Genetically modified microorganisms for producing 3-hydroxyadipic acid and / or α-hydromuconic acid and methods for producing said chemicals

Abstract

Disclosed are: a genetically modified microorganism capable of producing 3-hydroxyhexanedioic acid and / or (E)-hex-2-enedioic acid at a high yield; and a production method for 3-hydroxyhexanedioic acid and / or (E)-hex-2-enedioic acid using said genetically modified microorganism. The genetically modified microorganism has: the ability to produce 3-hydroxyhexanedioic acid and / or (E)-hex-2-enedioic acid; an enhanced enzyme activity that catalyzes a reaction that reduces 3–oxoadiphyl-CoA and generates 3–hydroxyadiphyl–CoA; and has lost or reduced the dicarboxylic acid discharge carrier function.

Description

[Technical Field]

[0001] The present invention relates to a genetically modified microorganism that produces 3-hydroxyadipic acid and / or α-hydromuconic acid at a high level, and a method for producing 3-hydroxyadipic acid and / or α-hydromuconic acid using the genetically modified microorganism. [Background technology]

[0002] 3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid) and α-hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid) are dicarboxylic acids with six carbon atoms. They can be polymerized with polyhydric alcohols to form polyesters, and with polyamines to form polyamides. Lactamized compounds obtained by adding ammonia to the terminals of these compounds can also be used as polyamide raw materials.

[0003] Patent Document 1, a document related to the production of a six-carbon dicarboxylic acid using a microorganism, describes a method for producing 3-hydroxyadipate, α-hydromuconic acid, and / or adipic acid using a polypeptide that exhibits excellent activity in catalyzing the reduction of 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, and describes that the biosynthetic pathway for these substances involves an enzymatic reaction that reduces 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. Furthermore, Patent Document 1 only describes reactions in the biosynthetic pathway within the cell, and does not describe the transport of 3-hydroxyadipate, α-hydromuconic acid, and / or adipic acid outside the cell.

[0004] Furthermore, Patent Document 2 describes a method for producing dicarboxylic acids using a bacterium in which the expression levels of dicarboxylic acid exporter genes yjjP, yjjB, yeeA, and ynfM are increased, and exemplified by succinic acid as a dicarboxylic acid with four carbon atoms and adipic acid as a dicarboxylic acid with six carbon atoms. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] EP 3719121 A1 [Patent Document 2] Japanese Patent Application Publication No. 2017-216881 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to provide a genetically modified microorganism for producing 3-hydroxyadipic acid and / or α-hydromuconic acid in high yields by further modifying a dicarboxylic acid exporter based on a genetically modified microorganism into which a 3-oxoadipyl-CoA reductase gene has been introduced or expression of the gene has been enhanced to enhance the enzyme activity, and to provide a genetically modified microorganism for producing 3-hydroxyadipic acid and / or α-hydromuconic acid in high yields, as well as a method for producing substances using the modified microorganism. [Means for solving the problem]

[0007] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered that, contrary to expectations based on the prior art, genetically modified microorganisms in which the function of dicarboxylic acid exporters has been deleted or reduced have excellent production ability of 3-hydroxyadipic acid and / or α-hydromuconic acid, and thus completed the present invention.

[0008] That is, the present invention provides the following.

[0009] (1) A genetically modified microorganism that has the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid and has enhanced activity of an enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA, and in which the function of a dicarboxylic acid exporter is deleted or reduced. (2) The genetically modified microorganism according to (1), wherein the deficiency or reduction in the function of the dicarboxylic acid exporter is caused by the deficiency or reduction in the function of YjjP or a homolog thereof and / or YjjB or a homolog thereof. (3) The genetically modified microorganism according to (2), in which the yjjP gene or a homologue thereof and / or the yjjB gene or a homologue thereof is / are disrupted or deleted. (4) The genetically modified microorganism according to (1), wherein the deficiency or reduction in the function of the dicarboxylic acid exporter is caused by the deficiency or reduction in the function of YeeA or a homolog thereof and / or YnfM or a homolog thereof. (5) The genetically modified microorganism according to (4), in which the yeeA gene or a homologue thereof and / or the ynfM gene or a homologue thereof is disrupted or deleted. (6) The genetically modified microorganism according to any one of (1) to (5), wherein the microorganism belongs to the genus Escherichia or Serratia. (7) A genetically modified microorganism according to any one of (1) to (6), into which a gene encoding an enzyme that catalyzes a reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA has been introduced. (8) A method for producing 3-hydroxyadipic acid and / or α-hydromuconic acid, comprising a step of culturing the genetically modified microorganism according to any one of (1) to (7). [Effects of the Invention]

[0010] The genetically modified microorganism can produce 3-hydroxyadipic acid and / or α-hydromuconic acid at a higher yield than a parent strain microorganism in which the dicarboxylic acid exporter gene has not been modified. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, 3-hydroxyadipate and α-hydromuconic acid will be abbreviated as 3HA and HMA, respectively. 3-oxoadipyl-CoA will be abbreviated as 3OA-CoA, 3-hydroxyadipyl-CoA as 3HA-CoA, and 2,3-dehydroadipyl-CoA as HMA-CoA. The enzyme that catalyzes the reduction of 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA will be referred to as "3-oxoadipyl-CoA reductase." When the yjjP and yjjB genes form an operon under the presence of a single promoter, this will be referred to as yjjPB. The proteins encoded by the yjjP and yjjB genes will be referred to as YjjP and YjjB, respectively, and their complex will be referred to as YjjPB. In addition, the protein encoded by the yeeA gene may be referred to as YeeA, and the protein encoded by the ynfM gene may be referred to as YnfM.

[0012] The microorganism of the present invention has been genetically modified to eliminate or reduce the function of a dicarboxylic acid exporter. "Deleting or reducing the function of a dicarboxylic acid exporter" means deleting or reducing dicarboxylic acid export activity. The method for deleting or reducing the function is not particularly limited. For example, the gene can be disrupted or deleted by subjecting a dicarboxylic acid exporter gene or its homologous gene (hereinafter collectively referred to as "dicarboxylic acid exporter gene"), or the nucleotide sequence of the promoter or terminator region of the gene, to gene mutation treatment using a gene mutagen or ultraviolet irradiation, or by deleting part or all of the nucleotide sequence using site-directed mutagenesis, or by introducing a frameshift mutation into the nucleotide sequence, or by inserting a stop codon into the nucleotide sequence. Gene disruption or deletion can also be achieved by using genetic recombination techniques to remove all or part of the nucleotide sequence of a dicarboxylic acid exporter gene or the nucleotide sequence of the promoter or terminator region of the gene, or by replacing it with another nucleotide sequence. Among these, the method of deleting part or all of the nucleotide sequence of a dicarboxylic acid exporter gene is preferred.

[0013] Dicarboxylic acid exporter genes whose functions are deleted or reduced in the present invention can be easily obtained, for example, by performing a BLAST (Basic Local Alignment Search Tool) search on public databases such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes) using the amino acid sequence of a known dicarboxylic acid exporter as a query sequence and referencing genes encoding the matching sequence. Alternatively, dicarboxylic acid exporter genes can be obtained by PCR using oligonucleotides prepared based on the nucleotide sequence of a known dicarboxylic acid exporter gene as primers and the genomic DNA of another organism as a template.

[0014] The amino acid sequence identity between a known dicarboxylic acid exporter and a homologous protein of the carrier is specifically 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more.

[0015] In the present invention, "sequence identity" refers to the percentage of identical amino acids or bases in the entire overlapping amino acid sequence (including the amino acid that serves as the translation initiation point) or base sequence (including the initiation codon) in the optimal alignment of two amino acid sequences or base sequences, with or without gaps introduced between them, and is calculated using formula (1). In formula (1), the length of the shorter sequence to be compared is 100 amino acids or 300 bases or more; sequence identity is not defined for sequences shorter than 100 amino acids or 300 bases. Sequence identity can be easily determined, for example, using default parameters in BLAST. Sequence identity can also be determined using a similar function built into software such as Genetyx.

[0016] Sequence identity (%) = number of matches (gaps not counted) / length of shorter sequence (length excluding gaps at both ends) × 100... Equation (1).

[0017] The dicarboxylic acid exporters whose functions are deleted or reduced in the present invention are preferably YjjP or a homolog thereof, YjjB or a homolog thereof, YeeA or a homolog thereof, and / or YnfM or a homolog thereof.

[0018] YjjP and YjjB are proteins putatively expressed as putative succinate exporters. Specific examples include YjjP (NCBI Protein ID: NP_418784, SEQ ID NO: 2) and YjjB (NCBI Protein ID: NP_418783, SEQ ID NO: 4) derived from Escherichia coli str. K-12 substr. MG1655, and a YjjP homologue (SEQ ID NO: 6) and a YjjB homologue (SEQ ID NO: 8) derived from Serratia grimesii NBRC13537. Genes encoding these proteins include yjjP (NCBI Gene ID: 948812, SEQ ID NO: 1) and yjjB (NCBI Gene ID: 948811, SEQ ID NO: 3) derived from Escherichia coli str. K-12 substr. MG1655, and a YjjB homologue (SEQ ID NO: 8) derived from Serratia grimesii NBRC13537. Examples include the yjjP homolog (SEQ ID NO: 5) and yjjB homolog (SEQ ID NO: 7) derived from the NBRC13537 strain. The sequence identity between the amino acid sequences of SEQ ID NOs: 2 and 6 calculated using the Genetyx function (% Identity Matrix) according to the above formula (1) was 71.1%. The sequence identity between the amino acid sequences of SEQ ID NOs: 4 and 8 calculated was 75.8%.

[0019] It is known that YjjP and YjjB have dicarboxylic acid export activity when they coexist (Biosci Biotechnol Biochem 2017 Sep;81(9):1837-1844). In particular, JP 2017-216881 A discloses that modifying a bacterium to increase expression of the dicarboxylic acid exporter genes yjjP and yjjB can improve the bacterium's ability to produce dicarboxylic acids. The document also discloses that the dicarboxylic acids produced by the bacterium include succinic acid (having four carbon atoms) and adipic acid (having six carbon atoms). Furthermore, it discloses that YjjP and YjjB derived from Escherichia coli and Enterobacter aerogenes, both of the Enterobacteriaceae family, actually possess dicarboxylic acid export activity. Therefore, a person skilled in the art would expect that if dicarboxylic acid export activity is deleted or reduced in a microorganism capable of producing a six-carbon dicarboxylic acid, the microorganism's ability to produce a six-carbon dicarboxylic acid would be reduced. However, as shown in the Examples of the present specification, the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid, which are dicarboxylic acids having six carbon atoms, is improved by deleting the functions of YjjP and YjjB. Contrary to the expectations of those skilled in the art, when dicarboxylic acid excretion activity is deleted or reduced in a microorganism capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid, the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid can be increased.

[0020] YeeA is a protein predicted to be a putative transporter, and specific examples thereof include YeeA derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Protein ID: NP_416512, SEQ ID NO: 91), and examples of genes encoding these proteins include yeeA derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Gene ID: 946545, SEQ ID NO: 90).

[0021] YnfM is a protein predicted to be a putative membrane transport protein, and specific examples thereof include YnfM derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Protein ID: NP_416113, SEQ ID NO: 93), and examples of genes encoding these proteins include ynfM derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Gene ID: 946138, SEQ ID NO: 92).

[0022] YeeA and YnfM are known to have dicarboxylic acid export activity (Biosci Biotechnol Biochem 2017 Sep;81(9):1837-1844). In particular, JP 2017-216881 A discloses that modifying a bacterium to increase expression of the dicarboxylic acid exporter gene yeeA or ynfM can improve the bacterium's ability to produce dicarboxylic acids. The publication also describes the dicarboxylic acids produced, such as succinic acid (having four carbon atoms) and adipic acid (having six carbon atoms). Furthermore, it has been disclosed that YeeA and YnfM derived from Pantoea ananatis and Enterobacter aerogenes, both of the Enterobacteriaceae family, actually possess dicarboxylic acid export activity. Therefore, a person skilled in the art would expect that if dicarboxylic acid export activity were deleted or reduced in a microorganism capable of producing a six-carbon dicarboxylic acid, the microorganism's ability to produce a six-carbon dicarboxylic acid would be reduced. However, as shown in the Examples of the present specification, the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid, which are dicarboxylic acids having six carbon atoms, is improved by deleting the functions of YeeA and YnfM. Contrary to the expectations of those skilled in the art, when dicarboxylic acid excretion activity is deleted or reduced in a microorganism capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid, the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid can be increased.

[0023] The YjjP homolog, YjjB homolog, YeeA homolog, or YnfM homolog whose function is to be deleted or reduced in the present invention preferably has succinate export activity. Whether the above proteins have succinate export activity can be confirmed, for example, by introducing the above homolog gene into the Escherichia coli AFP184 strain (WO 2005 / 116227) capable of producing succinic acid and verifying that succinic acid productivity is improved.

[0024] In the present invention, methods for enhancing the activity of the enzyme (3-oxoadipyl-CoA reductase) that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA include introducing a gene encoding the enzyme into a host microorganism, increasing the copy number of the gene, or modifying the promoter region or ribosome binding sequence upstream of the coding region of the gene. These methods may be performed alone or in combination. The method for introducing the gene is not particularly limited, and may include incorporating the gene into an expression vector capable of autonomous replication in a microorganism and then introducing it into a host microorganism, or integrating the gene into the genome of the microorganism.

[0025] The gene to be introduced may be one or more types, and gene introduction and enhanced expression may be combined.

[0026] When a gene encoding an enzyme to be expressed in the present invention is integrated into an expression vector or the genome of a host microorganism, the expression vector or nucleic acid to be integrated into the genome preferably comprises a promoter, a ribosome binding sequence, a coding region of the gene, and a transcription termination sequence. It may also contain a gene that controls promoter activity.

[0027] The promoter used in the present invention is not particularly limited as long as it can express the enzyme in the host microorganism, and examples thereof include the gap promoter, trp promoter, lac promoter, tac promoter, and T7 promoter.

[0028] The expression vector used in the present invention to introduce a gene or enhance expression is not particularly limited as long as it is capable of autonomous replication in the microorganism, and examples include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above-mentioned vectors.

[0029] In the present invention, when introducing a gene or enhancing its expression using a nucleic acid for genome integration, the gene can be introduced using site-specific homologous recombination. The method of site-specific homologous recombination is not particularly limited, and examples include a method using λ Red recombinase and FLP recombinase (Proc Natl Acad Sci USA. 2000 Jun 6;97(12):6640-6645.) and a method using λ Red recombinase and the sacB gene (Biosci Biotechnol Biochem. 2007 Dec;71(12):2905-11.).

[0030] The method for introducing an expression vector or a nucleic acid to be integrated into a genome is not particularly limited as long as it is a method for introducing a nucleic acid into a microorganism, and examples thereof include the calcium ion method (Journal of Molecular Biology, 53, 159 (1970)) and the electroporation method (NM Calvin, PC Hanawalt. J. Bacteriol, 170 (1988), pp. 2796-2801).

[0031] Reaction Scheme 1 below shows an example of the reaction pathway required to produce 3-hydroxyadipyl-CoA and / or α-hydromuconic acid. Here, Reaction A shows the reaction of producing 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA. Reaction B shows the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. Reaction C shows the reaction of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction D shows the reaction of producing 3-hydroxyadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction E shows the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA.

[0032] [ka]

[0033] When a microorganism has the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid, it is known that the microorganism has an enzyme that catalyzes at least reaction A in the biosynthetic pathway shown in Reaction Scheme 1 above (WO 2019 / 107516, JP 2013-535203 A, U.S. Patent Application Publication No. 2011 / 0124911 A1).

[0034] The reaction for producing 3-hydroxyadipyl-CoA and α-hydromuconic acid preferably has the biosynthetic pathway shown in Reaction Scheme 1. That is, when the genetically modified microorganism of the present invention has the ability to produce 3-hydroxyadipyl-CoA, the host microorganism for the genetically modified microorganism preferably has the ability to produce 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (reaction A), the ability to reduce 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA (reaction B), and the ability to produce 3-hydroxyadipyl-CoA from 3-hydroxyadipyl-CoA (reaction D). Furthermore, when the genetically modified microorganism of the present invention has the ability to produce α-hydromuconic acid, the host microorganism for the genetically modified microorganism preferably has the ability to produce 3-oxoadipyl-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (reaction A), the ability to reduce 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA (reaction B), the ability to produce 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA (reaction C), and the ability to produce α-hydromuconic acid from 2,3-dehydroadipyl-CoA (reaction E).

[0035] By using a microorganism having these biosynthetic pathways as a host microorganism, deleting or reducing the function of YjjP or a homolog thereof, YjjB or a homolog thereof, YeeA or a homolog thereof, and / or YnfM or a homolog thereof, and enhancing the activity of the enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA, it is possible to obtain a genetically modified microorganism that produces high amounts of 3-hydroxyadipate and / or α-hydromuconic acid.

[0036] Microorganisms that inherently have the ability to produce 3-hydroxyadipic acid include the following microorganisms:

[0037] Escherichia genus, including Escherichia fergusonii and Escherichia coli. Genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia nematodiphila. The genus Pseudomonas, including Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens. Hafnia genus, such as Hafnia alvei. Corynebacterium spp. such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, Corynebacterium glutamicum. Bacillus genus, including Bacillus badius, Bacillus magaterium, and Bacillus roseus. Streptomyces genus such as Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus. Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus oxalaticus. Acinetobacter genus, including Acinetobacter baylyi and Acinetobacter radioresistens. The genus Alcaligenes, such as Alcaligenes faecalis. Nocardioides genus, such as Nocardioides albus. Brevibacterium genus, such as Brevibacterium iodinum. Delftia genus, such as Delftia acidovorans. The genus Shimwellia, including Shimwellia blattae. Aerobacter genus, such as Aerobacter cloacae. Rhizobium species such as Rhizobium radiobacter 。

[0038] Among the microorganisms that inherently have the ability to produce 3-hydroxyadipic acid, microorganisms belonging to the genus Escherichia or Serratia are preferably used in the present invention.

[0039] Microorganisms that are presumed to inherently have the ability to produce α-hydromuconic acid include the following microorganisms:

[0040] Escherichia genus, including Escherichia fergusonii and Escherichia coli. Genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia nematodiphila. The genus Pseudomonas, including Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens. Hafnia genus, such as Hafnia alvei. Bacillus genus, such as Bacillus badius. Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus numazuensis, Cupriavidus oxalaticus. Acinetobacter genus, including Acinetobacter baylyi and Acinetobacter radioresistens. The genus Alcaligenes, such as Alcaligenes faecalis. Delftia genus, such as Delftia acidovorans. The genus Shimwellia, including Shimwellia blattae.

[0041] Among the microorganisms that inherently have the ability to produce α-hydromuconic acid, microorganisms belonging to the genus Escherichia or Serratia are preferably used in the present invention.

[0042] If the genetically modified microorganism of the present invention does not originally have the ability to produce 3-hydroxyadipic acid, this ability can be imparted by introducing into the microorganism an appropriate combination of nucleic acids encoding enzymes that catalyze reactions A, B, and D.Furthermore, if the genetically modified microorganism of the present invention does not originally have the ability to produce α-hydromuconic acid, this ability can be imparted by introducing into the microorganism an appropriate combination of nucleic acids encoding enzymes that catalyze reactions A, B, C, and E.

[0043] Microorganisms that can be used as hosts for obtaining genetically modified microorganisms in the present invention are not particularly limited as long as they are microorganisms that can be genetically modified, but microorganisms belonging to the genera Escherichia, Serratia, Hafnia, Psuedomonas, Corynebacterium, Bacillus, Streptomyces, Cupriavidus, Acinetobacter, Alcaligenes, Brevibacterium, Delftia, Shimwellia, Aerobacter, Rhizobium, Thermobifida, Clostridium, Schizosaccharomyces, Kluyveromyces, Pichia and Candida are preferred, microorganisms belonging to the genera Escherichia, Serratia, Hafnia and Pseudomonas are more preferred, and microorganisms belonging to the genus Escherichia or Serratia are particularly preferred.

[0044] Specific examples of the enzyme that catalyzes reaction B in which 3-oxoadipyl-CoA is reduced to produce 3-hydroxyadipyl-CoA include the polypeptides shown in (a) to (c) below. (a) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 (b) a polypeptide consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 in which one or several amino acids have been substituted, deleted, inserted, and / or added, and which has an enzyme activity that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. (c) a polypeptide having 70% or more sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 and having the activity of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA.

[0045] Furthermore, enzymes classified as 3-hydroxyacyl-CoA dehydrogenases in EC 1.1.1.35 and enzymes classified as 3-hydroxybutyryl-CoA dehydrogenases in EC 1.1.1.157 can also be used as enzymes having 3-oxoadipyl-CoA reductase activity. Specifically, PaaH derived from Pseudomonas putida KT2440 strain (NCBI Protein ID: NP_745425.1), PaaH derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Protein ID: NP_415913.1), DcaH derived from Acinetobacter baylyi ADP1 strain (NCBI Protein ID: CAG68533.1), PaaH derived from Serratia plymuthica NBRC102599 strain (NCBI Protein ID: WP_063197120), and a polypeptide derived from Serratia nematodiphila DSM21420 strain (NCBI Protein ID: WP_033633399.1) are also exemplified as enzymes that catalyze the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. Among these, the polypeptides described in (a) to (c) above are preferred.

[0046] In the present invention, the polypeptide has the enzyme activity of 3-oxoadipyl-CoA reductase and is composed of an amino acid sequence set forth in any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22, in which one or several amino acids have been substituted, deleted, inserted, and / or added. The range of "one or several" is preferably 10 or fewer, more preferably 5 or fewer, particularly preferably 4 or fewer, and most preferably 1 or 2 or fewer. When an amino acid is substituted, the activity of the polypeptide is more likely to be maintained if it is substituted with an amino acid having similar properties (so-called conservative substitution). In other words, when an amino acid is substituted, physiological activity is often maintained even when it is substituted with an amino acid having similar properties, and therefore, in the case of substitution, substitution with an amino acid having similar properties is preferred. In other words, the 20 types of amino acids that make up natural proteins can be grouped into groups with similar properties, such as neutral amino acids with low polarity side chains (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp), and substitutions between these amino acids often do not change the properties of the polypeptide.

[0047] For a polypeptide used in the present invention that has 70% or more sequence identity to any of the amino acid sequences of SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 and has the enzyme activity of 3-oxoadipyl-CoA reductase, the preferred range of sequence identity is 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and even more preferably 99% or more.

[0048] The polypeptides set forth in SEQ ID NOS: 10, 12, 14, 16, 18, 20, and 22 in (a) all share a consensus sequence 1 consisting of the 24 amino acid residues set forth in SEQ ID NOS: 23 at amino acid residues 15 to 38 from the N-terminus (hereinafter, for convenience, the amino acid residue position from the N-terminus may be represented by "aa." Thus, for example, amino acid residues 15 to 38 from the N-terminus may be simply represented as "15-38a.a."). In consensus sequence 1, Xaa is any amino acid residue, but 13a.a. is preferably phenylalanine or leucine, 15a.a. is preferably leucine or glutamine, 16a.a. is preferably lysine or asparagine, 17a.a. is glycine or serine, more preferably glycine, 19a.a. is preferably proline or arginine, and 21a.a. is preferably leucine, methionine, or valine. Consensus sequence 1 corresponds to the NAD+-binding residue and its surrounding amino acid residues. As shown in Biochimie. 2012 Feb;94(2):471-8, the NAD+-binding residue is characterized in that the 24th amino acid residue in consensus sequence 1 is aspartic acid, whereas in consensus sequence 1 it is asparagine. It is believed that by having consensus sequence 1, the polypeptides set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 exhibit excellent enzymatic activity as 3-oxoadipyl-CoA reductase.

[0049] The polypeptides described in (b) and (c) also preferably have consensus sequence 1 consisting of the 24 amino acid residues shown in SEQ ID NO: 23 within 1 to 200 a.a. The position of the consensus sequence is more preferably within 1 to 150 a.a., and even more preferably within 1 to 100 a.a.

[0050] The nucleic acid encoding the polypeptide of the present invention described in (a) to (c) may contain a sequence that allows an additional peptide or protein to be added to the N-terminus and / or C-terminus of the polypeptide. Examples of such peptides or proteins include those containing secretory signal sequences, transport proteins, binding proteins, tag peptides for purification, fluorescent proteins, etc. With regard to such peptides or proteins, those skilled in the art can select peptides or proteins having the desired function depending on the purpose and add them to the polypeptide of the present invention. Note that sequence identity of amino acid sequences does not include such peptides or proteins.

[0051] Nucleic acids encoding the polypeptides set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 are not particularly limited as long as they are nucleotide sequences that can be translated into the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22, and can be determined with reference to the codons (standard genetic code) corresponding to each amino acid. In this case, the nucleotide sequence may be redesigned to use codons that are commonly used in the host microorganism used in the present invention.

[0052] Specific examples of nucleic acid base sequences encoding polypeptides having the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14, 16, 18, 20, and 22 include the base sequences set forth in SEQ ID NOs: 9, 11, 13, 15, 17, 19, and 21, respectively.

[0053] In the present invention, whether a polypeptide encoded by a certain nucleic acid has 3-oxoadipyl-CoA reductase activity can be determined by preparing the following transformant strains A and B, and conducting a culture test. If 3-hydroxyadipate or α-hydromuconic acid is confirmed in the culture medium, the nucleic acid is determined to encode a polypeptide having 3-oxoadipyl-CoA reductase activity. The method for determination is explained using the biosynthetic pathway shown in Reaction Scheme 1 above.

[0054] Transformant A has enzymes that catalyze reactions A, D, and E. Transformant B has enzymes that catalyze reactions A, C, D, and E.

[0055] First, transformant A is prepared. Plasmids expressing enzymes that catalyze reactions A, D, and E are constructed. Reactions D and E can be catalyzed by the same enzyme. The plasmids are then introduced into Escherichia coli BL21(DE3), a microbial strain that lacks the ability to produce either 3-hydroxyadipic acid or α-hydromuconic acid. An expression plasmid containing a nucleic acid encoding a polypeptide to be tested for enzymatic activity downstream of an appropriate promoter is then introduced into the resulting transformant to obtain transformant A. Transformant A is then cultured, and the culture medium is checked for the presence of 3-hydroxyadipic acid. If 3-hydroxyadipic acid is detected in the culture medium, transformant B is then prepared. Transformant B is obtained by introducing a plasmid expressing an enzyme that catalyzes reaction C into transformant A. Transformant B is then cultured, and the culture medium is checked for the presence of α-hydromuconic acid. If it can be confirmed that the culture medium after cultivation contains α-hydromuconic acid, it will be understood that the 3-hydroxyadipate produced by transformant strain A and the α-hydromuconic acid produced by transformant strain B were produced via 3-hydroxyadipyl-CoA, and therefore the target polypeptide is determined to have 3-oxoadipyl-CoA reductase activity.

[0056] As a specific method for the above, for example, the method described in WO2019 / 107516 can be used.

[0057] The activity of 3-oxoadipyl-CoA reductase can be calculated by measuring the amount of 3-hydroxyadipyl-CoA produced by purified 3-oxoadipyl-CoA reductase using 3-oxoadipyl-CoA prepared from 3-oxoadipate by enzymatic reaction as a substrate. The specific method is as follows.

[0058] 3-Oxoadipic acid can be prepared by known methods (for example, the method described in Reference Example 1 of WO2017 / 099209).

[0059] Preparation of 3-oxoadipyl-CoA solution: PCR was performed using the genomic DNA of Pseudomonas putida KT2440 strain as a template according to standard methods to amplify the full-length nucleic acid encoding CoA transferase (pcaI and pcaJ, NCBI Gene IDs: 1046613 and 1046612). The primer sequences used in this PCR are, for example, SEQ ID NOS: 24 and 25. The amplified fragment was inserted into the KpnI site of the E. coli expression vector pRSF-1b (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid was then introduced into E. coli BL21(DE3), and expression of the enzyme was induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. The enzyme was then purified from the culture medium using the histidine tag to obtain a CoA transferase solution. Using this solution, an enzyme reaction solution for preparing 3-oxoadipyl-CoA was prepared with the following composition. After the reaction was allowed to proceed at 25°C for 3 minutes, the enzyme was removed by treatment with a UF membrane (Amicon Ultra-0.5 mL 10K, manufactured by Merck Millipore), and the resulting permeate was used as a 3-oxoadipyl-CoA solution.

[0060] (Enzyme reaction solution) 100 mM Tris-HCl (pH 8.2) 10mM MgCl2 0.5mM succinyl-CoA 5mM 3-oxoadipic acid sodium salt 2 μM CoA transferase.

[0061] Confirmation of 3-oxoadipyl-CoA reductase activity: PCR was performed using the genomic DNA of the target microbial strain as a template according to standard methods to amplify the full-length nucleic acid encoding 3-oxoadipyl-CoA reductase. The amplified fragment was inserted into the BamHI site of the E. coli expression vector pACYCDuet-1 (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid was then introduced into E. coli BL21(DE3), and expression of the enzyme was induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. Subsequently, the culture medium was purified using the histidine tag to obtain a 3-oxoadipyl-CoA reductase solution. 3-oxoadipyl-CoA reductase activity was confirmed by preparing an enzyme reaction solution of the following composition using this enzyme solution and measuring the amount of 3-hydroxyadipyl-CoA produced at 25°C.

[0062] (Enzyme reaction solution) 100 mM Tris-HCl (pH 8.2) 10mM MgCl2 150μL / mL 3-oxoadipyl-CoA solution 0.5mM NADH 1mM dithiothreitol 10 μM 3-oxoadipyl-CoA reductase.

[0063] Next, specific examples of enzymes that catalyze reactions A and C to E are shown. As the enzyme that catalyzes reaction A to produce 3-oxoadipyl-CoA, for example, acyltransferase (β-ketothiolase) can be used. Although there is no particular limitation on the classification of acyltransferases based on EC numbers, acyltransferases classified in EC2.3.1.- are preferred, and specific examples include enzymes classified as 3-oxoadipyl-CoA thiolases in EC2.3.1.174, enzymes classified as acetyl-CoA C-acetyltransferases in EC2.3.1.9, and enzymes classified as acetyl-CoA C-acyltransferases in EC2.3.1.16. Among these, PaaJ (NCBI Protein ID: NP_415915) derived from Escherichia coli str. K-12 substr. MG1655 strain, PcaF (NCBI Protein ID: NP_743536) derived from Pseudomonas putida KT2440 strain, and the like can be preferably used.

[0064] Whether the above-mentioned acyltransferase can produce 3-oxoadipyl-CoA using succinyl-CoA and acetyl-CoA as substrates can be confirmed by combining the 3-oxoadipyl-CoA production reaction by the purified acyltransferase with the reduction reaction by purified 3-oxoadipyl-CoA reductase using 3-oxoadipyl-CoA as a substrate, and measuring the amount of NADH reduction associated with the reduction of 3-oxoadipyl-CoA. Specific measurement methods are, for example, as follows.

[0065] Confirmation of acyltransferase activity: PCR was performed using the genomic DNA of the target microbial strain as a template according to standard methods to amplify the full-length nucleic acid encoding the acyltransferase. The amplified fragment was inserted into the SacI site of the E. coli expression vector pACYCDuet-1 (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid was then introduced into E. coli BL21(DE3), and expression of the enzyme was induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. Subsequently, the culture medium was purified using the histidine tag to obtain an acyltransferase solution. Acyltransferase activity was confirmed by preparing an enzyme reaction solution of the following composition using the enzyme solution and measuring the decrease in absorbance at 340 nm associated with the oxidation of NADH at 30°C.

[0066] 100 mM Tris-HCl (pH 8.0) 10mM MgCl2 0.1mM succinyl-CoA 0.2mM acetyl-CoA 0.2mM NADH 1mM dithiothreitol 10μg / mL 3-oxoadipyl-CoA reductase 5 μg / mL acyltransferase.

[0067] Whether or not an enzyme possessed by a microorganism has acyltransferase activity can be confirmed by carrying out the above-mentioned measurement using CFE instead of purified acyltransferase. A specific measurement method for Escherichia coli is as follows, for example.

[0068] Preparation of CFE: One loopful of E. coli MG1655 strain to be tested for activity was inoculated into 5 mL of medium (medium composition: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride) adjusted to pH 7, and cultured with shaking at 30°C for 18 hours. The resulting culture was added to 5 mL of medium (medium composition: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 2.5 mM ferulic acid, 2.5 mM p-coumaric acid, 2.5 mM benzoic acid, 2.5 mM cis,cis-muconic acid, 2.5 mM protocatechuic acid, 2.5 mM catechol, 2.5 mM 3OA, 2.5 mM 3-hydroxyadipic acid, 2.5 mM α-hydromuconic acid, 2.5 mM adipic acid, 2.5 mM phenylethylamine) adjusted to pH 7, and cultured with shaking at 30°C for 3 hours.

[0069] The resulting culture medium was washed by adding 10 mL of 0.9% sodium chloride, centrifuging the cells, and removing the supernatant three times. The washed cells were suspended in 1 mL of Tris-HCl buffer (100 mM Tris-HCl (pH 8.0) and 1 mM dithiothreitol). Glass beads (0.1 mm diameter) were added to the resulting suspension, and the cells were disrupted using an ultrasonic disrupter at 4°C. The disrupted cells were centrifuged, and 0.5 mL of the resulting supernatant was passed through a UF membrane (Amicon Ultra-0.5 mL 10K, Merck Millipore). After removing the permeate, 0.4 mL of Tris-HCl buffer was added. This process was repeated three times to remove low-molecular-weight contaminants. The resulting solution was then resuspended in Tris-HCl buffer to a volume of 0.1 mL, which was used as CFE. 0.05 mL of this CFE was added to a total of 0.1 mL of enzyme reaction solution in place of purified enzyme, and enzyme activity was confirmed.

[0070] The enzyme that catalyzes reaction C to produce 2,3-dehydroadipyl-CoA can be, for example, enoyl-CoA hydratase. While the enoyl-CoA hydratases are not particularly limited by EC number, enoyl-CoA hydratases classified under EC number 4.2.1.- are preferred, and specific examples include enzymes classified under EC 4.2.1.17 as enoyl-CoA hydratases or 2,3-dehydroadipyl-CoA hydratases. Among these, PaaF (NCBI Protein ID: NP_415911) derived from Escherichia coli str. K-12 substr. MG1655 and PaaF (NCBI Protein ID: NP_745427) derived from Pseudomonas putida KT2440 are preferred.

[0071] Because reactions catalyzed by enoyl-CoA hydratase are generally reversible, the activity of enoyl-CoA hydratase to catalyze the reaction of 3-hydroxyadipyl-CoA as a substrate to produce 2,3-dehydroadipyl-CoA can be confirmed by detecting the 3-hydroxyadipyl-CoA produced by using purified enoyl-CoA hydratase with 2,3-dehydroadipyl-CoA prepared from α-hydromuconic acid by enzymatic reaction as a substrate. Specific measurement methods are, for example, as follows.

[0072] Preparation of α-hydromuconic acid: α-hydromuconic acid is prepared by the method described in Reference Example 1 of WO2016 / 199858.

[0073] Preparation of 2,3-dehydroadipyl-CoA solution: PCR was performed using the genomic DNA of Pseudomonas putida KT2440 strain as a template according to standard methods to amplify the full-length nucleic acid encoding CoA transferase (pcaI and pcaJ, NCBI Gene IDs: 1046613 and 1046612). The primer sequences used in this PCR are, for example, SEQ ID NOs: 24 and 25. The amplified fragment was inserted into the KpnI site of the E. coli expression vector pRSF-1b (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid was then introduced into E. coli BL21(DE3), and expression of the enzyme was induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. The enzyme was then purified from the culture medium using the histidine tag to obtain a CoA transferase solution. Using this solution, an enzyme reaction solution for preparing 2,3-dehydroadipyl-CoA was prepared with the following composition. After reacting at 30°C for 10 minutes, the enzyme was removed by treatment with a UF membrane (Amicon Ultra-0.5mL 10K, manufactured by Merck Millipore), and the resulting permeate was used as a 2,3-dehydroadipyl-CoA solution.

[0074] Enzyme reaction solution for preparing 2,3-dehydroadipyl-CoA 100 mM Tris-HCl (pH 8.0) 10mM MgCl2 0.4mM succinyl-CoA 2mM α-hydromuconic acid sodium salt 20 μg / mL CoA transferase.

[0075] Confirmation of enoyl-CoA hydratase activity: According to standard methods, PCR is performed using the genomic DNA of the target microbial strain as a template to amplify the full-length nucleic acid encoding enoyl-CoA hydratase. The amplified fragment is inserted into the NdeI site of the E. coli expression vector pET-16b (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid is then introduced into E. coli BL21(DE3), and expression of the enzyme is induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. Subsequently, the culture medium is purified using the histidine tag to obtain an enoyl-CoA hydratase solution. Enoyl-CoA hydratase activity can be confirmed by preparing an enzyme reaction solution of the following composition using this solution, reacting at 30°C for 10 minutes, then removing the enzyme by treatment with a UF membrane (Amicon Ultra-0.5mL 10K, Merck Millipore), and detecting 3-hydroxyadipyl-CoA in the resulting permeate using a high-performance liquid chromatography-tandem mass spectrometer (LC-MS / MS) (Agilent).

[0076] 100 mM Tris-HCl (pH 8.0) 10mM MgCl2 300μL / mL 2,3-dehydroadipyl-CoA solution 1mM dithiothreitol 20μg / mL enoyl-CoA hydratase.

[0077] Whether the enzyme originally expressed in the host microorganism used in the present invention has enoyl-CoA hydratase activity can be confirmed by adding 0.05 mL of CFE instead of purified enoyl-CoA hydratase to a total volume of 0.1 mL of enzyme reaction solution and performing the above-mentioned measurement. A specific example of a method for preparing CFE for E. coli is as described in the method for confirming acyltransferase activity.

[0078] The enzymes that catalyze reaction D to produce 3-hydroxyadipic acid and reaction E to produce α-hydromuconic acid can be, for example, CoA transferase or acyl-CoA hydrolase, with CoA transferase being preferred.

[0079] CoA transferases are not particularly limited in classification by EC number, but are preferably CoA transferases classified under EC number 2.8.3.-, and specific examples include enzymes classified under EC 2.8.3.6 as CoA transferases or acyl-CoA transferases.

[0080] In the present invention, the term "CoA transferase" refers to an enzyme having catalytic activity (CoA transferase activity) for a reaction that produces a carboxylic acid and succinyl-CoA using acyl-CoA and succinic acid as substrates.

[0081] As the enzymes that catalyze reaction D to produce 3-hydroxyadipic acid and reaction E to produce α-hydromuconic acid, PcaI and PcaJ (NCBI Protein IDs: NP_746081 and NP_746082) derived from Pseudomonas putida KT2440 strain can be preferably used.

[0082] Because the enzymatic reaction is reversible, CoA transferase activity using 3-hydroxyadipyl-CoA or 2,3-dehydroadipyl-CoA as a substrate can be confirmed by detecting the 3-hydroxyadipyl-CoA or 2,3-dehydroadipyl-CoA produced by using purified CoA transferase with 3-hydroxyadipate and succinyl-CoA, or α-hydromuconic acid and succinyl-CoA as substrates. Specific measurement methods are, for example, as follows.

[0083] Preparation of 3-hydroxyadipic acid: 3-hydroxyadipic acid is prepared by the method described in Reference Example 1 of WO2016 / 199856.

[0084] Confirmation of CoA transferase activity using 3-hydroxyadipic acid as a substrate: PCR is performed using the genomic DNA of the target microbial strain as a template according to standard methods to amplify the full-length nucleic acid encoding the CoA transferase. The amplified fragment is inserted into the KpnI site of the E. coli expression vector pRSF-1b (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid is then introduced into E. coli BL21(DE3), and expression of the enzyme is induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. The CoA transferase solution is then purified from the culture medium using the histidine tag. Using this solution, an enzyme reaction solution of the following composition was prepared, and after reacting at 30°C for 10 minutes, the enzyme was removed by treatment with a UF membrane (Amicon Ultra-0.5mL 10K, Merck Millipore). 3-Hydroxyadipyl-CoA in the permeate was detected and confirmed using a high-performance liquid chromatography-tandem mass spectrometer (LC-MS / MS) (Agilent).

[0085] 100 mM Tris-HCl (pH 8.0) 10mM MgCl2 0.4mM succinyl-CoA 2mM 3-hydroxyadipic acid sodium salt 20 μg / mL CoA transferase.

[0086] Preparation of α-hydromuconic acid: α-hydromuconic acid is prepared by the method described in Reference Example 1 of WO2016 / 199858.

[0087] Confirmation of CoA transferase activity using α-hydromuconic acid as a substrate: PCR was performed using the genomic DNA of the target microbial strain as a template according to standard methods to amplify the full-length nucleic acid encoding the CoA transferase. The amplified fragment was inserted into the KpnI site of the E. coli expression vector pRSF-1b (Merck Millipore) in the same frame as the histidine tag sequence. The plasmid was then introduced into E. coli BL21(DE3), and expression of the enzyme was induced using isopropyl-β-thiogalactopyranoside (IPTG) according to standard methods. The CoA transferase solution was then purified from the culture medium using the histidine tag. Using this solution, an enzyme reaction solution of the following composition was prepared, and after reacting at 30°C for 10 minutes, the enzyme was removed by treatment with a UF membrane (Amicon Ultra-0.5mL 10K, Merck Millipore). 2,3-Dehydroadipyl-CoA in the permeate was detected and confirmed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Agilent).

[0088] 100 mM Tris-HCl (pH 8.0) 10mM MgCl2 0.4mM succinyl-CoA 2mM α-hydromuconic acid sodium salt 20 μg / mL CoA transferase.

[0089] Whether the enzyme originally expressed in the host microorganism used in the present invention has CoA transferase activity can be confirmed by adding 0.05 mL of CFE instead of purified CoA transferase to a total volume of 0.1 mL of enzyme reaction solution and performing the above-mentioned measurement. A specific example of a method for preparing CFE for E. coli is as described in the method for confirming acyltransferase activity.

[0090] In the present invention, when a nucleic acid encoding any one of acyltransferase, 3-oxoadipyl-CoA reductase, enoyl-CoA hydratase, and CoA transferase is introduced into a host microorganism, the nucleic acid may be artificially synthesized based on the amino acid sequence of the enzyme present in a database, or may be isolated from nature. When artificially synthesized, the frequency of codon usage corresponding to each amino acid may be changed to suit the host microorganism into which it is introduced.

[0091] When a nucleic acid encoding the enzyme is isolated from nature, the organism that serves as the gene source is not particularly limited, and examples thereof include the genus Acinetobacter, such as Acinetobacter baylyi and Acinetobacter radioresistens; the genus Aerobacter, such as Aerobacter cloacae; the genus Alcaligenes, such as Alcaligenes faecalis; the genus Bacillus, such as Bacillus badius, Bacillus magaterium, and Bacillus roseus; the genus Brevibacterium, such as Brevibacterium iodinum; the genus Corynebacterium, such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, and Corynebacterium glutamicum; Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus numazuensis, and Cupriavidus oxalaticus and other strains of Cupriavidus, Delftia and other strains of Delftia acidovorans, Escherichia and other strains of Escherichia fergusonii, Hafnia and other strains of Hafnia alvei, Microbacterium and other strains of Microbacterium ammoniaphilum, Nocardioides and other strains of Nocardioides albus, Planomicrobium and other strains of Planomicrobium okeanokoites, Pseudomonas and other strains of Pseudomonas azotoformans, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas putida, and Pseudomonas reptilivora, Rhizobium and other strains of Rhizobium radiobacter, and RhodosporidiumRhodosporidium species such as Rhodosporidium toruloides, Saccharomyces species such as Saccharomyces cerevisiae, Serratia species such as Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesii, Serratia nematodiphila, Serratia odorifera, and Serratia plymuthica, Shimwellia species such as Shimwellia blattae, Streptomyces species such as Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus, and Streptomyces vinaceus, Yarrowia species such as Yarrowia lipolytica, Yersinia species such as Yersinia ruckeri, Euglena species such as Euglena gracilis, and Thermobifida Examples of the genera include the genus Thermobifida, such as Acinetobacter, Corynebacterium, Escherichia, Pseudomonas, Serratia, Euglena, and Thermobifida.

[0092] The microorganism of the present invention is preferably deficient in pyruvate kinase function in order to enhance the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid.

[0093] Pyruvate kinase is classified as EC 2.7.1.40 and is an enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate, converting it to pyruvate and ATP. Specific examples include PykF (NCBI Protein ID: NP_416191, SEQ ID NO: 27) and PykA (NCBI Protein ID: NP_416368, SEQ ID NO: 28) derived from Escherichia coli str. K-12 substr. MG1655, and PykF (SEQ ID NO: 30) and PykA (SEQ ID NO: 31) derived from Serratia grimesii NBRC13537. When the microorganism used in the present invention has two or more genes encoding pyruvate kinase, it is preferable to eliminate the functions of all pyruvate kinases. Whether the polypeptide encoded by the gene possessed by the microorganism used in the present invention is pyruvate kinase can be determined by performing a BLAST search on public databases such as NCBI and KEGG.

[0094] The microorganism of the present invention preferably has enhanced phosphoenolpyruvate carboxykinase activity in order to enhance the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid.

[0095] Phosphoenolpyruvate carboxykinase is classified as EC 4.1.1.49 and is an enzyme that catalyzes the reaction of phosphoenolpyruvate, carbon dioxide, and ADP to produce oxaloacetate and ATP. Specific examples include Pck (NCBI Protein ID: NP_417862, SEQ ID NO: 32) derived from Escherichia coli str. K-12 substr. MG1655, and PckA_1 (SEQ ID NO: 33) and PckA_2 (SEQ ID NO: 34) derived from Serratia grimesii NBRC13537.

[0096] Physiologically, phosphoenolpyruvate carboxykinase is responsible for the key reaction in the production of glucose from fatty acids in gluconeogenesis. The reaction catalyzed by phosphoenolpyruvate carboxykinase is reversible, but in the production of 3-hydroxyadipate and / or α-hydromuconic acid, the reaction proceeds in the direction of converting phosphoenolpyruvate and carbon dioxide to oxaloacetate.

[0097] Whether the polypeptide encoded by the enzyme gene used in the present invention is phosphoenolpyruvate carboxykinase can be determined by performing a BLAST search on websites such as NCBI and KEGG. The genetically modified microorganism of the present invention is cultured in a medium, preferably a liquid medium, containing a carbon source that can be utilized by ordinary microorganisms as a fermentation raw material. In addition to the carbon source that can be utilized by the genetically modified microorganism, a medium containing an appropriate amount of a nitrogen source, inorganic salts, and, as necessary, organic trace nutrients such as amino acids and vitamins is used. Either natural or synthetic media can be used as long as they contain the above nutrient sources.

[0098] The fermentation raw material is a raw material that can be metabolized by the genetically modified microorganism. "Metabolism" refers to the conversion of a chemical compound that the microorganism has taken in from outside the cell or that is produced from another chemical compound within the cell into another chemical compound through an enzymatic reaction. Sugars are preferably used as carbon sources. Specific examples of sugars include glucose. ,centre Monosaccharides such as lucotose, galactose, mannose, xylose, and arabinose, and sugars formed by combining these monosaccharides Sucrose, etc. Examples of such sugars include disaccharides, polysaccharides, and sugars containing these, such as starch saccharified solution, molasses, and cellulose-containing biomass saccharified solution.

[0099] The carbon sources listed above may be used alone or in combination, but it is particularly preferable to culture in a medium containing glucose. When adding a carbon source, the concentration of the carbon source in the medium is not particularly limited and can be set appropriately depending on the type of carbon source, etc. The preferred concentration of glucose is 5 to 300 g / L.

[0100] Nitrogen sources that can be used in culturing the genetically modified microorganism include, for example, ammonia gas, ammonia water, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources, such as oil cakes, soybean hydrolysate, casein hydrolysate, other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermentation bacteria and their hydrolysates, etc. The concentration of the nitrogen source in the medium is not particularly limited, but is preferably 0.1 to 50 g / L.

[0101] As inorganic salts used in culturing the genetically modified microorganism, for example, phosphate salts, magnesium salts, calcium salts, iron salts, manganese salts, etc. can be added as appropriate.

[0102] The culture conditions for the genetically modified microorganism to produce 3-hydroxyadipic acid and / or α-hydromuconic acid are set by appropriately adjusting or selecting the medium with the above-mentioned component composition, culture temperature, stirring speed, pH, aeration amount, inoculation amount, etc. depending on the type of the genetically modified microorganism and external conditions, etc.

[0103] The pH range for cultivation is not particularly limited as long as the genetically modified microorganism can grow, but is preferably pH 5 to 8, more preferably pH 5.5 to 6.8.

[0104] There are no particular limitations on the range of aeration conditions during culture as long as 3-hydroxyadipic acid and / or α-hydromuconic acid can be produced. However, in order to grow the microbial mutant well, it is preferable that oxygen remain in the liquid or gas phase in the culture vessel at least at the start of culture.

[0105] If foaming occurs during liquid culture, an antifoaming agent such as mineral oil, silicone oil, or a surfactant can be added to the medium as appropriate.

[0106] After 3-hydroxyadipic acid and / or α-hydromuconic acid have been produced in the culture of the microorganism to a recoverable amount, the produced product can be recovered. The produced product can be recovered, for example, isolated, by stopping the culture when the accumulated amount has reached an appropriate level and collecting a fermentation product from the culture in accordance with a general method. Specifically, the product can be isolated from the culture by separating the microbial cells by centrifugation, filtration, or the like, followed by column chromatography, ion exchange chromatography, activated carbon treatment, crystallization, membrane separation, distillation, or the like. More specifically, examples of such methods include, but are not limited to, a method of adding an acid component to a salt of the product and recovering the precipitate; a method of concentrating the culture using a reverse osmosis membrane, evaporator, or the like to remove water and increase the concentration of the product, followed by cooling crystallization or adiabatic crystallization to precipitate crystals of the product and / or a salt of the product, and then obtaining crystals of the product and / or a salt of the product by centrifugation, filtration, or the like; a method of adding alcohol to the culture to convert the product into an ester, recovering the ester of the product by distillation, and then obtaining the product by hydrolysis. These recovery methods can be appropriately selected and optimized depending on the physical properties of the product, etc. [Example]

[0107] The present invention will be specifically described below with reference to examples.

[0108] Reference example 1 Construction of plasmids expressing enzymes that catalyze the reaction from acetyl-CoA and succinyl-CoA to produce 3OA-CoA and coenzyme A (reaction A), the reaction from 3OA-CoA to produce 3HA-CoA (reaction B), and the reaction from 3HA-CoA to produce 3-hydroxyadipic acid (reaction D) and α-hydromuconic acid (reaction E) from HMA-CoA. The autonomously replicable vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166: 175-176) in E. coli was digested with XhoI to obtain pBBR1MCS-2 / XhoI. To incorporate a constitutive expression promoter into this vector, primers (SEQ ID NOS: 36 and 37) were designed to PCR amplify a 200-b region (SEQ ID NOS: 35) upstream of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR was performed according to standard methods. The resulting fragment and pBBR1MCS-2 / XhoI were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the nucleotide sequence was confirmed by standard methods. The resulting plasmid was designated pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was digested with ScaI to obtain pBBR1MCS-2::Pgap / ScaI. To amplify the gene encoding the enzyme catalyzing reaction A, primers (SEQ ID NOS: 39 and 40) were designed to PCR amplify the full-length acyltransferase gene pcaF (NCBI Gene ID: 1041755, SEQ ID NOS: 38) using the genomic DNA of Pseudomonas putida KT2440 strain as a template, and PCR was performed according to standard methods. The resulting fragment and pBBR1MCS-2::Pgap / ScaI were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by a standard method. The plasmid was designated pBBR1MCS-2::AT. Subsequently, pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT / HpaI.To amplify the genes encoding the enzymes catalyzing reactions D and E, primers were designed (SEQ ID NOs: 43 and 44) ​​to amplify a continuous sequence containing the full length of the CoA transferase genes pcaI and pcaJ (NCBI Gene IDs: 1046613 and 1046612; SEQ ID NOs: 41 and 42) using the genomic DNA of Pseudomonas putida KT2440 as a template, and PCR was performed according to standard methods. The resulting fragment and pBBR1MCS-2::AT / HpaI were ligated using the In-Fusion HD Cloning Kit and introduced into the E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by standard methods. The plasmid was designated pBBR1MCS-2::ATCT.

[0109] pBBR1MCS-2::ATCT was digested with ScaI to obtain pBBR1MCS-2::ATCT / ScaI. To amplify the nucleic acid encoding the polypeptide of SEQ ID NO: 10, primers (SEQ ID NOs: 45 and 46) for amplifying the nucleic acid of SEQ ID NO: 9 were designed using the genomic DNA of Serratia marcescens ATCC13880 strain as a template, and PCR was performed according to standard methods. The resulting fragment and pBBR1MCS-2::ATCT / ScaI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into the Escherichia coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by standard methods. This plasmid was designated pBBR1MCS-2::ATCTOR.

[0110] Reference example 2 Construction of a plasmid to express the enzyme that catalyzes the reaction (reaction C) that produces HMA-CoA from 3HA-CoA The expression vector pMW119 (Nippon Gene Co., Ltd.), which is capable of autonomous replication in E. coli, was digested with SacI to obtain pMW119 / SacI. To incorporate a constitutive expression promoter into this vector, primers (SEQ ID NOs: 47 and 48) were designed to PCR amplify a 200-b region (SEQ ID NO: 35) upstream of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR was performed according to standard methods. The resulting fragment and pMW119 / SacI were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) and introduced into the E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the nucleotide sequence was confirmed using standard methods. The resulting plasmid was designated pMW119::Pgap. Next, pMW119::Pgap was digested with SphI to obtain pMW119::Pgap / SphI. To amplify the gene encoding the enzyme catalyzing reaction C, primers (SEQ ID NOs: 50 and 51) were designed to PCR amplify the full-length enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 49) using the genomic DNA of Pseudomonas putida KT2440 strain as a template, and PCR was performed according to standard methods. The resulting fragment and pMW119::Pgap / SphI were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into the Escherichia coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by standard methods. The resulting plasmid was designated pMW119::EH.

[0111] Example 1 Construction of Serratia mutants lacking the function of YjjPB homologues To eliminate the function of the YjjPB homologue in Serratia spp., we constructed a Serratia spp. mutant lacking the yjjPB homologue gene.

[0112] The yjjPB homologue gene was deleted according to the method described in Proc Natl Acad Sci USA. 2000 Jun 6;97(12):6640-6645.

[0113] PCR was performed using pKD4, which contains a kanamycin resistance gene used as a marker for gene deletion and an FRT (FLP recognition target) sequence used to eliminate the kanamycin resistance gene, as a template. A 1.6 kb PCR fragment for the deletion of the yjjPB homolog gene was obtained by using oligonucleotides SEQ ID NOs: 52 and 53 as primers. The λ-red recombinase expression plasmid pKD46 was introduced into Serratia grimesii NBRC13537 to obtain an ampicillin-resistant strain. The resulting strain was inoculated into 5 mL of LB medium containing 500 μg / mL ampicillin and cultured at 30°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 50 mL of LB medium containing 500 μg / mL ampicillin and 50 mM arabinose and cultured at 30°C for 2 hours with a rotary swirl. The culture was cooled on ice for 20 minutes and then washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol and mixed with 5 μL of the PCR fragment. The suspension was then placed in an electroporation cuvette and cooled on ice for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-Rad) at 3 kV, 200 Ω, and 25 μF. Immediately after electroporation, 1 mL of SOC medium was added and the mixture was incubated at 30°C for 2 hours with shaking. The entire mixture was plated on LB agar medium containing 25 μg / mL kanamycin and incubated at 30°C for 1 day. Colony direct PCR was performed using the resulting kanamycin-resistant strains, and the deletion of the target gene and the insertion of the kanamycin resistance gene were confirmed based on the band lengths. Oligonucleotides of SEQ ID NOs: 54 and 56 were used as primers.

[0114] The kanamycin-resistant strain was then inoculated into 5 mL of LB medium and subcultured twice at 37°C to eliminate pKD46 and obtain an ampicillin-sensitive strain. The FLP recombinase expression plasmid pCP20 was introduced into the ampicillin-sensitive strain, again yielding an ampicillin-resistant strain. The resulting strain was subcultured twice at 40°C, followed by colony direct PCR, and the loss of the kanamycin resistance gene was confirmed based on the band length. Oligonucleotides with sequence numbers 55 and 56 were used as primers. The resulting strain was ampicillin-sensitive, confirming the loss of pCP20. The resulting strain was designated SgΔyjjPB.

[0115] Example 2 Construction of Serratia mutants lacking the function of YjjPB homologues and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E The plasmids prepared in Reference Example 1 were introduced into SgΔyjjPB prepared in Example 1 to prepare mutant Serratia microorganisms.

[0116] SgΔyjjPB was inoculated into 5 mL of LB medium and cultured at 30°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 5 mL of LB medium and cultured at 30°C for 2 hours with shaking. The culture was cooled on ice for 20 minutes, and then the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol, mixed with 1 μL of pBBR1MCS-2::ATCTOR, and then cooled on ice in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF). Immediately, 1 mL of SOC medium was added and cultured at 30°C for 1 hour with shaking. 50 μL of the culture was plated on LB agar containing 25 μg / mL kanamycin and incubated at 30°C for 1 day. The resulting strains were designated SgΔyjjPB / 3HA.

[0117] Reference example 3 Construction of Serratia mutants carrying the YjjPB homologues and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E pBBR1MCS-2::ATCTOR was introduced into Serratia grimesii NBRC13537 in the same manner as in Example 2. The resulting strain was designated Sg / 3HA.

[0118] Example 3 Production of 3-hydroxyadipic acid using Serratia mutants lacking the function of YjjPB homologues and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E. Using the Serratia genus mutant microorganism prepared in Example 2, a production test of 3-hydroxyadipic acid was carried out.

[0119] One platinum loop of the mutant prepared in Example 2 was inoculated into 5 mL of medium I (Bacto tryptone (manufactured by Difco Laboratories) 10 g / L, Bacto yeast extract (manufactured by Difco Laboratories) 5 g / L, sodium chloride 5 g / L, kanamycin 25 μg / mL) (φ18 mm glass test tube with aluminum stopper) adjusted to pH 7, and cultured with shaking at 30°C and 120 min-1 for 24 hours. 0.25 mL of the culture medium was added to 5 mL of Medium II (glucose 50 g / L, ammonium sulfate 1 g / L, potassium phosphate 50 mM, magnesium sulfate 0.025 g / L, iron sulfate 0.0625 mg / L, manganese sulfate 2.7 mg / L, calcium chloride 0.33 mg / L, sodium chloride 1.25 g / L, Bacto tryptone 2.5 g / L, Bacto yeast extract 1.25 g / L, kanamycin 25 μg / mL) adjusted to pH 6.5 (φ18 mm glass test tube, aluminum stopper), and the mixture was cultured statically at 30°C.

[0120] Quantitative analysis of substrates and products The supernatant obtained by centrifuging the culture medium to remove the bacterial cells was passed through a Millex-GV membrane (0.22 μm, PVDF, manufactured by Merck). The permeate was analyzed by the following method to quantify the concentrations of 3-hydroxyadipic acid and other products accumulated in the culture supernatant, as well as the sugars remaining unused in the medium. Furthermore, the yields of 3-hydroxyadipic acid and succinic acid calculated based on the results using the following formula (2) are shown in Table 1. Yield (%) = amount of product produced (mol) / amount of sugar consumed (mol) × 100 Equation (2).

[0121] Quantitative analysis of 3-hydroxyadipic acid by LC-MS / MS HPLC: 1290 Infinity (Agilent Technologies) Column: Synergi hydro-RP (Phenomenex), length 100 mm, inner diameter 3 mm, particle size 2.5 μm Mobile phase: 0.1% formic acid aqueous solution / methanol = 70 / 30 Flow rate: 0.3mL / min Column temperature: 40℃ LC detector: 1260DAD VL+ (210nm) MS / MS: Triple-Quad LC / MS (Agilent Technologies) Ionization method: ESI negative mode.

[0122] Quantitative analysis of organic acids by HPLC HPLC: LC-10A (Shimadzu Corporation) Column: Shim-pack SPR-H (Shimadzu GLC), length 250 mm, inner diameter 7.8 mm, particle size 8 μm Shim-pack SCR-101H (Shimadzu GLC) Length: 250 mm, inner diameter: 7.8 mm, particle size: 10 μm Mobile phase: 5mM p-toluenesulfonic acid Reaction solution: 5 mM p-toluenesulfonic acid, 0.1 mM EDTA, 20 mM Bis-Tris Flow rate: 0.8mL / min Column temperature: 45℃ Detector: CDD-10Avp (Shimadzu Corporation).

[0123] Quantitative analysis of sugars and alcohols by HPLC HPLC: Shimadzu Prominence (Shimadzu Corporation) Column: Shodex Sugar SH1011 (Showa Denko K.K.), length 300 mm, inner diameter 8 mm, particle size 6 μm Mobile phase: 0.05M sulfuric acid aqueous solution Flow rate: 0.6mL / min Column temperature: 65℃ Detector: RID-10A (Shimadzu Corporation).

[0124] Comparative Example 1 Production of 3-hydroxyadipic acid using a Serratia mutant that is functionally intact and has been transformed with plasmids expressing enzymes catalyzing reactions A, B, D, and E. The mutants prepared in Reference Example 3 were cultured in the same manner as in Example 3. The concentrations of 3-hydroxyadipic acid and other products accumulated in the culture supernatant, and the concentrations of sugars remaining unused in the medium, were quantified. Furthermore, the yields of 3-hydroxyadipic acid and succinic acid calculated based on the results using formula (2) are shown in Table 1.

[0125] Comparing the results of Comparative Example 1 with those of Example 3, it was found that the yield of 3-hydroxyadipic acid was improved by deleting the function of the YjjPB homologue of a microorganism of the genus Serratia.

[0126] [Table 1]

[0127] Example 4 Construction of Escherichia mutants lacking YjjPB function To eliminate the function of YjjPB in Escherichia, we constructed a mutant Escherichia lacking the yjjPB gene.

[0128] The yjjPB gene was deleted according to the method described in Proc Natl Acad Sci USA. 2000 Jun 6;97(12):6640-6645.

[0129] PCR was performed using pKD4 as a template and oligonucleotides (SEQ ID NOs: 57 and 58) as primers to obtain a 1.6 kb PCR fragment for the deletion of yjjPB. The FRT recombinase expression plasmid pKD46 was introduced into Escherichia coli str. K-12 substr. MG1655 to obtain an ampicillin-resistant strain. The resulting strain was inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and cultured at 37°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 50 mL of LB medium containing 100 μg / mL ampicillin and 50 mM arabinose and cultured at 37°C for 2 hours with a rotary swirl. The culture was cooled on ice for 20 minutes and then washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol and mixed with 5 μL of the PCR fragment. The suspension was then placed in an electroporation cuvette and cooled on ice for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-Rad) at 3 kV, 200 Ω, and 25 μF. Immediately after electroporation, 1 mL of SOC medium was added and the mixture was incubated at 37°C for 2 hours with shaking. The entire mixture was plated on LB agar medium containing 25 μg / mL kanamycin and incubated at 37°C for 1 day. Colony direct PCR was performed using the resulting kanamycin-resistant strains, and the deletion of the target gene and the insertion of the kanamycin resistance gene were confirmed based on the band lengths. Oligonucleotides of SEQ ID NOs: 54 and 60 were used as primers.

[0130] The kanamycin-resistant strain was then inoculated into 5 mL of LB medium and subcultured twice at 40°C to eliminate pKD46 and obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain, and an ampicillin-resistant strain was again obtained. The resulting strain was subcultured twice at 43°C, and then colony direct PCR was performed to confirm the loss of the kanamycin resistance gene based on the band length. Oligonucleotides of SEQ ID NOs: 59 and 60 were used as primers. The resulting strain was ampicillin-sensitive, confirming that pCP20 had been lost. The resulting strain was designated EcΔyjjPB.

[0131] Example 5 Construction of Escherichia mutants lacking YjjPB function and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E The plasmids prepared in Reference Example 1 were introduced into EcΔyjjPB prepared in Example 4 to prepare Escherichia genus microorganism mutants.

[0132] EcΔyjjPB was inoculated into 5 mL of LB medium and cultured at 37°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 5 mL of LB medium and cultured at 37°C for 2 hours with shaking. The culture was cooled on ice for 20 minutes, and then the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol, mixed with 1 μL of pBBR1MCS-2::ATCTOR, and then cooled on ice in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF). Immediately, 1 mL of SOC medium was added and cultured at 37°C for 1 hour with shaking. 50 μL of the culture was plated on LB agar containing 25 μg / mL kanamycin and incubated at 37°C for 1 day. The resulting strain was designated EcΔyjjPB / 3HA.

[0133] Reference example 4 Construction of Escherichia mutants carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E, and which are functionally intact in YjjPB. pBBR1MCS-2::ATCTOR was introduced into Escherichia coli str. K-12 substr. MG1655 in the same manner as in Example 5. The resulting strain was designated Ec / 3HA.

[0134] Example 6 Production of 3-hydroxyadipic acid using Escherichia mutants lacking the function of YjjPB and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E. The mutants prepared in Example 5 were cultured in the same manner as in Example 3. The concentrations of 3-hydroxyadipic acid and other products accumulated in the culture supernatant, as well as the sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid and succinic acid calculated based on these values ​​using equation (2) are shown in Table 2.

[0135] Comparative Example 2 Production of 3-hydroxyadipic acid using Escherichia mutants that are functionally intact and have been transformed with plasmids expressing enzymes that catalyze reactions A, B, D, and E. The mutants prepared in Reference Example 4 were cultured in the same manner as in Example 3. The concentrations of 3-hydroxyadipic acid and other products accumulated in the culture supernatant, and the sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid and succinic acid calculated based on these values ​​using formula (2) are shown in Table 2.

[0136] Comparing the results of Comparative Example 2 with those of Example 6, it was found that the yield of 3-hydroxyadipic acid was improved by deleting the function of YjjPB in Escherichia microorganisms.

[0137] [Table 2]

[0138] Example 7 Construction of Serratia mutants lacking the function of YjjPB homologues and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E The plasmid pMW119::EH prepared in Reference Example 2 was introduced into the Serratia mutant microorganism prepared in Example 2 to prepare a Serratia mutant microorganism.

[0139] SgΔyjjPB / 3HA was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and cultured at 30°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and cultured at 30°C for 2 hours with shaking. The culture was cooled on ice for 20 minutes, and then the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol and mixed with 1 μL of pMW119::EH. The suspension was then cooled on ice for 10 minutes in an electroporation cuvette. Electroporation was performed using a "Gene pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF). Immediately afterwards, 1 mL of SOC medium was added and the cells were cultured at 30°C for 1 hour with shaking. 50 μL of the culture was spread onto an LB agar medium containing 500 μg / mL of ampicillin and 25 μg / mL of kanamycin and incubated for 1 day at 30° C. The resulting strain was designated SgΔyjjPB / HMA.

[0140] Reference example 5 Construction of Serratia mutants carrying the YjjPB homologues and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E pMW119::EH was introduced into Sg / 3HA in the same manner as in Example 7. The resulting strain was designated Sg / HMA.

[0141] Example 8 Production of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia mutants lacking the function of YjjPB homologues and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutant prepared in Example 7 was cultured in the same manner as in Example 3, except that a medium containing 25 μg / mL kanamycin and 500 μg / mL ampicillin was used. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the sugars remaining unused in the medium, were quantified. Quantification of α-hydromuconic acid was performed using LC-MS / MS under the same conditions as for 3-hydroxyadipic acid. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated using equation (2) based on these values ​​are shown in Table 3.

[0142] Comparative Example 3 Production of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia mutants that are functionally intact and have been transformed with plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutants prepared in Reference Example 5 were cultured in the same manner as in Example 8. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated based on these values ​​using equation (2) are shown in Table 3.

[0143] Comparing the results of Comparative Example 3 with those of Example 8, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were improved by deleting the function of the YjjPB homologue of a microorganism of the genus Serratia.

[0144] [Table 3]

[0145] Example 9 Construction of Escherichia mutants lacking YjjPB function and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E The plasmid pMW119::EH prepared in Reference Example 2 was introduced into the Escherichia mutant microorganism prepared in Example 5 to prepare an Escherichia mutant.

[0146] EcΔyjjPB / 3HA was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and cultured at 37°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and cultured at 30°C for 2 hours with shaking. The culture was cooled on ice for 20 minutes, and then the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol and mixed with 1 μL of pMW119::EH. The suspension was then cooled on ice for 10 minutes in an electroporation cuvette. Electroporation was performed using a "Gene pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF). Immediately afterwards, 1 mL of SOC medium was added and the cells were cultured at 37°C for 1 hour with shaking. 50 μL of the culture was spread onto an LB agar medium containing 100 μg / mL of ampicillin and 25 μg / mL of kanamycin and incubated for 1 day at 37° C. The resulting strain was designated EcΔyjjPB / HMA.

[0147] Reference example 6 Construction of Escherichia mutants carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E, which are functionally intact in YjjPB. pMW119::EH was introduced into Ec / 3HA in the same manner as in Example 9. The resulting strain was designated Ec / HMA.

[0148] Example 10 Production of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia mutants lacking the function of YjjPB and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E The mutant prepared in Example 9 was cultured in the same manner as in Example 6, except that a medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin was used. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the sugars remaining unused in the medium, were quantified. Quantification of α-hydromuconic acid was performed using LC-MS / MS under the same conditions as for 3-hydroxyadipic acid. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated using equation (2) based on these values ​​are shown in Table 4.

[0149] Comparative Example 4 Production tests of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia mutants that are functionally intact and have been transformed with plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutant prepared in Reference Example 6 was cultured in the same manner as in Example 10. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated based on these values ​​using equation (2) are shown in Table 4.

[0150] Comparing the results of Comparative Example 4 with those of Example 10, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were improved by deleting the function of YjjPB in Escherichia microorganisms.

[0151] [Table 4]

[0152] Example 11 Construction of Serratia mutants lacking the functions of YjjPB homologues and pyruvate kinase, and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E We generated a Serratia mutant lacking pyruvate kinase function by deleting the pykF and pykA genes encoding pyruvate kinase in Serratia.

[0153] Construction of a pykF-deficient Serratia mutant Genetic recombination was carried out in the same manner as in Example 1, except that SgΔyjjPB was used as the target for deleting pykF, oligo DNAs of SEQ ID NOs: 61 and 62 were used as primers to obtain a PCR fragment for deleting pykF (SEQ ID NO: 29), oligo DNAs of SEQ ID NOs: 54 and 64 were used in colony direct PCR to confirm pykF deletion and insertion of the kanamycin resistance gene, and oligo DNAs of SEQ ID NOs: 63 and 64 were used in colony direct PCR to confirm loss of the kanamycin resistance gene. The resulting strain was designated SgΔyjjPB,pykF.

[0154] Construction of a pykA-deficient Serratia mutant Genetic recombination was carried out in the same manner as in Example 1, except that SgΔyjjPB,pykF was used as the target for deleting pykA, oligo DNAs of SEQ ID NOs: 66 and 67 were used as primers to obtain a PCR fragment for deleting pykA (SEQ ID NO: 65), oligo DNAs of SEQ ID NOs: 54 and 68 were used in colony direct PCR to confirm pykA deletion and insertion of the kanamycin resistance gene, and oligo DNAs of SEQ ID NOs: 68 and 69 were used in colony direct PCR to confirm loss of the kanamycin resistance gene. The resulting strain was designated SgΔyjjPB,pykFA.

[0155] Construction of Serratia mutants carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E pBBR1MCS-2::ATCTOR prepared in Reference Example 1 was introduced into SgΔyjjPB,pykFA in the same manner as in Example 2. Next, pMW119::EH was introduced into the resulting strain in the same manner as in Example 7. The resulting strain was designated SgΔyjjPB,pykFA / HMA.

[0156] Reference example 7 Construction of Serratia mutants that are functionally intact, have a pyruvate kinase defect, and are transfected with plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. Genetic recombination was carried out in the same manner as in Example 11, except that Serratia grimesii NBRC13537 was used as the target for deleting pykF and pykA. The resulting strain was designated SgΔpykFA / HMA.

[0157] Example 12 Production of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia mutants lacking the functions of YjjPB homologues and pyruvate kinase and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutant prepared in Example 11 was cultured in the same manner as in Example 8. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated based on these values ​​using equation (2) are shown in Table 5.

[0158] Comparative Example 5 Production tests of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia mutants that are functionally intact, functionally deficient in pyruvate kinase, and transfected with plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutant prepared in Reference Example 7 was cultured in the same manner as in Example 12. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated based on these values ​​using equation (2) are shown in Table 5.

[0159] Comparing the results of Comparative Example 5 with those of Example 12, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were further improved by deleting the functions of the YjjPB homolog and pyruvate kinase in Serratia microorganisms.

[0160] [Table 5]

[0161] Example 13 Construction of Escherichia mutants defective in YjjPB and pyruvate kinase functions We generated Escherichia mutants lacking pyruvate kinase function by deleting the pykF and pykA genes encoding pyruvate kinase in Escherichia.

[0162] Construction of Escherichia mutants lacking pykF Genetic recombination was carried out in the same manner as in Example 4, except that EcΔyjjPB was used as the target for deleting pykF, oligo DNAs of SEQ ID NOs: 70 and 71 were used as primers to obtain a PCR fragment for deleting pykF (NCBI Gene ID: 946179, SEQ ID NO: 26), oligo DNAs of SEQ ID NOs: 54 and 73 were used in colony direct PCR to confirm pykF deletion and insertion of the kanamycin resistance gene, and oligo DNAs of SEQ ID NOs: 72 and 73 were used in colony direct PCR to confirm loss of the kanamycin resistance gene. The resulting strain was designated EcΔyjjPB,pykF.

[0163] Construction of Escherichia mutants lacking pykA Genetic recombination was carried out in the same manner as in Example 4, except that EcΔyjjPB,pykF was used as the target for PykA deletion, oligo DNAs of SEQ ID NOs: 75 and 76 were used as primers to obtain a PCR fragment for deleting pykA (NCBI Gene ID: 946527, SEQ ID NO: 74), oligo DNAs of SEQ ID NOs: 54 and 78 were used in colony direct PCR to confirm pykA deletion and insertion of the kanamycin resistance gene, and oligo DNAs of SEQ ID NOs: 77 and 78 were used in colony direct PCR to confirm loss of the kanamycin resistance gene. The resulting strain was designated EcΔyjjPB,pykFA.

[0164] Reference example 8 Construction of Escherichia mutants that are functionally intact in YjjPB, functionally deficient in pyruvate kinase, and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. Genetic recombination was carried out in the same manner as in Example 13, except that Escherichia coli str. K-12 substr. MG1655 was used as the target for deleting pykF and pykA. The resulting strain was designated EcΔpykFA / HMA.

[0165] Example 14 Production of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia mutants lacking the functions of YjjPB and pyruvate kinase and carrying plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutant prepared in Example 13 was cultured in the same manner as in Example 10. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated based on these values ​​using equation (2) are shown in Table 6.

[0166] Comparative Example 6 Production tests of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia mutants that are functionally intact in YjjPB, functionally deficient in pyruvate kinase, and transfected with plasmids expressing enzymes catalyzing reactions A, B, C, D, and E. The mutants prepared in Reference Example 8 were cultured in the same manner as in Example 14. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated based on these values ​​using equation (2) are shown in Table 6.

[0167] Comparing the results of Comparative Example 6 with those of Example 14, it was found that the yields of 3-hydroxyadipic acid and α-hydromuconic acid were further improved by deleting the functions of YjjPB and pyruvate kinase in Escherichia microorganisms.

[0168] [Table 6]

[0169] Reference example 9 Construction of plasmids to enhance the function of YjjPB The expression vector pCDF-1b (Merck Millipore), which can autonomously replicate in E. coli, was digested with HindIII and XbaI to obtain pCDF-1b / HindIII,XbaI. To incorporate yjjP, yjjB, and their promoter regions, primers (SEQ ID NOs: 80 and 81) were designed to PCR amplify yjjP, yjjB, and their promoter region (SEQ ID NO: 79) using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR was performed according to standard methods. The resulting fragment and pCDF-1b / HindIII,XbaI were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the nucleotide sequence was confirmed by standard methods. The resulting plasmid was designated pCDF::yjjPB.

[0170] Reference example 10 Construction of Escherichia mutants carrying plasmids expressing enzymes that enhance the function of YjjPB and catalyze reactions A, B, C, D, and E The plasmid pCDF::yjjPB prepared in Reference Example 9, or pCDF-1b as a control, was introduced into the Escherichia mutant microorganism prepared in Reference Example 6 to prepare Escherichia mutants.

[0171] Ec / HMA was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin and cultured at 37°C for 1 day with shaking. 0.5 mL of the culture was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin and cultured at 30°C for 2 hours with shaking. The culture was cooled on ice for 20 minutes, and then the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol, mixed with 1 μL of pCDF::yjjPB or pCDF-1b, and then cooled on ice in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF), followed immediately by the addition of 1 mL of SOC medium and incubation at 37°C for 1 hour with shaking. 50 μL of the medium was plated on LB agar medium containing 25 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin and incubated at 37°C for 1 day. The resulting strains were designated Ec / HMA, yjjPB, and Ec / HMA, pCDF, respectively.

[0172] Reference example 11 Production of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia mutants carrying enhanced YjjPB function and plasmids expressing enzymes catalyzing reactions A, B, C, D, and E The mutants prepared in Reference Example 10 were cultured in the same manner as in Example 10, except that a medium containing 25 μg / mL of kanamycin, 100 μg / mL of ampicillin, and 50 μg / mL of streptomycin was used. The concentrations of 3-hydroxyadipic acid, α-hydromuconic acid, and other products accumulated in the culture supernatant, as well as the concentrations of sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid, α-hydromuconic acid, and succinic acid calculated from these values ​​using equation (2) are shown in Table 7.

[0173] Comparing the results of Reference Example 11, it was found that enhancing the function of YjjPB in Escherichia microorganisms improved the yield of succinic acid and reduced the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

[0174] [Table 7]

[0175] Example 15 Construction of Escherichia mutants lacking YeeA function To eliminate the function of YeeA in Escherichia bacteria, we constructed a mutant Escherichia bacteria lacking the yeeA gene.

[0176] The yeeA gene was deleted in the same manner as in Example 4, except that the oligoDNAs of SEQ ID NOs: 82 and 83 were used as primers for amplifying the kanamycin resistance gene and FRT sequence using pKD4 as a template, the oligoDNAs of SEQ ID NOs: 54 and 85 were used as primers for confirming introduction of the kanamycin resistance gene, and the oligoDNAs of SEQ ID NOs: 84 and 85 were used as primers for confirming loss of the kanamycin resistance gene. The resulting strain was designated EcΔyeeA.

[0177] Example 16 Construction of Escherichia mutants lacking YeeA function and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E In the same manner as in Example 5, the plasmid prepared in Reference Example 1 was introduced into EcΔyeeA prepared in Example 15 to prepare a mutant Escherichia microorganism. The resulting strain was designated EcΔyeeA / 3HA.

[0178] Example 17 Production of 3-hydroxyadipic acid using Escherichia mutants lacking YeeA function and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E. The mutant prepared in Example 16 was cultured in the same manner as in Example 6. The concentrations of 3-hydroxyadipic acid and other products accumulated in the culture supernatant, as well as the sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid and succinic acid calculated based on these values ​​using Equation (2) are shown in Table 8.

[0179] Comparing the results of Comparative Example 2 with those of Example 17, it was found that the yield of 3-hydroxyadipic acid was improved by deleting the function of YeeA in Escherichia microorganisms.

[0180] [Table 8]

[0181] Example 18 Construction of Escherichia mutants lacking YnfM function To eliminate the function of YnfM in Escherichia bacteria, we constructed a mutant Escherichia bacteria lacking the YnfM gene.

[0182] The ynfM gene was deleted in the same manner as in Example 4, except that the oligoDNAs of SEQ ID NOs: 86 and 87 were used as primers to amplify the kanamycin resistance gene and FRT sequence using pKD4 as a template, the oligoDNAs of SEQ ID NOs: 54 and 89 were used as primers to confirm introduction of the kanamycin resistance gene, and the oligoDNAs of SEQ ID NOs: 88 and 89 were used as primers to confirm loss of the kanamycin resistance gene. The resulting strain was designated EcΔynfM.

[0183] Example 19 Construction of Escherichia mutants lacking YnfM function and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E The plasmid prepared in Reference Example 1 was introduced into EcΔynfM prepared in Example 18 to prepare a mutant Escherichia microorganism in the same manner as in Example 5. The resulting strain was designated EcΔynfM / 3HA.

[0184] Example 20 3-Hydroxyadipic acid production test using Escherichia mutants lacking YnfM function and carrying plasmids expressing enzymes catalyzing reactions A, B, D, and E The mutant prepared in Example 19 was cultured in the same manner as in Example 6. The concentrations of 3-hydroxyadipic acid and other products accumulated in the culture supernatant, as well as the sugars remaining unused in the medium, were quantified. The yields of 3-hydroxyadipic acid and succinic acid calculated based on these values ​​using Equation (2) are shown in Table 9.

[0185] Comparing the results of Comparative Example 2 with those of Example 20, it was found that the yield of 3-hydroxyadipic acid was improved by deleting the YnfM function of Escherichia microorganisms.

[0186] [Table 9]

Claims

1. A genetically modified microorganism having the ability to produce 3-hydroxyadipate and / or α-hydromuconic acid, into which a gene encoding an enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA has been introduced, and in which the gene encoding a dicarboxylic acid exporter has been disrupted or deleted.

2. The genetically modified microorganism according to claim 1, wherein the disruption or deletion of the gene encoding the dicarboxylic acid exporter is the disruption or deletion of the yjjP gene and / or the yjjB gene.

3. The genetically modified microorganism according to claim 1 , wherein the disruption or deletion of the gene encoding the dicarboxylic acid exporter is the disruption or deletion of the yeeA gene and / or the ynfM gene.

4. The genetically modified microorganism according to any one of claims 1 to 3, wherein the microorganism is a microorganism belonging to the genus Escherichia or Serratia.

5. A method for producing 3-hydroxyadipic acid and / or α-hydromuconic acid, comprising a step of culturing the genetically modified microorganism according to any one of claims 1 to 4.

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