sequence of AccuD1 from Corynebacterium glutamicum R strain is described in Sequence ID No. 99, and the nucleotide sequence of the accD1 gene encoding AccuD1 is described in Sequence ID No. 100. The amino acid sequence of AccuA from E. coli is described in Sequence ID No. 101, and the nucleotide sequence of the accA gene encoding AccuA is described in Sequence ID No. 102. The amino acid sequence of AccuB from E. coli is described in Sequence ID No. 103, and the nucleotide sequence of the accB gene encoding AccuB is described in Sequence ID No. 104. The amino acid sequence of AccuC from E. coli is described in Sequence ID No. 105, and the nucleotide sequence of the accC gene encoding AccuC is described in Sequence ID No. 106. The amino acid sequence of AccuD from E. coli is described in Sequence ID No. 107, and the nucleotide sequence of the accD gene encoding AccuD is described in Sequence ID No. 108.

[0023] The microbial hosts relating to this disclosure include, in one or more embodiments, microbial hosts in which the expression levels of an enzyme having 2-oxoglutaric acid dehydrogenase activity (activity to produce succinyl-CoA from 2-oxoglutaric acid) and an enzyme having acetyl-CoA carboxylase activity are increased, in order to further improve the productivity of 6HH; a bacterium having an enzyme having activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity, in which the expression level of the enzyme having acetyl-CoA carboxylase activity is increased; and a microbial host that overexpresses, can improve, or can induce overexpression of an enzyme having activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity. The expression levels of the enzymes can be adjusted or improved as appropriate by those skilled in the art, for example, by using a suitable promoter. In one or more embodiments, the microbial host according to this disclosure may further include a microorganism capable of producing succinyl-CoA and malonyl-CoA, into which at least one of the genes encoding an enzyme having the activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity, a gene encoding an enzyme having acetyl-CoA carboxylase activity, or a gene encoding an enzyme having the activity to produce succinyl-CoA from 2-oxoglutaric acid and an enzyme having acetyl-CoA carboxylase activity has been introduced. From the viewpoint of further improving 6HH productivity, it is preferable that a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced.

[0024] The microbial hosts relating to this disclosure are not particularly limited and may include, for example, coryneform bacteria, Escherichia coli (Escherichia species, especially Escherichia corydoras), aromatic compound-utilizing bacteria, adipic acid-utilizing bacteria, and yeasts. 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. Examples of coryneform bacteria in one or more embodiments include Corynebacterium species, Brevibacterium species, Arthrobacter species, Mycobacterium species, and Micrococcus species. Examples of aromatic compound-utilizing bacteria in one or more embodiments include Pseudomonas species and Rosebacter species. Examples of adipic acid-utilizing bacteria include those of the genus Thermobifida in one or more embodiments.

[0025] The microbial host relating to this disclosure is not particularly limited and may be, for example, a bacterium of the genus Corynebacterium. Examples of Corynebacterium bacteria in one or more embodiments include Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerance, and Corynebacterium alkanolyticum. The microbial host relating to this disclosure is not particularly limited and may be, for example, Corynebacterium glutamicum, from the viewpoint of further improving the productivity of 6HH. In terms of further improving the productivity of 6HH, the microbial host according to this disclosure is preferably Corynebacterium glutamicum in which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced in one or more embodiments, and more preferably Corynebacterium glutamicum in which the accBC and accD1 genes have been modified to be expressibly incorporated into the chromosome.

[0026] The microbial host according to this disclosure is not particularly limited and may be, for example, Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), or ATCC13869 (DSM1412), or transformants thereof. In one or more embodiments, Corynebacterium glutamicum R into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced is preferred as the microbial host according to this disclosure from the viewpoint of further improving the productivity of 6HH, and Corynebacterium glutamicum R into which the accBC and accD1 genes have been modified to be expressibly incorporated into the chromosome is more preferred.

[0027] [Transformants of the Disclosure] In one embodiment, the Disclosure relates to transformants for producing 6HH, wherein a microbial host capable of producing succinyl-CoA and malonyl-CoA is expressedly introduced into a microbial host capable of producing succinyl-CoA and malonyl-CoA, comprising a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme active in producing adipic acid semialdehyde from adipyl-CoA. In another embodiment, the Disclosure relates to transformants in which the microbial host is Corynebacterium glutamicum capable of producing succinyl-CoA and malonyl-CoA. Transformants of the Disclosure can produce 6HH. Therefore, in one or more embodiments, transformants of the Disclosure can also be called transformants capable of producing 6HH. In one or more embodiments of the present disclosure, the transformants can produce 6HH from succinyl-CoA and malonyl-CoA by genes (A) and (B), as shown in Figure 1, and preferably the transformants can produce succinyl-CoA and malonyl-CoA from sugars (e.g., glucose, etc.) in vivo, and produce 6HH from the succinyl-CoA and malonyl-CoA.

[0028] [Gene (A)] Gene (A) in this disclosure is a gene encoding an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. In one or more embodiments, gene (A) is a gene encoding a homolog of 3-oxoacylACP synthase II having 3-oxoadipyl-CoA synthase activity. The homolog of 3-oxoadipyl-CoA synthase II having 3-oxoadipyl-CoA synthase activity (EC 2.3.1.179) is an enzyme that catalyzes the reaction to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. The enzyme encoded by gene (A) has a higher activity in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA than in producing succinyl-CoA and malonyl-CoA from 3-oxoadipyl-CoA.

[0029] The origin of gene (A) is not particularly limited, and in one or more embodiments, examples include the genus Streptomyces. The names (designations) of the gene-derived organisms shown in this disclosure are examples, and synonymous alternative names may exist.

[0030] Examples of Streptomyces species in one or more embodiments include Streptomyces alboniger, Streptomyces violaceoruber, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces microflavus, Streptomyces mediolani, and Streptomyces flavofungini. An enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces alboniger is, in one or more embodiments, a polypeptide (PamA) consisting of the amino acid sequence shown in SEQ ID NO: 1. An example of a gene encoding the polypeptide (pamA gene) consisting of the amino acid sequence shown in SEQ ID NO: 1 is the nucleotide sequence shown in SEQ ID NO: 8. The nucleotide sequence of SEQ ID NO: 8 is a codon-optimized nucleotide sequence for Corynebacterium glutamicum. In one or more embodiments, an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces violaceoruber is a polypeptide (NonU) consisting of the amino acid sequence shown in SEQ ID NO: 2. An example of the gene encoding the polypeptide (nonU gene) consisting of the amino acid sequence shown in SEQ ID NO: 2 is the nucleotide sequence shown in SEQ ID NO: 9. The nucleotide sequence of SEQ ID NO: 9 is a codon-optimized nucleotide sequence for Corynebacterium glutamicum. In one or more embodiments, an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces lincolnensis is a polypeptide (PamA) consisting of the amino acid sequence shown in SEQ ID NO: 3. An example of the gene encoding the polypeptide (pamA gene) consisting of the amino acid sequence shown in SEQ ID NO: 3 is the nucleotide sequence shown in SEQ ID NO: 10.In one or more embodiments, an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces phaeochromogenes is a polypeptide (PamA) consisting of the amino acid sequence shown in SEQ ID NO: 4. An example of the gene encoding the polypeptide (pamA gene) consisting of the amino acid sequence shown in SEQ ID NO: 4 is the nucleotide sequence shown in SEQ ID NO: 11. In one or more embodiments, an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces microflavus is a polypeptide (PamA) consisting of the amino acid sequence shown in SEQ ID NO: 5. An example of the gene encoding the polypeptide (pamA gene) consisting of the amino acid sequence shown in SEQ ID NO: 5 is the nucleotide sequence shown in SEQ ID NO: 12. In one or more embodiments, an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces mediolani is a polypeptide (PamA) consisting of the amino acid sequence shown in SEQ ID NO: 6. An example of the gene encoding the polypeptide (pamA gene) consisting of the amino acid sequence shown in SEQ ID NO: 6 is the nucleotide sequence shown in SEQ ID NO: 13. In one or more embodiments, an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA derived from Streptomyces flavofungini is a polypeptide (PamA) consisting of the amino acid sequence shown in SEQ ID NO: 7. An example of the gene encoding the polypeptide (pamA gene) consisting of the amino acid sequence shown in SEQ ID NO: 7 is the nucleotide sequence shown in SEQ ID NO: 14.

[0031] DNA encoding polypeptides or homologs thereof, consisting of the amino acid sequences shown in Sequence IDs 1, 2, 3, 4, 5, 6, and 7, is not particularly limited in its base sequence, as long as the encoded amino acid sequences are identical.

[0032] Gene (A) is not particularly limited and may be any of the following genes: (a), (b), (c), and (d). (a) A gene encoding a polypeptide having or comprising the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7; (b) A gene encoding a polypeptide having or comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (c) A gene encoding a polypeptide having or comprising an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (d) 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: 1, 2, 3, 4, 5, 6, or 7, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.

[0033] Gene (A) is not particularly limited and may be any of the following genes: (e), (f), (g), and (h): (e) A gene having the nucleotide sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14; (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14, and encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, or a gene consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14, and encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (g) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, as shown in sequence number 8, 9, 10, 11, 12, 13, or 14, and encoding a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; or a gene consisting of a nucleotide sequence in which 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, as shown in sequence number 8, 9, 10, 11, 12, 13, or 14, and encoding a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA;(h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, or 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.

[0034] As gene (A), in one or more embodiments, from the viewpoint of further improving the productivity of 6HH, a gene encoding an enzyme having 3-oxoadipyr-CoA synthase activity derived from any of the species Streptomyces alboniger, Streptomyces violaceoruber, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces microflavus, Streptomyces mediolani, and Streptomyces flavofungini is preferred, a gene encoding an enzyme having 3-oxoadipyr-CoA synthase activity derived from any of the species Streptomyces alboniger, Streptomyces lincolnensis, and Streptomyces flavofungini is more preferred, and a gene encoding an enzyme having 3-oxoadipyr-CoA synthase activity derived from any of the species Streptomyces alboniger and Streptomyces lincolnensis is even more preferred.

[0035] In one or more embodiments, the polypeptide encoded by gene (A) is preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, more preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3, or 7, and even more preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 3.

[0036] [Gene (B)] Gene (B) in this disclosure is a gene encoding an enzyme having the activity to produce adipic acid semialdehyde from adipyl-CoA. In one or more embodiments, gene (B) includes genes encoding a homolog of acetaldehyde dehydrogenase having adipic acid semialdehyde dehydrogenase activity and a homolog of acyl-CoA reductase. The homolog of acetaldehyde dehydrogenase having adipic acid semialdehyde dehydrogenase activity (EC: 1.2.1.10) and the homolog of acyl-CoA reductase (EC: 1.2.1.50) are enzymes that catalyze the reaction to produce adipic acid semialdehyde from adipyl-CoA.

[0037] The origin of gene (B) is not particularly limited, and in one or more embodiments, examples include the genera Lactobacillus, Citrobacter, Marinobacter, and Clostridium. The names of the genera and species of the organisms from which the genes are derived shown in this disclosure are examples, and synonymous names may exist.

[0038] Examples of the genus Lactobacillus include Lactobacillus brevis in one or more embodiments. Examples of the genus Citrobacter include Citrobacter koseri in one or more embodiments. Examples of the genus Marinobacter include Marinobacter hydrocarbonoclasticus in one or more embodiments. Examples of the genus Clostridium include Clostridium kluyveri in one or more embodiments. Examples of the genus Lactobacillus brevis include a polypeptide (EutE) consisting of the amino acid sequence shown in SEQ ID NO: 15 in one or more embodiments. Examples of the genus Clostridium include Clostridium kluyveri in one or more embodiments. Examples of the homolog of acetaldehyde dehydrogenase derived from Lactobacillus brevis include a polypeptide (EutE) consisting of the amino acid sequence shown in SEQ ID NO: 15 in one or more embodiments. Examples of the gene encoding the polypeptide (eutE gene) consisting of the amino acid sequence shown in SEQ ID NO: 15 include the nucleotide sequence shown in SEQ ID NO: 18 in one or more embodiments. Examples of the homolog of acetaldehyde dehydrogenase derived from Citrobacter koseri include a polypeptide (PduP) consisting of the amino acid sequence shown in SEQ ID NO: 16 in one or more embodiments. An example of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16 (pduP gene) is the nucleotide sequence shown in SEQ ID NO: 19. In one or more embodiments, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 17 (Acr) is used as a homolog of acyl-CoA reductase derived from Marinobacter hydrocarbonoclasticus. An example of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 17 (acr gene) is the nucleotide sequence shown in SEQ ID NO: 20. In one or more embodiments, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 115 (SucD) is used as a homolog of acyl-CoA reductase derived from Clostridium kluyveri. An example of a gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 115 (sucD gene) is the nucleotide sequence shown in SEQ ID NO: 116.In one or more embodiments, a homolog of the acyl-CoA reductase derived from Clostridium kluyveri is sucD derived from Clostridium kluyveri, as described in Patent Document 1.

[0039] The DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15, 16, or 17, or its homolog thereof, is not particularly limited in its base sequence, as long as the encoded amino acid sequence is the same.

[0040] Gene (B) is not particularly limited and may be any of the following genes: (a), (b), (c), and (d): (a) a gene encoding a polypeptide having or consisting of the amino acid sequence shown in SEQ ID NO: 15, 16, or 17; (b) a gene encoding a polypeptide having or consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, 16, or 17, and encoding a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; (c) a gene encoding a polypeptide having or consisting of an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, and encoding a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; (d) 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: 15, 16, or 17, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA.

[0041] Gene (B) is not particularly limited and may be any of the following genes: (e), (f), (g), and (h): (e) A gene having the nucleotide sequence shown in SEQ ID NO: 18, 19, or 20, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 18, 19, or 20; (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 18, 19, or 20, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 18, 19, or 20, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA; (g) A gene having a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 18, 19, or 20, in which 100 nucleotides constitute one unit, and each unit contains deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in Sequence ID No. 18, 19, or 20, in which 100 nucleotides constitute one unit, and each unit contains deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; (h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 18, 19, or 20, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 18, 19, or 20, and which hybridizes under stringent conditions, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA.

[0042] In one or more embodiments, the gene (B) is preferably a gene encoding an enzyme having adipic acid semialdehyde dehydrogenase activity derived from any of the species Lactobacillus brevis, Citrobacter koseri, and Marinobacter hydrocarbonoclasticus, from the viewpoint of further improving the productivity of 6HH, and more preferably a gene encoding an enzyme having adipic acid semialdehyde dehydrogenase activity derived from any of the species Lactobacillus brevis and Citrobacter koseri.

[0043] In one or more embodiments, the polypeptide encoded by gene (B) is preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, 16, or 17, and more preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 15 or 16, from the viewpoint of further improving the productivity of 6HH.

[0044] The transformants of this disclosure, in terms of further improving the productivity of 6HH, in one or more embodiments include at least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, the pamA gene from Streptomyces phaeochromogenes, the pamA gene from Streptomyces microflavus, the pamA gene from Streptomyces mediolani, and the pamA gene from Streptomyces flavofungini, along with the eutE gene from Lactobacillus brevis, the pduP gene from Citrobacter koseri, and Marinobacter A transformant obtained by expressively introducing at least one selected from the group consisting of acr genes derived from hydrocarbonoclasticus into Corynebacterium glutamicum is preferable, a transformant obtained by expressively introducing into Corynebacterium glutamicum in which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum in which accBC and accD1 genes have been modified to be expressively integrated into the chromosome, and even more preferably into Corynebacterium glutamicum in which accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressively integrated into the chromosome.In terms of further improving the productivity of 6HH, the transformants of the present disclosure, in one or more embodiments, include at least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, and the pamA gene from Streptomyces phaeochromogenes, as well as the eutE gene from Lactobacillus brevis and Citrobacter A transformant obtained by expressively introducing at least one of the pduP genes derived from koseri into Corynebacterium glutamicum is preferably a transformant obtained by expressively introducing a gene encoding an enzyme having acetyl-CoA carboxylase activity into Corynebacterium glutamicum, more preferably a transformant obtained by modifying Corynebacterium glutamicum in which the accBC and accD1 genes are expressively integrated into the chromosome, and even more preferably a transformant obtained by modifying Corynebacterium glutamicum in which the accBC and accD1 genes derived from Corynebacterium glutamicum are expressively integrated into the chromosome.In one or more embodiments, from the perspective of further improving 6HH productivity, the transformant of the present disclosure is a transformant obtained by expressibly introducing at least one selected from the group consisting of the pamA gene derived from Streptomyces alboniger, the nonU gene derived from Streptomyces violaceoruber, and the pamA gene derived from Streptomyces lincolnensis, and at least one of the eutE gene derived from Lactobacillus brevis and the pduP gene derived from Citrobacter koseri into Corynebacterium glutamicum. Preferable examples include transformants obtained by said expressible introduction into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced; more preferable examples include transformants obtained by said expressible introduction into Corynebacterium glutamicum modified to chromosomally integrate accBC and accD1 genes in an expressible manner; and still more preferable examples include transformants obtained by said expressible introduction into Corynebacterium glutamicum modified to chromosomally integrate accBC and accD1 genes derived from Corynebacterium glutamicum in an expressible manner. In one or more embodiments, from the perspective of further improving 6HH productivity, the transformant of the present disclosure is a transformant obtained by expressibly introducing the pamA gene derived from Streptomyces alboniger or the nonU gene derived from Streptomyces violaceoruber, and the eutE gene derived from Lactobacillus brevis or the pduP gene derived from Citrobacter koseri into Corynebacterium glutamicum. Preferable examples include transformants obtained by said expressible introduction into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced; more preferable examples include transformants obtained by said expressible introduction into Corynebacterium glutamicum modified to chromosomally integrate accBC and accD1 genes in an expressible manner; and still more preferable examples include transformants obtained by said expressible introduction into Corynebacterium glutamicum modified to chromosomally integrate accBC and accD1 genes derived from Corynebacterium glutamicum in an expressible manner.

[0045] The transformants of this disclosure are, in terms of further improving the productivity of 6HH, transformants obtained by expressively introducing into Corynebacterium glutamicum, in one or more embodiments, a polypeptide having an amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7 or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence represented by SEQ ID NO: 15, 16, or 17 or an amino acid sequence having 90% or more identity with said sequence, preferably into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum into which accBC and accD1 genes have been modified to be expressably integrated into the chromosome, and even more preferably into Corynebacterium glutamicum into which accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressably integrated into the chromosome. The transformants of this disclosure are, in terms of further improving the productivity of 6HH, transformants obtained by expressively introducing into Corynebacterium glutamicum, in one or more embodiments, a polypeptide having an amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, or 7 or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence represented by SEQ ID NO: 15, 16, or 17 or an amino acid sequence having 90% or more identity with said sequence, preferably into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum into which accBC and accD1 genes have been modified to be expressably integrated into the chromosome, and even more preferably into Corynebacterium glutamicum into which accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressably integrated into the chromosome.The transformants of this disclosure are, in terms of further improving the productivity of 6HH, transformants obtained by expressively introducing into Corynebacterium glutamicum, in one or more embodiments, a polypeptide having an amino acid sequence shown in SEQ ID NO: 1, 3, or 7 or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence shown in SEQ ID NO: 15 or 16 or an amino acid sequence having 90% or more identity with said sequence, preferably into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum into which accBC and accD1 genes have been modified to be expressably integrated into the chromosome, and even more preferably into Corynebacterium glutamicum into which accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressably integrated into the chromosome. The transformants of this disclosure are, in terms of further improving the productivity of 6HH, transformants obtained by expressively introducing into Corynebacterium glutamicum, in one or more embodiments, a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 3 or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having the amino acid sequence shown in SEQ ID NO: 15 or 16 or an amino acid sequence having 90% or more identity with said sequence, preferably into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum into which accBC and accD1 genes have been modified to be expressably integrated into the chromosome, and even more preferably into Corynebacterium glutamicum into which accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressably integrated into the chromosome.

[0046] In one or more embodiments, the transformants of this disclosure have been introduced to expressibly encode a gene that has the activity of producing 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA, or the expression level of said enzyme has been increased. Examples of the enzyme that has the activity of producing 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA include, in one or more embodiments, 3-Hydroxyadipyl-CoA dehydrogenase (EC: 1.1.1.157). Examples of 3-Hydroxyadipyl-CoA dehydrogenase (3-hydroxyadipyl-CoA dehydrogenase) include, in one or more embodiments, PaaH, and PaaH derived from Escherichia coli may be used. Examples of said enzyme include, in one or more embodiments, enzymes derived from microorganisms other than the genus Burkholderia. In one or more embodiments, the transformants of this disclosure have been introduced to expressibly encode a gene having the activity of producing 2,3-dehydroxyadipyl-CoA from 3-hydroxyadipyl-CoA, or the expression level of said enzyme has been increased. Examples of the enzyme having the activity of producing 2,3-dehydroxyadipyl-CoA from 3-hydroxyadipyl-CoA include, in one or more embodiments, 2,3-dehydroadipyl-CoA hydratase (EC: 4.2.1.17). Examples of 2,3-dehydroadipyl-CoA hydratase (EC: 4.2.1.17) include, in one or more embodiments, PaaF, and PaaF derived from Escherichia coli may be used. Examples of said enzyme include, in one or more embodiments, enzymes derived from microorganisms other than the genus Burkholderia. The paaH gene encoding PaaH and the paaF gene encoding PaaF may, in one or more embodiments, form an operon. In one or more embodiments, the transformant of this disclosure may have an expressible paaHF gene, obtained by cloning the paaH and paaF genes as an operon, introduced into it.The amino acid sequence of PaaH derived from Escherichia coli is set forth in SEQ ID NO: 109, and the nucleotide sequence of the paaH gene encoding PaaH is set forth in SEQ ID NO: 110. The amino acid sequence of PaaF derived from Escherichia coli is set forth in SEQ ID NO: 111, and the nucleotide sequence of the paaF gene encoding PaaF is set forth in SEQ ID NO: 112.

[0047] In one or more embodiments of the present disclosure, the transformant of the present disclosure has a gene encoding an enzyme having an activity of producing adipyl-CoA from 2,3-dehydroadipyl-CoA introduced therein in an expressible manner, or the expression level of the enzyme is increased, and from the viewpoint of further improving 6HH productivity, it is preferable that the expression level of the enzyme is increased. As an enzyme having an activity of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA, in one or more embodiments, Trans-2-enoyl-CoA reductase (EC: 1.3.1.44) and the like can be mentioned. As Trans-2-enoyl-CoA reductase, in one or more embodiments, Ter and the like can be mentioned, and Ter derived from Treponema denticola can be used. The amino acid sequence of Ter derived from Treponema denticola is set forth in SEQ ID NO: 113, and the nucleotide sequence of the ter gene encoding Ter is set forth in SEQ ID NO: 114.

[0048] Although it is not an essential component of the transformant of the present disclosure, from the viewpoint of further improving 6HH productivity, in one embodiment, one, two or more or all of the following enzymes (1) to (8) may optionally have reduced function or loss of function, and it is preferable that all enzymes have reduced function or loss of function: (1) lactate dehydrogenase; (2) pyruvate quinone oxidoreductase; (3) phosphate acetyltransferase; (4) acetate kinase; (5) succinyl-CoA synthetase; (6) malate dehydrogenase; (7) acetyl-CoA C-acetyltransferase; (8) 3-oxoadipyl-CoA thiolase.

[0049] In one or more embodiments of the transformants of the present disclosure, for the benefit of further improving the productivity of 6HH, a portion of the genes encoding one or more or all of the enzymes (1) to (8) above may be destroyed or deleted, and a portion of all eight genes, including the gene encoding lactate dehydrogenase (e.g., ldhA gene), the gene encoding pyruvate quinone oxidoreductase (e.g., pqo gene), the gene encoding phosphate acetyltransferase (e.g., pta gene), the gene encoding acetate kinase (e.g., ack gene), the gene encoding succinyl-CoA synthetase (e.g., sucCD gene), the gene encoding malate dehydrogenase (e.g., mdh gene), the gene encoding acetyl-CoA C-acetyltransferase thiolase, and the gene encoding 3-oxoadipyr-CoA thiolase, may be destroyed or deleted.

[0050] In one embodiment, the transformants of the present disclosure may have enhanced 6HH production from sugar by increasing the production of succinyl-CoA and / or malonyl-CoA, which are endogenous precursors that serve as reaction substrates for the introduced gene (A).

[0051] This disclosure relates, in other embodiments, to vectors for introducing the above-mentioned gene (A) and / or gene (B) into a microorganism. The vectors relating to this disclosure are not particularly limited and may be, for example, plasmids.

[0052] Construction of a vector for transformants The introduction of the above gene into a microbial host can be carried out by amplifying the gene described above by PCR, cloning it into a suitable vector that can be amplified in a microbial host such as a Corynebacterium, and incubating the microbial host in the presence of the vector. Examples of promoters in one or more embodiments include the promoter of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (also called "glyceraldehyde 3-phosphate dehydrogenase") derived from Corynebacterium glutamicum R (PgapA), the promoter of the mdh gene encoding maleate dehydrogenase (Pmdh), and the promoter of the ldhA gene encoding lactate dehydrogenase (PldhA), among which PgapA is preferred. Examples of terminators in one or more embodiments include the rrnB T1T2 terminator of the E. coli rRNA operon, the trpA terminator of E. coli, and the trp terminator of Brevibacterium lactofermentum, among which the rrnB T1T2 terminator is preferred.

[0053] [Preparation of Transformants] The transformation method can be any known method without limitation. Such known methods include, in one or more embodiments, 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 Corynebacterium, the electric pulse method is preferred. The electric pulse method can be carried out by known methods [for example, 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)].

[0054] Disruption or Mutation of Host Chromosome Genes: When the microbial host is a Corynebacterium, genes encoding competitive biosynthetic pathways, repressors of biosynthetic pathways, or efflux transporters may be disrupted or deleted as needed. The function of enzyme proteins encoded by specific genes may be improved by introducing mutations into the chromosome. By ligating DNA fragments before and after the target gene to create a DNA fragment in which the entire target gene is deleted, and transforming the bacteria with this DNA to induce homologous recombination on the chromosome, the target gene on the chromosome can be completely deleted. 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. By transforming the bacteria with DNA containing this gene and inducing homologous recombination between the deletion-type gene and the gene on the chromosome, the target gene on the chromosome can be replaced with the deletion-type or disruption-type gene. Even if enzyme proteins encoded by the deletion-type or disruption-type gene are produced, they have a different three-dimensional structure from wild-type enzyme proteins and their function is reduced or absent. Furthermore, mutations can be introduced at specific locations on a chromosome by inducing homologous recombination between a gene fragment into which a specific mutation has been introduced and the chromosomal region in question. Gene deletion or disruption by gene substitution using such homologous recombination is already established, and includes methods such as using plasmids containing temperature-sensitive origins of replication, conjugate-transferable plasmids, and suicide vectors that do not have origins of replication in the host (U.S. Patent No. 6,303,383, and Japanese Patent Publication No. 05-007491, etc.). Markerless chromosomal gene transfer vector pCRA725 is a plasmid that cannot replicate in Corynebacterium glutamicum R. In the case of single crossover strains with homologous regions on chromosomes introduced into plasmid pCRA725, kanamycin resistance is exhibited due to the expression of the kanamycin resistance gene on pCRA725, and lethality in sucrose-containing medium is exhibited due to the expression of the sacR-sacB gene of Bacillus subtilis. In contrast, in the case of double crossover strains, kanamycin sensitivity is exhibited due to the loss of the kanamycin resistance gene on pCRA725, and growth in sucrose-containing medium is exhibited due to the loss of the sacR-sacB gene.Therefore, markerless chromosome gene transfection strains exhibit kanamycin sensitivity and growth in sucrose-containing media.

[0055] Microbial Growth The transformants of this disclosure are preferably grown by culturing under aerobic conditions in one or more embodiments. The culture conditions for growth are, in one or more embodiments, a temperature of about 25°C to about 38°C and a duration of about 12 hours to about 48 hours. The culture medium for growth is, in one or more embodiments, a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts and other nutrients. The pH of the medium is, in one or more embodiments, about 5 to about 8.

[0056] Examples of culture media include Medium A [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)], Medium BT [Omumasaba, CA et al., Corynebacterium glutamicum glyceraldehyde-3-phosphate dehydrogenase isoforms with opposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103 (2004)], and Medium CGXII [Hoffmann, J. et al., Hyaluronic acid production with Corynebacterium glutamicum: effect of media composition on yield and molecular weight. J. Appl. Microbiol. 117:663-678 (2014)]. When the host is E. coli, the culture medium may be LB medium or the like in one or more embodiments.

[0057] Examples of carbon sources in one or more embodiments 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, or gluconic acid; and alcohols such as ethanol or propanol. Hydrocarbons such as normal paraffin may also be used if desired. One carbon source may be used alone, or two or more may be used in combination. The concentration of these carbon sources in the growth medium is about 0.1 (w / v%) to about 10 (w / v%) in one or more embodiments.

[0058] Examples of nitrogen sources in one or more embodiments include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, and ammonium acetate, as well as 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 as nitrogen sources. One type of nitrogen source may be used alone, or two or more types may be used in combination. The nitrogen source concentration in the growth medium varies depending on the nitrogen compound used, but in one or more embodiments, it is approximately 0.1 (w / v%) to approximately 10 (w / v%).

[0059] Examples of inorganic salts in one or more embodiments 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 individually or in mixtures 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 approximately 0.01 (w / v%) to approximately 1 (w / v%).

[0060] Other nutrients may include, in one or more embodiments, meat extract, peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, skim soybean hydrochloride hydrolysate, and extracts or decomposition products thereof of animals, plants, or microorganisms. The concentration of nutrients in the culture medium varies depending on the nutrients 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. Examples of vitamins may include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, and nicotinic acid.

[0061] [Method for Producing 6HH] In other embodiments, this disclosure relates to a method for producing 6HH. More specifically, the method for producing 6HH according to this disclosure includes, in one embodiment, a step of culturing the transformant according to this disclosure to produce 6-hydroxyhexanoic acid.

[0062] In one or more embodiments, the manufacturing method of the present disclosure may include culturing the transformant of the present disclosure in a culture medium. In one or more embodiments, the culture medium preferably contains sugars as a carbon source (raw material). In one or more embodiments, the culture medium may also contain other nutrients other than sugars, such as vitamins, yeast extract, and dried yeast.

[0063] In one or more embodiments, the culture medium may be a nutrient medium or an inorganic salt medium, with the nutrient medium being preferred and the liquid nutrient medium being more preferred. In one or more embodiments, the nutrient medium may be CGXII medium or A medium, with CGXII medium containing sugars (preferably glucose) being preferred in order to further improve 6HH productivity. In one or more embodiments, the inorganic salt medium may be BT medium. In one or more embodiments, the culture medium may preferably contain inorganic salts. In one or more embodiments, the inorganic salts may include monopotassium phosphate, dipotassium phosphate, magnesium sulfate, sodium chloride, calcium chloride, nickel chloride, ferrous nitrate, manganese sulfate, zinc sulfate, copper sulfate, cobalt sulfate, calcium carbonate, urea, ammonium sulfate, and ferrous sulfate, among which urea, ammonium sulfate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, calcium chloride, ferrous sulfate, manganese sulfate, zinc sulfate, copper sulfate, and nickel chloride are preferred. The concentration of inorganic salts in the culture medium varies depending on the inorganic salt used, but in one or more embodiments, it is approximately 0.01 (w / v%) to approximately 1 (w / v%).

[0064] Examples of sugars in one or more embodiments include glucose, fructose, mannose, xylose, arabinose, galactose, sucrose, maltose, lactose, cellobiose, xylobiose, trehalose, and mannitol. The concentration of sugars in the culture medium is approximately 0.1 (w / v%) to 20 (w / v%) in one or more embodiments, 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, there may be one type of sugar or a combination of two or more types.

[0065] Reaction conditions In one or more embodiments, the reaction conditions include reducing conditions and aerobic conditions (for example, conditions with controlled dissolved oxygen concentration). In one or more embodiments, conditions with controlled dissolved oxygen concentration include conditions in which the dissolved oxygen in the culture medium is 15% or less, preferably 14% or less, 13% or less, 12% or less, 11% or less, or 10% or less, more preferably less than 10%.

[0066] The reaction temperature (the survival temperature of the transformants during the reaction) is approximately 15°C to approximately 50°C in one or more embodiments. 6HH can be efficiently produced within this temperature range. For further improvement of 6HH 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. Similarly, the reaction temperature is 49°C or lower, 45°C or lower, 40°C or lower, or 35°C or lower.

[0067] In one or more embodiments, the pH of the culture medium is preferably about 6 to about 8, and more preferably about 7. It is preferable to control the pH of the culture medium to about 6 to about 8 or about 7 using an aqueous ammonia solution and an aqueous sodium hydroxide solution, etc., with a pH controller built into the culture device (for example, Bio Jr.8 manufactured by Able Co., Ltd.) while carrying out the reaction.

[0068] The reaction time is not particularly limited and can be determined as appropriate. In one or more embodiments, the reaction time is about 1 to 7 days, and is preferably about 1 to 3 days. In one or more embodiments, the culture may be batch, fed-batch, or continuous, with the batch method being preferred.

[0069] In one or more embodiments, the manufacturing method of the present disclosure preferably includes changing the pH of the culture medium in the 6HH production process, and more preferably changing the pH of the culture medium to the acidic side (for example, pH 6.8 or lower), in order to further improve the productivity of 6HH. In one or more embodiments, the manufacturing method of the present disclosure preferably includes changing the pH of a culture medium with a pH of about 7 to 8 to about 6.8 or lower, and then further culturing the transformants of the present disclosure in a culture medium with a pH of about 6.7 or lower, in order to further improve the productivity of 6HH. In one or more embodiments, the pH change is preferably made between 24 and 48 hours from the start of cultivation. Examples of the pH to be changed (acidic pH) include pH 5 to pH 6.8, pH 5.5 to pH 6.7, or pH 6.0 to pH 6.5 in one or more embodiments. Examples of the pH for further cultivation after the pH change include pH 5 to pH 6.7, pH 5.5 to pH 6.7, or pH 6.0 to pH 6.5 in one or more embodiments. pH can be measured by the method described in the examples.

[0070] In order to further improve 6HH productivity, the manufacturing method of the present disclosure may, in one or more embodiments, include a first production step of culturing the transformant of the present disclosure in a medium with a pH of about 7 to 8 to produce 6HH, a step of changing the pH of the medium to about 6.8 or less, and a second production step of culturing the transformant of the present disclosure in a medium with a pH of about 6.7 or less to produce 6HH. In one or more embodiments, the first production step may include producing adipic acid as a byproduct. In one or more embodiments, the second production step may include producing 6HH using the adipic acid produced as a byproduct in the first production step as a raw material. In one or more embodiments, the culturing time in the first production step is 24 to 48 hours. In one or more embodiments, the step of changing the pH is preferably performed after culturing for 24 to 48 hours in the first production step. In one or more embodiments, the culturing time in the second production step is 12 to 72 hours, or 20 to 48 hours.

[0071] In order to further improve 6HH productivity, the manufacturing method of the present disclosure may, in one or more embodiments, include culturing the transformant of the present disclosure under conditions in which the pH of the culture medium is controlled. In order to further improve 6HH productivity, the manufacturing method of the present disclosure may, in one or more embodiments, include culturing the transformant of the present disclosure under conditions in which the culture medium contains sugars and the pH of the culture medium is controlled to about 7 to 8 (first culture), changing the pH of the culture medium to about 6.8 or less, and further culturing the transformant of the present disclosure under conditions in which the pH of the culture medium is controlled to about 6.7 or less (second culture). By performing the first culture under conditions in which the pH of the culture medium is controlled to about 7 to 8, the productivity of adipic acid can be improved compared to 6HH in one or more embodiments. Then, by changing the pH of the culture medium and performing the second culture under conditions in which the pH of the culture medium is controlled to about 6.7 or less, 6HH can be produced using the sugars and adipic acid in the culture medium as raw materials in one or more embodiments. These measures can further improve the productivity of 6HH. In the first culture, in one or more embodiments, the pH of the culture medium is controlled to about 7 to 7.5, or about 7. In one or more embodiments, the culture time in the first culture is 24 to 48 hours. In the second culture, in one or more embodiments, the pH of the culture medium is controlled to about 6.8 or less, or about 6.7 or less, and from the viewpoint of further improving 6HH, it is preferable to control it to about 5.5 to 6.5, or about 6. In one or more embodiments, the culture time in the second culture is 12 to 72 hours, or 20 to 48 hours.

[0072] In one or more embodiments, the 6HH production process may include the production of adipic acid as a by-product. In one or more embodiments, the 6HH production process may include the production of 6HH using the adipic acid produced as a by-product as a raw material.

[0073] In one or more embodiments, the manufacturing method of the present disclosure may include a step of separating and recovering the 6HH obtained in the production process. In one or more embodiments, the manufacturing method of the present disclosure may include a step of purifying the 6HH obtained in the production process. Examples of purification methods in one or more embodiments include membrane separation and distillation.

[0074] [Method for Producing Adipic Acid] In one or more embodiments, the transformants of the present disclosure may have the ability to produce adipic acid in addition to 6HH. Therefore, in one or more embodiments, the transformants of the present disclosure may be used for the production of adipic acid. In other embodiments, the present disclosure relates to a method for producing adipic acid, which includes a step of culturing the transformants of the present disclosure to produce adipic acid. Adipic acid may be used as a raw material for 6,6-nylon and as a raw material for 1,6-hexanediol, which is used as a raw material for polyester and polyurethane, etc.

[0075] The method for producing adipic acid according to this disclosure can be carried out in the same manner as the method for producing 6HH according to this disclosure, except that the substance to be produced is adipic acid. In one or more embodiments, adipic acid can be a by-product of the 6HH production process.

[0076] In one or more embodiments, the method for producing adipic acid of the present disclosure may include separating and recovering the adipic acid obtained in the production step. Alternatively, in one or more embodiments, the method for producing adipic acid of the present disclosure may include separating and recovering adipic acid from a product containing 6HH and adipic acid obtained in the method for producing 6HH of the present disclosure. In one or more embodiments, the production method of the present disclosure may include a step of purifying the adipic acid obtained in the production step.

[0077] [Transformant for Adipic Acid Production] In further embodiments, the present disclosure relates to a transformant for producing adipic acid, obtained by introducing a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA into a microbial host capable of producing succinyl-CoA and malonyl-CoA, wherein the microbial host is Corynebacterium glutamicum. The transformant of this embodiment can be used in one or more embodiments of the method for producing adipic acid of the present disclosure.

[0078] The transformants of this disclosure, in terms of further improving adipic acid productivity, in one or more embodiments include the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, the pamA gene from Streptomyces phaeochromogenes, the pamA gene from Streptomyces microflavus, the pamA gene from Streptomyces mediolani, and Streptomyces A transformant obtained by expressibly introducing at least one selected from the group consisting of pamA genes derived from flavofungini into Corynebacterium glutamicum is preferably a transformant obtained by expressibly introducing a gene encoding an enzyme having acetyl-CoA carboxylase activity into Corynebacterium glutamicum, more preferably a transformant obtained by expressibly introducing accBC and accD1 genes into Corynebacterium glutamicum that has been modified to allow expression of accBC and accD1 genes into the chromosome, and even more preferably a transformant obtained by expressibly introducing accBC and accD1 genes derived from Corynebacterium glutamicum into the chromosome.The transformants of this disclosure are, in terms of further improving the productivity of adipic acid, transformants obtained by expressively introducing at least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, and the pamA gene from Streptomyces phaeochromogenes into Corynebacterium glutamicum in one or more embodiments. Preferably, the transformants are obtained by expressively introducing a gene encoding an enzyme having acetyl-CoA carboxylase activity into Corynebacterium glutamicum in which a gene encoding an enzyme has been introduced; more preferably, into Corynebacterium glutamicum that has been modified so that accBC and accD1 genes can be expressively incorporated into the chromosome; and even more preferably, into Corynebacterium glutamicum that has been modified so that accBC and accD1 genes derived from Corynebacterium glutamicum can be expressively incorporated into the chromosome. The transformants of this disclosure are, in terms of further improving the productivity of adipic acid, transformants obtained by expressively introducing at least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, and the pamA gene from Streptomyces lincolnensis into Corynebacterium glutamicum in one or more embodiments. Preferably, transformants obtained by expressively introducing a gene encoding an enzyme having acetyl-CoA carboxylase activity into Corynebacterium glutamicum, more preferably into Corynebacterium glutamicum modified to allow expression of accBC and accD1 genes into the chromosome, and even more preferably into Corynebacterium glutamicum modified to allow expression of accBC and accD1 genes derived from Corynebacterium glutamicum into the chromosome.The transformants of this disclosure, in terms of further improving the productivity of adipic acid, are transformants obtained by expressibly introducing the pamA gene derived from Streptomyces alboniger or the nonU gene derived from Streptomyces violaceoruber into Corynebacterium glutamicum in one or more embodiments, preferably into Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum into which accBC and accD1 genes have been modified to be expressibly integrated into the chromosome, and even more preferably into Corynebacterium glutamicum into which accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressibly integrated into the chromosome.

[0079] The transformants of this disclosure are, in terms of further improving the productivity of adipic acid, transformants obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7, or an amino acid sequence having 90% or more identity with said sequence, into Corynebacterium glutamicum in one or more embodiments, preferably a transformant obtained by expressively introducing a polypeptide into Corynebacterium glutamicum in which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably a transformant obtained by expressively introducing a polypeptide into Corynebacterium glutamicum in which the accBC and accD1 genes have been modified to be expressably integrated into the chromosome, and even more preferably a transformant obtained by expressively introducing a polypeptide into Corynebacterium glutamicum in which the accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressably integrated into the chromosome. The transformants of this disclosure are, in terms of further improving the productivity of adipic acid, transformants obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 7, or an amino acid sequence having 90% or more identity with said sequence, into Corynebacterium glutamicum in one or more embodiments, preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 7, or an amino acid sequence having 90% or more identity with said sequence, into Corynebacterium glutamicum, preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 7, or an amino acid sequence having 90% or more identity with said sequence into Corynebacterium glutamicum, and even more preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 7 into Corynebacterium glutamicum, or a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 7, or an amino acid sequence having 90% or more identity with said sequence into Corynebacterium glutamicum, in one or more embodiments.The transformants of this disclosure are, in terms of further improving the productivity of adipic acid, transformants obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3, or 7 or an amino acid sequence having 90% or more identity with said sequence into Corynebacterium glutamicum in one or more embodiments, and more preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3, or 7 or an amino acid sequence having 90% or more identity with said sequence into Corynebacterium glutamicum in one or more embodiments, from the viewpoint of further improving the productivity of adipic acid, into Corynebacterium glutamicum in one or more embodiments, and more preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3, or 7 into Corynebacterium glutamicum in one or more embodiments, from the viewpoint of further improving the productivity of adipic acid into one or more embodiments, and more preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3, or 7 or an amino acid sequence having 90% or more identity with said sequence into Corynebacterium glutamicum in one or more embodiments, from the viewpoint of further improving the productivity of adipic acid in one or more embodiments, and more preferably a transformant obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, 3, or 7 into Corynebacterium glutamicum in one or more embodiments, from the viewpoint of further improving the productivity of adipic acid in one or more embodiments, and more preferably a transformant obtained by expressively introducing The transformants of this disclosure are, in terms of further improving the productivity of adipic acid, transformants obtained by expressively introducing a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 3 or an amino acid sequence having 90% or more identity with said sequence into Corynebacterium glutamicum in one or more embodiments, and more preferably transformants obtained by expressively introducing a polypeptide into Corynebacterium glutamicum in which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, more preferably into Corynebacterium glutamicum that has been modified so that accBC and accD1 genes can be expressively incorporated into the chromosome, and even more preferably into Corynebacterium glutamicum that has been modified so that accBC and accD1 genes derived from Corynebacterium glutamicum can be expressively incorporated into the chromosome.

[0080] In this embodiment, from the viewpoint of further improving the productivity of adipic acid, the transformant does not, in one or more embodiments, have an expressible gene (B) encoding an enzyme that has the activity to produce adipic acid semialdehyde from adipyl-CoA. In this embodiment, from the viewpoint of further improving the productivity of adipic acid, the transformant may, in one or more embodiments, have reduced function or loss of function of the enzyme that has the activity to produce adipic acid semialdehyde from adipyl-CoA.

[0081] In one or more embodiments of this embodiment, the transformant has been introduced in an expressible form a gene encoding an enzyme that has the activity to produce adipyl-CoA from 2,3-dehydroxyadipyl-CoA, or the expression level of said enzyme is improved, and it is preferable that the expression level of said enzyme is improved in order to further improve adipic acid productivity.

[0082] The contents of each document cited herein are incorporated by reference as constituting part of this disclosure.

[0083] The disclosure further relates to one or more embodiments described below. [A1] A transformant for producing 6HH obtained by introducing into a microbial host capable of producing succinyl-CoA and malonyl-CoA a gene (A) encoding an enzyme having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme having the activity to produce adipic acid semialdehyde from adipyl-CoA, wherein the microbial host is Corynebacterium glutamicum. [A2] The transformant according to [A1], wherein gene (A) is derived from at least one species selected from the group consisting of Streptomyces alboniger, Streptomyces violaceoruber, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces microflavus, Streptomyces mediolani, and Streptomyces flavofungini. [A3] The gene (A) is selected from the group consisting of (a), (b), (c), and (d) below, and is a transformant according to [A1] or [A2]. (a) A gene encoding a polypeptide having or consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. (b) A gene encoding a polypeptide having or consisting of an amino acid sequence that is 90% or more identical to the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.(c) A gene encoding a polypeptide having or consisting of an amino acid sequence in which 100 amino acids are considered as one unit, and 1 to 10 amino acids are deleted, substituted and / or added per unit, in the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. (d) 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: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. [A4] The gene (A) is selected from the group consisting of (e), (f), (g) and (h) below, and is a transformant according to any one of [A1] to [A3]. (e) A gene having the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, or a gene consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.(g) A gene having a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, wherein each unit consists of 100 nucleotides and has deletions, substitutions, and / or additions of 1 to 10 nucleotides, and is a gene encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, wherein each unit consists of 100 nucleotides and has deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, and is a gene encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. (h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. [A5] The transformant according to any one of [A1] to [A4], wherein the gene (B) is derived from at least one species selected from the group consisting of Lactobacillus brevis, Citrobacter koseri, and Marinobacter hydrocarbonoclasticus. [A6] The transformant according to any one of [A1] to [A5], wherein the gene (B) is selected from the group consisting of (a), (b), (c), and (d) below.(a) A gene encoding a polypeptide having or consisting of the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. (b) A gene encoding a polypeptide having or consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and which has the activity to produce adipic acid semialdehyde from adipyl-CoA. (c) A gene encoding a polypeptide having or consisting of an amino acid sequence in which 100 amino acids constitute one unit, and 1 to 10 amino acids are deleted, substituted, and / or added per unit, in the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and which has the activity to produce adipic acid semialdehyde from adipyl-CoA. (d) A gene that hybridizes under stringent conditions with a gene having a complementary nucleotide sequence to a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and which has the activity to produce adipic acid semialdehyde from adipyl-CoA. [A7] The gene (B) is selected from the group consisting of (e), (f), (g), and (h) below, and is a transformant according to any of [A1] to [A6]. (e) A gene having the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene consisting of a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA.(g) A gene having a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, in which 100 nucleotides constitute one unit, with 1 to 10 nucleotides deleted, substituted and / or added per unit, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, in which 100 nucleotides constitute one unit, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides deleted, substituted and / or added per unit, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA. (h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA. [A8] The transformant is the transformant according to any one of [A1] to [A7], wherein at least one of the enzymes selected from the group consisting of (1) to (8) below, preferably all of the enzymes (1) to (8) below, is impaired or has no function. (1) Lactate dehydrogenase (ldhA) (2) Pyruvate quinone oxidoreductase (pqo) (3) Phosphate acetyltransferase (pta) (4) Acetate kinase (ack) (5) Succinyl-CoA synthase (sucCD) (6) Malate dehydrogenase (mdh) (7) Acetyl-CoA C-acetyltransferase (8) 3-oxoadipyr-CoA thiolase.[A9] The microbial host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof, as described in any of [A1] to [A8]. [A10] The transformant according to any one of [A1] to [A9], wherein the microbial host is Corynebacterium glutamicum into which a gene encoding an enzyme having acetyl-CoA carboxylase activity has been introduced, preferably Corynebacterium glutamicum into which the accBC and accD1 genes have been modified to be expressibly integrated into the chromosome, more preferably Corynebacterium glutamicum into which the accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressibly integrated into the chromosome, and even more preferably Corynebacterium glutamicum R into which the accBC and accD1 genes derived from Corynebacterium glutamicum have been modified to be expressibly integrated into the chromosome. [A11] The transformant according to any one of [A1] to [A10], wherein at least one selected from the group consisting of (1) to (8) below has been introduced in an expressible manner. (1) At least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, the pamA gene from Streptomyces phaeochromogenes, the pamA gene from Streptomyces microflavus, the pamA gene from Streptomyces mediolani, and the pamA gene from Streptomyces flavofungini, and at least one selected from the group consisting of the eutE gene from Lactobacillus brevis, the pduP gene from Citrobacter koseri, and the acr gene from Marinobacter hydrocarbonoclasticus; (2) The pamA gene from Streptomyces alboniger, Streptomyces(3) At least one selected from the group consisting of the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, and the pamA gene from Streptomyces phaeochromogenes, and at least one of the eutE gene from Lactobacillus brevis and the pduP gene from Citrobacter koseri; (4) At least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, and the pamA gene from Streptomyces lincolnensis, and at least one of the eutE gene from Lactobacillus brevis and the pduP gene from Citrobacter koseri; (5) The pamA gene from Streptomyces alboniger or the nonU gene from Streptomyces violaceoruber, and the eutE gene from Lactobacillus brevis or the pduP gene from Citrobacter koseri; (5) A polypeptide having an amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence shown in SEQ ID NO: 15, 16, or 17, or an amino acid sequence having 90% or more identity with said sequence; (6) A polypeptide having an amino acid sequence shown in SEQ ID NO: 1, 2, 3, 4, or 7, or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence shown in SEQ ID NO: 15, 16, or 17, or an amino acid sequence having 90% or more identity with said sequence; (7) A polypeptide having an amino acid sequence shown in SEQ ID NO: 1, 3, or 7, or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence shown in SEQ ID NO: 15 or 16, or an amino acid sequence having 90% or more identity with said sequence;(8) A polypeptide having an amino acid sequence shown in SEQ ID NO: 1 or 3 or an amino acid sequence having 90% or more identity with said sequence, and a polypeptide having an amino acid sequence shown in SEQ ID NO: 15 or 16 or an amino acid sequence having 90% or more identity with said sequence. [A12] A transformant according to any one of [A1] to [A11], wherein (1) a gene encoding an enzyme having the activity of producing adipyl-CoA from 2,3-dehydroxyadipyl-CoA, preferably a gene encoding trans-2-enoyl-CoA reductase is introduced in an expressible form, or the expression level of said enzyme is improved, or (2) a polypeptide having an amino acid sequence shown in SEQ ID NO: 113 or an amino acid sequence having 90% or more identity with said sequence is introduced in an expressible form. [A13] A method for producing 6HH, comprising the step of culturing the transformant according to any one of [A1] to [A12] to produce 6HH. [A14] The method for producing 6HH according to [A13], comprising the step of changing the pH of the culture medium for culturing the transformant in the 6HH production step. [A15] The method for producing adipic acid according to [A13] or [A14], comprising culturing the transformant in a culture medium, preferably a culture medium containing sugars, more preferably a nutrient medium containing sugars, and even more preferably CGXII medium containing sugars (preferably glucose). [A16] The method for producing adipic acid according to [A15], comprising culturing the transformant under conditions where the dissolved oxygen in the culture medium is 15% or less, preferably 14% or less, 13% or less, 12% or less, 11% or less, or 10% or less, more preferably less than 10%. [B1] A method for producing adipic acid, comprising the step of culturing the transformant according to any one of [A1] to [A12] to produce adipic acid. [B2] A transformant for producing adipic acid, obtained by introducing a gene (A) encoding an enzyme that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA into a microbial host capable of producing succinyl-CoA and malonyl-CoA, wherein the microbial host is Corynebacterium glutamicum. [B3] The transformant according to [B2], wherein gene (A) is a gene defined in any of [A2] to [A4].[B4] The transformant according to [B2], wherein at least one selected from the group consisting of (1) to (8) below is introduced in an expressible manner. (1) At least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, the pamA gene from Streptomyces phaeochromogenes, the pamA gene from Streptomyces microflavus, the pamA gene from Streptomyces mediolani, and the pamA gene from Streptomyces flavofungini; (2) At least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, the pamA gene from Streptomyces lincolnensis, and the pamA gene from Streptomyces phaeochromogenes; (3) At least one selected from the group consisting of the pamA gene from Streptomyces alboniger, the nonU gene from Streptomyces violaceoruber, and the pamA gene from Streptomyces lincolnensis; (4) The pamA gene derived from Streptomyces alboniger or the nonU gene derived from Streptomyces violaceoruber; (5) A polypeptide having an amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, or an amino acid sequence having 90% or more identity with said sequence; (6) A polypeptide having an amino acid sequence represented by SEQ ID NO: 1, 2, 3, 4, or 7, or an amino acid sequence having 90% or more identity with said sequence; (7) A polypeptide having an amino acid sequence represented by SEQ ID NO: 1, 3, or 7, or an amino acid sequence having 90% or more identity with said sequence; (8) A polypeptide having an amino acid sequence represented by SEQ ID NO: 1 or 3, or an amino acid sequence having 90% or more identity with said sequence.[B5] A transformant according to any one of [B2] to [B4], wherein (1) a gene encoding an enzyme having the activity to produce adipyl-CoA from 2,3-dehydroxyadipyl-CoA, preferably a gene encoding trans-2-enoyl-CoA reductase is introduced in an expressible form, or the expression level of said enzyme is increased, or (2) a polypeptide having the amino acid sequence shown in SEQ ID NO: 113 or an amino acid sequence having 90% or more identity with said sequence is introduced in an expressible form. [B6] A transformant according to any one of [B2] to [B5], wherein the transformant does not have a gene (B) encoding an enzyme having the activity to produce adipic acid semialdehyde from adipyl-CoA in an expressible form, and / or the enzyme having the activity to produce adipic acid semialdehyde from adipyl-CoA is functionally impaired or has lost function. [B7] The transformant is the transformant according to any one of [B2] to [B6], wherein at least one enzyme selected from the group consisting of (1) to (8) below is impaired or lacks function: (1) lactate dehydrogenase (ldhA) (2) pyruvate quinone oxidoreductase (pqo) (3) phosphate acetyltransferase (pta) (4) acetate kinase (ack) (5) succinyl-CoA synthase (sucCD) (6) malate dehydrogenase (mdh) (7) acetyl-CoA C-acetyltransferase (8) 3-oxoadipyr-CoA thiolase. [B8] The microbial host is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032 (DSM20300), ATCC13869 (DSM1412), or a transformant thereof, as described in any of [B2] to [B7]. [B9] The microbial host is a microorganism defined in [A10], as described in any of [B2] to [B8]. [B10] (1) A gene encoding an enzyme having the activity to produce adipyl-CoA from 2,3-dehydroxyadipyl-CoA, preferably a gene encoding trans-2-enoyl-CoA reductase, is introduced in an expressible manner, or the expression level of the enzyme is increased,(2) A transformant according to any one of [B2] to [B9], wherein a polypeptide having the amino acid sequence shown in Sequence ID No. 113 or an amino acid sequence having 90% or more identity with said sequence is introduced in an expressible manner. [B11] A method for producing adipic acid, comprising the step of culturing the transformant according to any one of [B2] to [B10] to produce adipic acid. [B12] The method for producing adipic acid according to [B10] or [B11], comprising the step of changing the pH of the culture medium in which the transformant is cultured in the adipic acid production step.

[0084] The present disclosure will be further described below using examples. However, the present disclosure shall not be construed as being limited to the following examples.

[0085] Construction of 6HH-producing strains (1) Preparation and acquisition of chromosomal DNA The chromosomal DNA of Corynebacterium glutamicum R (FERM BP-18976), Escherichia coli HST02, Citrobacter koseri ATCC BAA895D-5, and Streptomyces lincolnensis JCM 4287 was prepared using a DNA genome extraction kit (illustra bacteria genomicPrep Mini Spin Kit, Cytiva) after culturing according to the information of the strain acquisition institution. The 3-oxoacyl ACP synthase II homolog enzyme genes of Streptomyces alboniger, Streptomyces violaceoruber, Streptomyces flavofungini, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces mediolani, and Streptomyces microflavus, the Trans-2-enoyl-CoA reductase gene of Treponema denticola, and the acetaldehyde dehydrogenase gene of Lactobacillus brevis were synthesized artificially.

[0086] (2) Construction of an adipyr-CoA production related gene expression plasmid A plasmid was constructed to produce adipyr-CoA from succinyl-CoA and malonyl-CoA. The paaH and paaF genes from E. coli were cloned as operons to encode genes that have the activity to produce 3-hydroxyadipyr-CoA from 3-oxoadipyr-CoA and genes that have the activity to produce 2,3-dehydroxyadipyr-CoA from 3-hydroxyadipyr-CoA, respectively. For the gene (A) encoding the enzyme that has the activity to produce 3-oxoadipyr-CoA from succinyl-CoA and malonyl-CoA, the pamA gene from Streptomyces alboniger or the nonU gene from Streptomyces violaceoruber was cloned after codon optimization of the gene synthesis. The codon-optimized genes are denoted as pamA(San) and nonU(Svu), respectively. The paaJ gene from Escherichia coli was cloned as a gene encoding an enzyme (EC.2.3.1.174) that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and acetyl-CoA. The paaHF operon from Escherichia coli was further ligated downstream of the pamA(San), nonU(Svu), or paaJ genes, and artificial operons pamA(San)-paaHF, nonU(Svu)-paaHF, or paaJ-paaHF were amplified by PCR. PaaJ is an enzyme in which the reverse reaction of decomposing 3-oxoadipyl-CoA into succinyl-CoA and acetyl-CoA is energetically dominant. The ter gene from Treponema denticola was cloned as a gene encoding an enzyme that has the activity to produce adipyl-CoA from 2,3-dehydroxyadipyl-CoA. The amplification and ligation of each gene were performed by PCR using a VeritiPro thermal cycler (Thermo Fisher Scientific, Inc.) and PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) as the reaction reagent. The primer sequences used to isolate each gene are shown in Table 1.

[0087] The PCR-amplified artificial operons pamA(San)-paaHF, nonU(Svu)-paaHF, paaJ-paaHF, or ter were introduced into the cloning vector pCRB209 [WO2012 / 033112] containing the PgapA promoter. Using plasmid pCRG57 as a template, a ter gene expression cassette containing the PgapA promoter and terminator was amplified by PCR and introduced into the cloning vector pCRB11, which uses a different origin of replication than pCRB209. The names of the introduced cloning vectors and the resulting plasmids are shown in Table 2.

[0088] (3) Construction of a 6HH production-related gene expression plasmid. The pamA(San) gene and the ter gene were ligated by PCR to produce adipyl-CoA. The gene (B) encoding the enzyme that has the activity to produce adipic acid semialdehyde from adipyl-CoA was obtained by amplified by PCR the genes eutE, pduP, and acr, which encode the acetaldehyde dehydrogenase homolog EutE from Lactobacillus brevis, the acetaldehyde dehydrogenase homolog PduP from Citrobacter koseri, and the acyl-CoA reductase homolog ACR from Marinobacter hydrocarbonoclasticus, respectively. The primer sequences used to isolate each gene are shown in Table 3.

[0089] The PCR-amplified artificial operon pamA(San)-ter and gene (B) (eutE, pduP, and acr) were introduced into the cloning vector pCRB209 [WO2012 / 033112] containing the PgapA promoter, respectively. Furthermore, a pamA(San)-ter artificial operon expression cassette containing the PgapA promoter and terminator was amplified by PCR using pCRG59 as a template and introduced into the cloning vector pCRB11. An expression cassette of the Lactobacillus brevis-derived acetaldehyde dehydrogenase homolog gene eutE, containing the PgapA promoter and terminator, was amplified by PCR using pCRG61 as a template. This was inserted into the restriction enzyme Sse3738I cleavage site of pCRG59. The constructed plasmid was named pCRG59w. The names of the introduced cloning vectors and the resulting plasmids are shown in Table 4.

[0090] (4) Construction of a plasmid for gene (A) discovery Gene (A), which encodes an enzyme that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, was cloned from Streptomyces alboniger, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces microflavus, Streptomyces mediolani, and Streptomyces flavofungini. Each gene was designated pamA(San), pamA(Sli), pamA(Sph), pamA(Smf), pamA(Smd), and pamA(Sff), and the ter gene from Treponema denticola was ligated downstream of each gene. The primer sequences used to isolate each gene are shown in Table 5.

[0091] Gene amplification was performed by PCR. A VeritiPro thermal cycler (Thermo Fisher Scientific, Inc.) was used for PCR, and PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.) was used as the reaction reagent. In Fusion Snap Assembly Master Mix (Takara Bio Inc.) was used for gene ligation and integration into the vector. Table 6 shows the names of the introduced cloning vectors and the resulting plasmids.

[0092] (5) Construction of a plasmid for disrupting the chromosomal genes of Corynebacterium glutamicum R strain. The DNA region necessary for markerless disruption of the chromosomal genes of Corynebacterium glutamicum R strain was amplified by PCR. Each PCR fragment can be ligated with phosphorylated primers. The obtained DNA fragments were introduced into the markerless chromosomal gene modification plasmid pCRA725. The primer sequences and the obtained plasmid are shown in Table 7.

[0093] (6) Construction of a plasmid for introducing Corynebacterium glutamicum R strain chromosome genes. The paaH and paaF genes from Escherichia coli were amplified by PCR and cloned into the cloning vector pCRB209. The accBC and accD genes from Corynebacterium glutamicum R strain, which encode acetyl-CoA carboxylase, were amplified by PCR and cloned into a cloning vector containing the Psod promoter. In addition, DNA fragments before and after the chromosomal site to which each gene was introduced were amplified by PCR, ligated, and cloned into the markerless chromosome gene modification vector pCRA725 [J. Mol. Microbiol. Biotechnol. 8:243-254 (2004), JP 2007-295809]. The primer sequences and the obtained plasmids are shown in Table 8.

[0094] Expression cassettes containing promoters and terminators for each gene were amplified by PCR using plasmids pCRG73, pCRG75, pCGR77, or pCRG79 as templates. Each gene expression cassette was then inserted onto chromosomal fragments cloned into plasmids pCRG74, pCRG76, pCGR78, or pCRG80, respectively. The primer sequences and resulting plasmids are shown in Table 9.

[0095] (7) Construction of chromosome-modified strains for 6HH production The markerless chromosome gene modification vector pCRA725 is a plasmid that cannot replicate within Corynebacterium glutamicum R. In the case of a single crossover strain with a homologous region on a chromosome introduced into plasmid pCRA725, it exhibits kanamycin resistance due to the expression of the kanamycin resistance gene on pCRA725 and lethality in 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, it exhibits kanamycin sensitivity due to the loss of the kanamycin resistance gene on pCRA725 and growth in sucrose-containing medium due to the loss of the sacR-sacB gene. Therefore, markerless chromosome-modified strains exhibit kanamycin sensitivity and growth in sucrose-containing medium. Genetically modified strains were constructed using the chromosome gene disruption plasmid described above by the method described above. Specifically, Corynebacterium glutamicum R ldhA knockout strain CRZ1 [Biotechnol Bioeng. Nov;110(11):2938-2948 (2013)] was used as the host strain, and each gene was disrupted using the chromosomal gene disruption plasmids listed in Table 7, as well as the sucCD gene disruption plasmid pCRG69, the cgR_653 gene disruption plasmid pCRG70, the cgR_2630 gene disruption plasmid pCRG71, and the cgR_2274 gene disruption plasmid pCRG72. Furthermore, the pta-ack gene, pqo gene, and mdh gene were also disrupted, and the constructed strain was named LHglc3055. The cgR_653 gene encodes acetyl-CoA C-acetyltransferase, and the cgR_2630 and cgR_2274 genes encode 3-oxoadipyr-CoA thiolase. Using the LHglc3055 strain as a host, the paaH and paaF genes were introduced into the chromosome using the E. coli-derived paaH gene plasmid pCRG81 and paaF gene plasmid pCRG82, respectively, to construct the LHglc3111 strain.Furthermore, using the LHglc3111 strain as a host, the accBC and accD1 genes were introduced into the chromosome using the Corynebacterium glutamicum R-derived accBC gene plasmid pCRG83 and the accD1 gene plasmid pCRG84 to construct the LHglc3306 strain. The outline of this chromosomal genetic recombination is summarized in Table 10.

[0096] (8) Construction of strains with 6HH production-related gene expression plasmids The above-mentioned pCRG54, pCRG55, or pCRG56 was introduced into LHglc3055 strain together with pCRG58. The above-mentioned plasmids pCRB209 (empty vector), pCRG61, pCRG62, or pCRG63 were introduced into LHglc3111 strain together with pCRG60. The above-mentioned plasmids pCRG59w, pCRG59, pCRG64, pCRG65, pCRG66, pCRG67, or pCRG68 were introduced into LHglc3306 strain. The outlines of these introduced strains are summarized in Table 11.

[0097] Production Experiment (1) Adipic Acid Production Experiment (Test Tube Culture) First, in order to investigate the ability to produce adipyl-CoA, a precursor of 6HH, the ability to produce adipic acid released from adipyl-CoA was investigated. Using the constructed bacterial strain (AZIW01-03), test tube culture was performed using a nutrient medium with glucose as the carbon source. Each bacterial strain was inoculated in 10 ml of CGXII-Glc liquid medium (CGXII liquid medium (MOPS 21g, (NH2)2CO 5g, (NH4)2SO4 5g, KH2PO4 1.0g, K2HPO4 1.0g, MgSO4・7H2O 1.0g, CaCl2 0.01g, FeSO4・7H2O 10mg, MnSO4・H2O 10mg, ZnSO4・7H2O 1mg, CuSO4 0.2mg, NiCl2・6H2O 0.02mg, thiamin 1mg, biotin 0.5mg, pantothenic acid 1mg, protocatechuate 0.003mg, yeast extract). The mixture (2g dissolved in 1L of distilled water) was cultured for 48 hours in a liquid medium (in a test tube) containing 4% glucose, 50μg / ml kanamycin, and 5μg / ml chloramphenicol. The adipic acid contained in the culture supernatant of each strain after culturing was quantified by LC-MS / MS analysis. As a result, all strains (AZIW01 and AZIW02) into which gene (A) was introduced produced adipic acid from sugar (glucose), and the production amount was more than 10 times higher than that of strain AZIW03 (Example 13), into which the paaJ gene derived from E. coli was introduced as gene (A). In particular, as shown in Figure 3, strain AZIW01 (Example 11), which used the pamA gene derived from Streptomyces alboniger as gene (A), had the highest production amount at 59 mg / L.

[0098] (2) 6HH production experiment (test tube culture) To screen for enzymes that have the activity to produce adipic acid semialdehyde from adipyl-CoA, the constructed bacterial strain (AZIW04-07) was used and cultured in test tubes using a nutrient medium with glucose as the carbon source. Each bacterial strain was inoculated in 10 ml of CGXII-Glc liquid medium (CGXII liquid medium (MOPS 21g, (NH2)2CO 5g, (NH4)2SO4 5g, KH2PO4 1.0g, K2HPO4 1.0g, MgSO4・7H2O 1.0g, CaCl2 0.01g, MgSO4・7H2O 1.0g, FeSO4・7H2O 10mg, MnSO4・H2O 10mg, ZnSO4・7H2O 1mg, CuSO4 0.2mg, NiCl2・6H2O 0.02mg, thiamin 1mg, biotin 0.5mg, pantothenic acid 1mg, protocatechuate 0.003mg, yeast extract). The strains were cultured for 48 hours in a liquid medium (in a test tube) containing 2g dissolved in 1L of distilled water, to which 4% glucose, 50μg / ml kanamycin, and 5μg / ml chloramphenicol were added. 6HH and adipic acid contained in the culture supernatant of each strain after culturing were quantified by LC-MS / MS analysis. As a result, all strains into which genes (A) and (B) were introduced (AZIW05 (Example 1), AZIW06 (Example 2), and AZIW07 (Example 3)) produced 6HH from sugar (glucose). In particular, as shown in Figure 4, the highest 6HH production was observed in strain AZIW05 (Example 1), which used the eutE gene derived from Lactobacillus brevis as gene (B), and strain AZIW06 (Example 2), which used the pduP gene derived from Citrobacter koseri. The bacterial strain AZIW04 (Example 14), in which gene (B) was not introduced, did not produce 6HH.

[0099] (3) 6HH Production Experiment (Fed Batch Culture) Using strain AZIW08 (Example 4) into which the 6HH production plasmid pCRG59w was introduced, test tube culture was performed using a glucose-based nutrient medium. The 6HH production capacity of each strain from glucose was investigated when cultured in a 200 ml jar fermenter under controlled conditions of temperature, pH, and dissolved oxygen concentration. The bacterial strain was inoculated in 10 ml of CGXII-Glc liquid medium (CGXII liquid medium (MOPS 21g, (NH2)2CO 5g, (NH4)2SO4 5g, KH2PO4 1.0g, K2HPO4 1.0g, MgSO4・7H2O 1.0g, CaCl2 0.01g, MgSO4・7H2O 1.0g, FeSO4・7H2O 10mg, MnSO4・H2O 10mg, ZnSO4・7H2O 1mg, CuSO4 0.2mg, NiCl2・6H2O 0.02mg, thiamin 1mg, biotin 0.5mg, pantothenic acid 1mg, protocatechuate 0.003mg, yeast extract). (2g dissolved in 1L of distilled water), then inoculated overnight in a liquid medium (in a test tube) containing 4% glucose and 50μg / ml kanamycin. Subsequently, 100 ml of production liquid medium (CGXII liquid medium ((NH2)2CO 2g, (NH4)2SO4 7g, KH2PO4 1.0g, K2HPO4 1.0g, MgSO4・7H2O 1.0g, CaCl2 0.01g, FeSO4・7H2O 10mg, MnSO4・H2O 10mg, ZnSO4・7H2O 1mg, CuSO4 0.2mg, NiCl2・6H2O 0.02mg, thiamin 1mg, biotin 0.5mg, pantothenic acid 1mg, protocatechuate 0.003mg, yeast extract 2g dissolved in 1 L of distilled water)) with 5% glucose added was inoculated so that the initial OD610 was 1, and the culture was maintained at 30°C and pH 7.0 (5N). Culture was performed in a 200 ml jar fermenter under the conditions of automatic adjustment by adding NH3 aqueous solution, and DO > 10% (automatically controlled by stirring speed control between 100 and 2000 rpm).48 hours after the start of cultivation, 6HH and adipic acid in the culture supernatant of each strain were quantified by LC-MS / MS analysis. The results showed that strain AZIW08 produced 0.2 g / L of 6HH and 0.7 g / L of adipic acid from sugar (glucose). Next, the pH of the medium was changed by stopping the automatic pH adjustment until the pH of the medium decreased to 6.0. After that, cultivation was further carried out under conditions of pH 6 (automatically adjusted by adding 5N NH3 aqueous solution). 72 hours after the start of jar cultivation (24 hours after pH adjustment), 6HH and adipic acid in the culture supernatant of each strain were quantified by LC-MS / MS analysis. The results showed that strain AZIW08 produced 1.2 g / L of 6HH and 0.4 g / L of adipic acid from sugar (glucose). From the above results, it can be said that by controlling the pH of the culture medium to neutral (pH 7) and culturing for a predetermined time (first culture), and then changing the pH of the culture medium to the acidic side and culturing further in that state (second culture), 6HH could be produced from adipic acid produced in the first culture in addition to glucose in the culture medium. Therefore, by changing the pH of the culture medium, the productivity of 6HH could be further improved. The pH of the culture medium was measured using the pH controller built into the culture device (Bio Jr.8, manufactured by Able Co., Ltd.).

[0100] (4) Search for PamA homologs (test tube culture) To screen for enzymes that have the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, test tube cultures were performed using a nutrient medium with glucose as the carbon source. Each strain (AZIW03, AZIW09-14) was placed in 10 ml of CGXII-Glc liquid medium (CGXII liquid medium (MOPS 21g, (NH2)2CO 5g, (NH4)2SO4 5g, KH2PO4 1.0g, K2HPO4 1.0g, MgSO4・7H2O 1.0g, CaCl2 0.01g, MgSO4・7H2O 1.0g, FeSO4・7H2O 10mg, MnSO4・H2O 10mg, ZnSO4・7H2O 1mg, CuSO4 0.2mg, NiCl2・6H2O 0.02mg, thiamin 1mg, biotin 0.5mg, pantothenic acid 1mg, protocatechuate 0.003mg, yeast extract). The cells were cultured for 48 hours in a liquid medium (in a test tube) prepared by dissolving 2 g in 1 L of distilled water, and then adding 4% glucose, 50 μg / ml kanamycin, and 5 μg / ml chloramphenicol. The adipic acid contained in the culture supernatant of each strain after culturing was quantified by LC-MS / MS analysis. As a result, all strains (AZIW09, AZIW10, AZIW11, AZIW12, AZIW13, and AZIW14) produced adipic acid from sugar (glucose). Among them, as shown in Figure 5, strain AZIW10 (Example 20), which used the PamA(Sli) gene derived from Streptomyces lincolnensis as gene (A), had the highest adipic acid production at 286 mg / L.

[0101] The following lists the compound names and their CAS numbers as shown in Figure 2 (a complete metabolic pathway diagram illustrating the biosynthetic pathway of 6HH). The "compound names" listed below are examples, and it goes without saying that synonymous names may exist. Furthermore, it goes without saying that the "CAS numbers" listed below do not necessarily exhaustively include all possible compounds.

[0102] The enzymes encoded by the genes shown in Figure 2 (a complete metabolic pathway diagram illustrating the biosynthetic pathway of 6HH) and their EC numbers are shown below. The "enzyme names" shown below are examples, and it goes without saying that synonymous names may exist. 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.

[0103]

Claims

1. A transformant for producing 6-hydroxyhexanoic acid (6HH), obtained by introducing into a microbial host capable of producing succinyl-CoA and malonyl-CoA a gene (A) encoding an enzyme active in producing 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme active in producing adipic acid semialdehyde from adipyl-CoA, wherein the microbial host is Corynebacterium glutamicum.

2. The transformant according to claim 1, wherein the gene (A) is derived from at least one species selected from the group consisting of Streptomyces alboniger, Streptomyces violaceoruber, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces microflavus, Streptomyces mediolani, and Streptomyces flavofungini.

3. The transformant according to claim 1, wherein the gene (A) is selected from the group consisting of (a), (b), (c), and (d) below. (a) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; (b) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (c) A gene encoding a polypeptide having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (d) 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: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.

4. The transformant according to claim 1, wherein the gene (A) is selected from the group consisting of (e), (f), (g), and (h) below. (e) A gene having the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (g) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, as shown in SEQ ID NO: 14, and encoding a polypeptide having the activity to produce 3-oxoadip(h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.

5. The transformant according to claim 1, wherein the gene (B) is derived from at least one species selected from the group consisting of Lactobacillus brevis, Citrobacter koseri, and Marinobacter hydrocarbonoclasticus.

6. The transformant according to claim 1, wherein gene (B) is selected from the group consisting of (a), (b), (c), and (d) below: (a) a gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; (b) a gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; (c) a gene encoding a polypeptide having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; (d) 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: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA.

7. The transformant according to claim 1, wherein gene (B) is selected from the group consisting of (e), (f), (g), and (h) below: (e) a gene having the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20; (f) a gene having a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and encoding a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and encoding a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA; (g) A gene having a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, in which 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA; or a gene consisting of a nucleotide sequence in the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, in which 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, and encoding a polypeptide that has the activity to produce adipic acid semialdehyde from adipyl-CoA; (h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA, or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20, and which encodes a polypeptide having the activity to produce adipic acid semialdehyde from adipyl-CoA.

8. The transformant according to claim 1, wherein at least one enzyme selected from the group consisting of (1) to (8) below is impaired or lacks function: (1) lactate dehydrogenase; (2) pyruvate quinone oxidoreductase; (3) phosphate acetyltransferase; (4) acetate kinase; (5) succinyl-CoA synthase; (6) malate dehydrogenase; (7) acetyl-CoA C-acetyltransferase; (8) 3-oxoadipyr-CoA thiolase.

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

10. A method for producing 6HH, comprising the step of culturing a transformant according to any one of claims 1 to 9 to produce 6-hydroxyhexanoic acid (6HH).

11. The manufacturing method according to claim 10, wherein the production step includes changing the pH of the culture medium in which the transformant is cultured.

12. The manufacturing method according to claim 11, wherein the production step comprises culturing the transformant in a culture medium containing sugars and having a pH controlled to near neutral, changing the pH of the culture medium to the acidic side, and culturing the transformant in a culture medium with a pH controlled to the acidic side.

13. A method for producing adipic acid, comprising the step of culturing the transformant according to any one of claims 1 to 9 to produce adipic acid.

14. A method for producing adipic acid, comprising the step of culturing a transformant according to any one of claims 1 to 9 in a culture medium containing sugars to produce adipic acid.

15. A transformant for producing adipic acid, obtained by introducing into a microbial host capable of producing succinyl-CoA and malonyl-CoA a gene (A) encoding an enzyme that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA, and a gene (B) encoding an enzyme that has the activity to produce adipic acid semialdehyde from adipic acid semialdehyde, wherein the microbial host is Corynebacterium glutamicum.

16. A transformant for producing adipic acid, obtained by introducing a gene (A) encoding an enzyme that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA into a microbial host capable of producing succinyl-CoA and malonyl-CoA, wherein the microbial host is Corynebacterium glutamicum.

17. The transformant according to claim 15 or 16, wherein the gene (A) is derived from at least one species selected from the group consisting of Streptomyces alboniger, Streptomyces violaceoruber, Streptomyces lincolnensis, Streptomyces phaeochromogenes, Streptomyces microflavus, Streptomyces mediolani, and Streptomyces flavofungini.

18. The transformant according to claim 15 or 16, wherein the gene (A) is selected from the group consisting of (a), (b), (c), and (d) below. (a) A gene encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; (b) A gene encoding a polypeptide having an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (c) A gene encoding a polypeptide having an amino acid sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted, substituted, and / or added per unit, with 100 amino acids forming one unit, and which has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (d) 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: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.

19. The transformant according to claim 15 or 16, wherein the gene (A) is selected from the group consisting of (e), (f), (g), and (h) below. (e) A gene having the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, or a gene consisting of the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; (f) A gene having a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and encoding a polypeptide that has the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; (g) A gene having a nucleotide sequence in which 100 nucleotides constitute one unit, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14 as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, with deletions, substitutions, and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides per unit, as shown in SEQ ID NO: 14, and encoding a polypeptide having the activity to produce 3-oxoadip(h) A gene that hybridizes under stringent conditions with a gene having a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA; or a gene that hybridizes under stringent conditions with a gene consisting of a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14, and which encodes a polypeptide having the activity to produce 3-oxoadipyl-CoA from succinyl-CoA and malonyl-CoA.

Images