JP2023144366A - GENETICALLY ENGINEERED MICROBE FOR PRODUCING 3-HYDROXYADIPIC ACID AND/OR α-HYDROMUCONIC ACID, AND METHOD FOR PRODUCING THE CHEMICAL - Google Patents

Abstract

To provide a novel genetically engineered microbe that can improve the accumulation levels of 3-hydroxyadipic acid and/or α-hydromuconic acid.SOLUTION: A genetically engineered microbe is a microbe having the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid. For the microbe, the reaction to form acetyl-CoA from pyruvic acid is enhanced, and the reaction to form carbon dioxide from formic acid is also enhanced.SELECTED DRAWING: None

Description

The present invention relates to a genetically modified microorganism that produces a high amount of 3-hydroxyadipic acid and/or α-hydromuconic acid, and a method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid using the genetically modified microorganism.

3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid) and α-hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid) are dicarboxylic acids having 6 carbon atoms. These can be used as polyesters by polymerizing with polyhydric alcohols, and as raw materials for polyamides by polymerizing with polyvalent amines. Furthermore, compounds obtained by adding ammonia to these terminals to form lactams can also be used as raw materials for polyamide.

As a document related to the production of 3-hydroxyadipic acid and/or α-hydromuconic acid using genetically modified microorganisms with modified metabolic pathways, Patent Document 1 describes the production of 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA. A method for producing 3-hydroxyadipic acid, α-hydromuconic acid and/or adipic acid using a polypeptide that exhibits excellent activity of catalyzing the reduction reaction to There is a description that the process involves an enzymatic reaction that reduces -oxoadipyl-CoA to 3-hydroxyadipyl-CoA. Furthermore, Patent Document 2 uses a polypeptide that exhibits an excellent activity of catalyzing the reduction reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, and genetically modified the pyruvate kinase function to be deleted. A process for the production of 3-hydroxyadipic acid, α-hydromuconic acid and/or adipic acid by microorganisms is described.

Patent Document 3 describes the production of 3-3 by a genetically modified microorganism that lacks the function of pyruvate kinase and further enhances the reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA and the activity of phosphoenolpyruvate carboxykinase. A method for producing hydroxyadipic acid, α-hydromuconic acid and/or adipic acid is described.

WO2019/107516 WO2020/230718 WO2020/230719

As mentioned above, in the production of 3-hydroxyadipic acid and/or α-hydromuconic acid using a microorganism capable of producing 3-hydroxyadipic acid and/or α-hydromuconic acid, the metabolic pathway of the microorganism is modified. Although a technology has been established to increase the productivity of 3-hydroxyadipic acid and/or α-hydromuconic acid by increasing the productivity of 3-hydroxyadipic acid and/or α-hydromuconic acid, it has not been known until now to improve the accumulated concentration of 3-hydroxyadipic acid and/or α-hydromuconic acid. Further improvements in productivity can be expected by modifying metabolism-related genes that contribute to

Therefore, in the present invention, we identify a hitherto unknown metabolism-related gene that contributes to increasing the accumulated concentration of 3-hydroxyadipic acid and/or α-hydromuconic acid, and modify the metabolic pathway in which the metabolism-related gene is involved. Thus, the present invention aims to provide a novel genetically modified microorganism that has an improved accumulation concentration of 3-hydroxyadipic acid and/or α-hydromuconic acid.

As a result of intensive studies to achieve the above object, the present inventors have found that acetyl-CoA can be extracted from pyruvate in the metabolic pathway by genetic modification in microorganisms capable of producing 3-hydroxyadipic acid and/or α-hydromuconic acid. In addition to strengthening the reaction that generates carbon dioxide from formic acid in the metabolic pathway, we have found that the accumulated concentration of 3-hydroxyadipic acid and/or α-hydromuconic acid can be increased by strengthening the reaction that generates carbon dioxide from formic acid. , we have completed the present invention.

That is, the present invention is comprised of the following (1) to (12).
(1) In microorganisms that have the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid, enhance the reaction that produces acetyl-CoA from pyruvic acid and enhance the reaction that produces carbon dioxide from formic acid. Genetically modified microorganisms.
(2) The enhancement of the reaction that produces acetyl-CoA from pyruvate is enhancement of the reaction catalyzed by a pyruvate dehydrogenase complex and/or the enhancement of the reaction catalyzed by pyruvate formate lyase. genetically modified microorganisms.
(3) The gene according to (2), wherein the reaction catalyzed by the pyruvate dehydrogenase complex is enhanced by increasing the expression level of the pyruvate dehydrogenase complex and/or enhancing the activity of the pyruvate dehydrogenase complex. Modified microorganisms.
(4) The genetically modified microorganism according to (3), wherein the expression level of the pyruvate dehydrogenase complex is increased by reducing the function of a pyruvate dehydrogenase complex transcription repressor.
(5) The genetically modified microorganism according to (2), wherein the reaction catalyzed by pyruvate formate lyase is enhanced by increasing the expression level of pyruvate formate lyase.
(6) The gene according to any one of (1) to (5), wherein the enhancement of the reaction that generates carbon dioxide from formic acid is an increase in the expression level of an enzyme that catalyzes the reaction that generates carbon dioxide from formic acid. Modified microorganisms.
(7) The genetically modified microorganism according to any one of (1) to (6), wherein the enhancement of the reaction for producing carbon dioxide from formic acid is enhancement of the reaction catalyzed by NADH-producing formate dehydrogenase.
(8) The genetically modified microorganism according to any one of (1) to (7), which further enhances the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA.
(9) The genetically modified microorganism according to any one of (1) to (8), which is a microorganism that does not metabolize sugar through the phosphoketolase pathway.
(10) A method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid, comprising the step of culturing the genetically modified microorganism according to any one of (1) to (9).
(11) A step of enhancing the reaction of a microorganism capable of producing 3-hydroxyadipic acid and/or α-hydromuconic acid to produce acetyl-CoA from pyruvic acid through genetic modification, and producing carbon dioxide from formic acid. A method for producing a genetically modified microorganism, which includes a step of enhancing a reaction to produce a genetically modified microorganism.
(12) The method for producing a genetically modified microorganism according to (11), further comprising a step of enhancing the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA by genetic modification.

Genetic modification that enhances the reaction of producing acetyl-CoA from pyruvic acid and the reaction of producing carbon dioxide from formic acid in a microorganism capable of producing 3-hydroxyadipic acid and/or α-hydromuconic acid. As a result, 3-hydroxyadipic acid and/or α-hydromuconic acid can be produced at a higher cumulative concentration than the parent microorganism strain in which the gene has not been modified.

Hereinafter, in this specification, 3-hydroxyadipic acid may be abbreviated as 3HA, and α-hydromuconic acid may be abbreviated as HMA. Further, 3-oxoadipyl-CoA may be abbreviated as 3OA-CoA, 3-hydroxyadipyl-CoA as 3HA-CoA, and 2,3-dehydroadipyl-CoA as HMA-CoA. In addition, phosphoenolpyruvate may be abbreviated as PEP. Furthermore, an enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA may be referred to as "3-oxoadipyl-CoA reductase." Further, a complex of proteins encoded by the aceE, aceF, and lpd genes is referred to as a pyruvate dehydrogenase complex, and may be abbreviated as PDHc. Furthermore, the aceE, aceF, and lpd genes may be collectively referred to as the PDHc gene group. Furthermore, pyruvate formate lyase and pyruvate formate lyase activating enzyme are collectively referred to as pyruvate formate lyase, and may be abbreviated as PFL. Furthermore, a nucleic acid encoding a functional polypeptide is sometimes referred to as a gene.

The genetically modified microorganism of the present invention can biosynthesize 3-hydroxyadipic acid and/or α-hydromuconic acid using acetyl-CoA and succinyl-CoA as intermediates as shown in the metabolic pathway below. It is well known that the metabolic pathway from glucose to acetyl-CoA is glycolysis, the metabolic pathway to succinyl-CoA is TCA cycle, and the metabolic pathway consists of a replenishment pathway that generates oxaloacetate from PEP and a reductive TCA cycle.

The metabolic pathway for producing 3-hydroxyadipic acid and/or α-hydromuconic acid from acetyl-CoA and succinyl-CoA, with the metabolites represented by chemical formulas, is shown below. Here, reaction A indicates a reaction for producing 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA. Reaction B shows the reduction of 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-hydroxyadipic acid from 3-hydroxyadipyl-CoA. Reaction E shows the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA. A method for creating microorganisms having the ability to produce enzymes that catalyze each reaction in the following metabolic pathways and 3-hydroxyadipic acid and/or α-hydromuconic acid using the following metabolic pathways is disclosed in WO2019/107516. detailed in.

One method of enhancing the reaction of producing acetyl-CoA from pyruvate in the present invention is to enhance the reaction catalyzed by the pyruvate dehydrogenase complex (PDHc). PDHc, which is the target of enhancement, has a catalytic activity that generates acetyl-CoA from pyruvate. For example, in bacteria such as Escherichia and Serratia, PDHc is a catalytic activity that generates acetyl-CoA from pyruvate. 1), dihydrolipoyl transacetylase (E2, EC2.3.1.12), and dihydrolipoyl dehydrogenase (E3, EC1.8.1.4), which are encoded by the aceE, aceF, and lpd genes, respectively. . PDHc collectively catalyzes the reactions that produce acetyl-CoA, CO ₂ , and NADH from pyruvate, coenzyme A, and NAD+. These PDHc gene groups form an operon, and the expression of the PDHc gene group is controlled by the PDHc transcription repressor (PdhR) encoded by the pdhR gene. In the presence of PdhR, transcription of the PDHc gene group is suppressed, so the expression level of PDHc decreases. On the other hand, in the absence of PdhR, transcription of the PDHc gene group is not inhibited, and the expression level of PDHc increases.

A specific example of PDHc is a BLAST search on public databases such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes). Examples include polypeptides that hit as DHc, and preferred specific examples include , Escherichia coli str. K-12 substr. AceE (NCBI-Protein ID: NP_414656), AceF (NCBI-Protein ID: NP_414657), Lpd (NCBI-Protein ID: NP_414658) derived from the MG1655 strain, and Serratia grimesii N AceE (SEQ ID NO: 1) and AceF (SEQ ID NO: 2) derived from BRC13537 strain ), Lpd (SEQ ID NO: 3), Klebsiella pneumoniae subsp. Examples include LpdA (NCBI-Protein ID: ABR75580) derived from P. pneumoniae MGH 78578 strain.

Specific examples of PDHc transcriptional repressors include polypeptides that are found as PDHc transcriptional repressors by BLAST searches on public databases such as NCBI and KEGG; preferred specific examples include Escherichia coli str. K-12 substr. Examples include PdhR derived from the MG1655 strain (NCBI-Protein ID: NP_414655) and PdhR derived from the Serratia grimesii NBRC13537 strain (SEQ ID NO: 4).

Examples of methods for enhancing the reaction catalyzed by PDHc include increasing the expression level of at least one or more subunits that constitute PDHc. Examples of methods for increasing the expression level of at least one or more subunits constituting PDHc include introducing at least one or more genes constituting the PDHc gene group from outside the host microbial cell into the microbial cell. Examples include methods of increasing the copy number of the gene group, or modifying the promoter region or ribosome binding sequence upstream of the coding region of the gene group. These methods may be performed alone or in combination. Furthermore, the expression level of PDHc can be increased by decreasing the function of a PDHc transcription repressor.

Another method of enhancing the reaction catalyzed by PDHc includes, for example, enhancing the activity of PDHc. In order to enhance the activity of PDHc, the catalytic activity of at least one enzyme constituting PDHc may be enhanced. A specific method for enhancing PDHc activity includes, for example, reducing sensitivity to NADH. Decreased sensitivity to NADH means, for example, that the KI value of the enzyme for NADH is twice or more higher than that of the control. PDHc with reduced sensitivity to NADH has been described by Kim et al., J. Bacteriol. 190:3851-3858 (2008)). E354K and/or H322Y mutants in K-12-derived Lpd (NCBI-Protein ID: NP_414658), Klebsiella pneumoniae-derived Lpd (NCBI-Protein ID: ABR75580), which is known to function in an anaerobic environment, or a part thereof It can be obtained by using The enzyme gene with enhanced catalytic activity may be replaced with the wild-type gene originally possessed by the microorganism used for production, or may coexist with it. Furthermore, the copy number of the enzyme gene may be increased, or the promoter region or ribosome binding sequence upstream of the coding region of the enzyme gene may be modified. These methods may be performed alone or in combination.

Another method of enhancing the reaction of producing acetyl-CoA from pyruvate in the present invention is to enhance the reaction catalyzed by pyruvate formate lyase. Pyruvate formate lyase, which is the target of enhancement, is not particularly limited as long as it has catalytic activity to generate acetyl-CoA from pyruvate. formate-lyase, EC2.3.1.54) functions in the presence of pyruvate-formate-lyase activating enzyme (EC1.97.1.4), which are encoded by the pflB and pflA genes, respectively. ing. Pyruvate formate lyase catalyzes the reaction that produces acetyl-CoA and formate from pyruvate and coenzyme A. pflB and pflA form an operon.

Specific examples of pyruvate formate lyase include polypeptides that are found as pyruvate formate lyase in a BLAST search on public databases such as NCBI and KEGG; preferred specific examples include Escherichia coli str. K-12 substr. PflB (NCBI-Protein ID: NP_415423) and PflA (NCBI-Protein ID: NP_415422) derived from MG1655 strain, and PflB (SEQ ID NO: 5) and PflA (SEQ ID NO: 6) derived from Serratia grimesii NBRC13537 strain. Examples include.

Examples of methods for enhancing the reaction catalyzed by pyruvate formate lyase include enhancing the catalytic activity of pyruvate formate lyase itself and increasing the expression level of pyruvate formate lyase. It is preferable to increase the expression level of lyase. Methods for increasing the expression level of pyruvate formate lyase include, for example, introducing the pyruvate formate lyase gene from outside the host microorganism into the host microorganism, increasing the copy number of the gene, or increasing the number of copies of the gene. Examples include methods of modifying the promoter region or ribosome binding sequence upstream of the coding region. These methods may be performed alone or in combination.

The present invention is characterized by enhancing the reaction of producing carbon dioxide from formic acid. One way to enhance the reaction that produces carbon dioxide from formic acid is to enhance the reaction catalyzed by the hydrogen formate lyase complex. The hydrogen formate lyase complex catalyzes the reaction that generates hydrogen during the production of carbon dioxide from formic acid. Preferred specific examples of the hydrogen formate lyase complex include formate dehydrogenase-H (EC 1.17.98.4) encoded by the fdhF gene in bacteria such as Escherichia, hycB, hycC, hycD, hycE, One example is the formate hydrogen lyase complex (EC 1.17.98.4), which is composed of seven polypeptide subunits encoded by the hycF and hycG genes.

Another method for enhancing the reaction of producing carbon dioxide from formic acid includes enhancing the reaction catalyzed by formate dehydrogenase O and/or formate dehydrogenase N. Formate dehydrogenase O and/or formate dehydrogenase N catalyze a reaction that reduces quinones in the process of producing carbon dioxide from formic acid. A preferred specific example of formate dehydrogenase O and/or formate dehydrogenase N is formate dehydrogenase O (formate dehydrogenase-O), which is composed of three polypeptide subunits encoded by fdoG, fdoH, and fdoI genes in bacteria such as Escherichia. EC 1.17.5.3), and formate dehydrogenase-N (EC 1.17.5.3), which is composed of three polypeptide subunits encoded by the fdnG, fdnH, and fdnI genes. can be mentioned.

Yet another method for enhancing the reaction that produces carbon dioxide from formic acid is to enhance the reaction catalyzed by NADH-producing formate dehydrogenase (EC 1.17.1.9). NADH-producing formate dehydrogenase catalyzes a reaction that generates carbon dioxide from formic acid and reduces NAD+ to NADH. A specific example of NADH-producing formate dehydrogenase is a polypeptide that is found as NADH-producing formate dehydrogenase in a BLAST search on public databases such as NCBI or KEGG, and a preferred specific example is FDH1 (derived from Candida boidinii strain). SEQ ID NO: 7) or homologues thereof.

Examples of methods for enhancing the reaction that generates carbon dioxide from formic acid include enhancing the catalytic activity of the enzyme itself that catalyzes the reaction, and increasing the expression level of the enzyme. It is preferable to increase it. Methods for increasing the expression level include, for example, introducing the enzyme gene from outside the host microorganism into the host microorganism, increasing the copy number of the gene, or increasing the promoter region upstream of the coding region of the gene. Examples include methods of modifying the ribosome binding sequence. These methods may be performed alone or in combination.

Further, in the present invention, it is preferable to enhance the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. Increasing the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA includes, for example, increasing the expression level of an enzyme that catalyzes this reaction. Methods for increasing the expression level include, for example, introducing the enzyme gene from outside the host microorganism into the host microorganism, increasing the copy number of the gene, or increasing the promoter region upstream of the coding region of the gene. Examples include methods of modifying the ribosome binding sequence. These methods may be performed alone or in combination, but the enzyme gene is transferred to the host microorganism's bacterial cells by the method described in WO2019/107516 (US Patent Application Publication No. 2020/291435A1). It is preferable to introduce it into the bacterial body from outside.

A specific example of 3-oxoadipyl-CoA reductase that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA is EC1.1.1.35 as 3-hydroxyacyl-CoA dehydrogenase. An enzyme classified as 3-hydroxybutyryl-CoA dehydrogenase, an enzyme classified as EC1.1.1.157, and an enzyme disclosed in WO2019/107516 (US Patent Application Publication No. 2020/291435A1). Enzymes that have 70% or more sequence identity to any of the amino acid sequences of SEQ ID NOs: 1 to 6 and 213, and have 3-oxoadipyl-CoA reductase activity. As specific examples, PaaH derived from Pseudomonas putida KT2440 strain (NCBI-Protein ID: NP_745425.1), Escherichia coli str. K-12 substr. PaaH derived from MG1655 strain (NCBI-Protein ID: NP_415913.1), DcaH derived from Acinetobacter baylyi ADP1 strain (NCBI-Protein ID: CAG68533.1), Serratia plymuthica PaaH (NCBI-Protein ID: WP_063197120) derived from NBRC102599 strain, Serratia marcescens ATCC13880 strain-derived polypeptide (NCBI-Protein ID: KFD11732.1).

Furthermore, from the viewpoint of productivity of 3-hydroxyadipic acid and/or α-hydromuconic acid, the genetically modified microorganism of the present invention is preferably a microorganism that does not metabolize sugar through the phosphoketolase pathway. Such microorganisms exist in the natural world and can be preferably used as a host when creating the genetic microorganism of the present invention that does not metabolize sugar through the phosphoketolase pathway. Furthermore, after creating the genetically modified microorganism of the present invention, a microorganism that does not metabolize sugar through the phosphoketolase pathway may be created by knocking out the phosphoketolase gene of the microorganism using a method well known to those skilled in the art.

By the way, US Patent Application Publication No. 2017/0298363A1 describes that adipic acid is produced using a non-natural microorganism that has improved energy efficiency by modifying the metabolism of a microorganism that has a phosphoketolase pathway. . This document states that while sugar metabolism in the glycolytic pathway produces NADH, sugar metabolism in the phosphoketolase pathway does not produce NADH. describes that the expression or activity of PDHc or pyruvate formate lyase and NAD(P)H-producing formate dehydrogenase is enhanced in order to improve NADH deficiency in the metabolism of the microorganism. Here, since adipic acid is a compound in a more reduced state than 3-hydroxyadipic acid and α-hydromuconic acid, those skilled in the art will understand that when producing 3-hydroxyadipic acid and/or α-hydromuconic acid, It is believed that enhancing the reaction of PDHc and/or pyruvate formate lyase will lead to an imbalance in redox balance and reduce the accumulated concentration of the compound. The effect of increasing the accumulated concentration of 3-hydroxyadipic acid and/or α-hydromuconic acid is an unexpected effect for those skilled in the art.

Examples of microorganisms that inherently have the ability to produce 3-hydroxyadipic acid include the following microorganisms.

Escherichia genus such as Escherichia fergusonii and Escherichia coli.

Serratia grimesii, Serratia ficaria, Serratia fonticoia, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia Serratia spp., such as A nematodiphila.

Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotoformans, Pseudomonas chlororaphis subsp. Psuedomonas spp., such as Psuedomonas aureofaciens.

Hafnia spp. such as Hafnia alvei.

Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, Corynebacterium glutamicum, etc. The genus Corynebacterium.

Bacillus genus such as Bacillus badius, Bacillus megaterium, 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 such as Acinetobacter baylyi and Acinetobacter radiores Istens.

Alcaligenes spp., such as Alcaligenes faecalis.

Genus Nocardioides such as Nocardioides albus.

Brevibacterium genus such as Brevibacterium iodinum.

Delftia spp., such as Delftia acidovorans.

Shimwellia genus such as Shimwellia blattae.

Aerobacter genus such as Aerobacter cloacae.

Rhizobium genus such as Rhizobium radiobacter.

Among the microorganisms that inherently have the ability to produce 3-hydroxyadipic acid, the present invention uses microorganisms that do not metabolize sugar through the phosphoketolase pathway, such as Escherichia, Serratia, Hafnia, Corynebacterium, Brevibacterium, Shimwellia, Microorganisms belonging to the genus Aerobacter are preferred, and microorganisms belonging to the genus Escherichia or Serratia are more preferably used.

Microorganisms that are presumed to have the ability to produce α-hydromuconic acid include the following microorganisms.

Escherichia genus such as Escherichia fergusonii and Escherichia coli.

Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serrati Serratia spp., such as A nematodiphila.

Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, Pseudomonas chlororaphis subsp. Psuedomonas spp., such as Psuedomonas aureofaciens.

Hafnia spp. such as Hafnia alvei.

Bacillus genus such as Bacillus badius.

Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus numazuensis, Cupriavidus oxalaticus.

Acinetobacter genus such as Acinetobacter baylyi and Acinetobacter radioresistens.

Alcaligenes spp., such as Alcaligenes faecalis.

Delftia spp., such as Delftia acidovorans.

Shimwellia genus such as Shimwellia blattae.

Among the microorganisms that inherently have the ability to produce α-hydromuconic acid, in the present invention, microorganisms of the genus Escherichia, Serratia, Hafnia, and Shimwellia, which do not metabolize sugar through the phosphoketolase pathway, are preferable, and the genus Escherichia or the genus Serratia is preferred. Microorganisms belonging to the following are more preferably used.

If the genetically modified microorganism of the present invention does not originally have the ability to produce 3-hydroxyadipic acid, appropriate combinations of nucleic acids encoding enzymes that catalyze reactions A, B, and D are introduced into the microorganism. In addition, if the ability to produce α-hydromuconic acid is not originally present, the enzyme encoding the enzyme that catalyzes reactions A, B, C, and E can be added. The ability to produce these can be imparted by introducing appropriate combinations of nucleic acids into microorganisms.

Microorganisms that can be used as hosts for obtaining genetically modified microorganisms in the present invention are not particularly limited as long as they can be genetically modified and have the ability to produce 3-hydroxyadipic acid and/or α-hydromuconic acid. The microorganisms may be microorganisms with or without the genus Escherichia, genus Serratia, genus Hafnia, genus Psuedomonas, genus Corynebacterium, genus Bacillus, genus Streptomyces, genus Cupriavidus, Acinetobact er, Alcaligenes, Brevibacterium, Delftia In the genera Shimwellia, Aerobacter, Rhizobium, Thermobifida, Clostridium, Schizosaccharomyces, Kluyveromyces, Pichia and Candida. Microorganisms belonging to the genus Escherichia, Serratia, Hafnia, and Pseudomonas are more preferred. Preferably, microorganisms belonging to the genus Escherichia or Serratia are particularly preferred.

The method of introducing a gene in order to create the genetically modified microorganism of the present invention is not particularly limited. Methods such as gene integration can be used.

The number of genes to be introduced may be one or more. Furthermore, gene introduction and expression enhancement may be combined.

When a gene to be expressed in the present invention is integrated into an expression vector or a host microorganism genome, the expression vector or nucleic acid for genome integration preferably comprises a promoter, a ribosome binding sequence, a gene to be expressed, and a transcription termination sequence. Furthermore, a gene that controls promoter activity may be included.

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

In the present invention, when introducing a gene or enhancing gene expression using an expression vector, there is no particular limitation as long as it can autonomously replicate in the microorganism, but examples include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector. , pRSF vector, pCDF vector, pACYC vector, and derivatives of the above-mentioned vectors.

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

The method for introducing the expression vector or the nucleic acid for genome integration is not particularly limited as long as it is a method for introducing nucleic acids into microorganisms, but examples include the calcium ion method (Journal of Molecular Biology, 53, 159 (1970)), the electroporation method ( NM Calvin, PC Hanawalt. J. Bacteriol, 170 (1988), pp. 2796-2801).

The genetically modified microorganism of the present invention is cultured in a medium, preferably a liquid medium, containing a carbon source that can be used by normal microorganisms as a fermentation raw material. In addition to the carbon source that can be used by the genetically modified microorganism, a medium containing a nitrogen source, an inorganic salt, and, if necessary, an appropriate amount of organic micronutrients such as amino acids and vitamins is used. Either a natural medium or a synthetic medium can be used as long as it contains the above-mentioned nutrient source.

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 taken in by a microorganism from outside the cell or produced from another chemical compound within the cell into another chemical compound through an enzymatic reaction. Saccharides can be preferably used as the carbon source. Specific examples of sugars include monosaccharides such as glucose, sucrose, fructose, galactose, mannose, xylose, and arabinose, disaccharides and polysaccharides in which these monosaccharides are bonded, and starch saccharification solutions containing these, molasses, and cellulose. Examples include containing biomass saccharification liquid.

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 appropriately set depending on the type of carbon source. The preferred concentration of glucose is 5-300 g/L.

Examples of the nitrogen source used for culturing the genetically modified microorganism include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other auxiliary organic nitrogen sources such as oil cake, soybean hydrolyzate, Casein decomposition products, other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermentation microbial cells and their hydrolysates, etc. can be used. The concentration of the nitrogen source in the medium is not particularly limited, but is preferably 0.1 to 50 g/L.

As the inorganic salts used for culturing the genetically modified microorganism, for example, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, etc. can be appropriately added and used.

The culture conditions of the genetically modified microorganism for producing 3-hydroxyadipic acid and/or α-hydromuconic acid include a medium with the above-mentioned component composition, culture temperature, stirring speed, pH, aeration amount, inoculation amount, etc. Adjust or select settings as appropriate depending on the type of modified microorganism and external conditions.

The pH range for culturing 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.

The range of aeration conditions during culture is not particularly limited as long as 3-hydroxyadipic acid and/or α-hydromuconic acid can be produced. Preferably, oxygen remains in the gas phase and/or liquid phase within.

If foaming occurs in liquid culture, antifoaming agents such as mineral oil, silicone oil, and surfactants can be appropriately added to the medium.

After 3-hydroxyadipic acid and/or α-hydromuconic acid are produced in recoverable amounts in the culture of the microorganism, the produced products can be recovered. Recovery, e.g. isolation, of the produced product can be carried out according to the general method of stopping the culture when the accumulated amount has increased to an appropriate level and collecting the fermentation product from the culture. . Specifically, after separating bacterial cells by centrifugation, filtration, etc., the product is isolated from the culture by column chromatography, ion exchange chromatography, activated carbon treatment, crystallization, membrane separation, distillation, etc. be able to. More specifically, methods include adding an acid component to the salt of the product and collecting the precipitate, and removing water from the culture using a reverse osmosis membrane or evaporator to increase the concentration of the product. After increasing the temperature, the product and/or salt crystals of the product are precipitated by cooling crystallization or adiabatic crystallization, and the product and/or salt crystals of the product are separated by centrifugation or filtration. Examples of methods include 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. It is not limited. Further, these recovery methods can be appropriately selected and optimized depending on the physical properties of the product.

The present invention will be specifically described below with reference to Examples. Note that various analyzes in this example were conducted according to the following methods.

[Quantitative analysis of 3-hydroxyadipic acid and α-hydromuconic acid]
The supernatant obtained by centrifuging the bacterial cells from the culture solution is subjected to membrane treatment using Millex-GV (0.22 μm, PVDF, manufactured by Merck), and the permeate is analyzed by LC-MS/MS under the following conditions. The accumulated concentrations of 3-hydroxyadipic acid and α-hydromuconic acid in the culture supernatant were quantified.
・HPLC: 1290Infinity (manufactured by Agilent Technologies)
Column: Synergi hydro-RP (manufactured by 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: 0.3 mL/min Column temperature: 40°C
LC detector: 1260DAD VL+ (210nm)
・MS/MS: Triple-Quad LC/MS (manufactured by Agilent Technologies)
Ionization method: ESI negative mode.

(Reference Example 1) Reaction to produce 3OA-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (reaction A), reaction to produce 3HA-CoA from 3OA-CoA (reaction B), 3HA-CoA to 3HA-CoA (reaction B), - Preparation of a plasmid expressing enzymes that catalyze the reaction of producing hydroxyadipic acid (reaction D) and the reaction of producing α-hydromuconic acid from HMA-CoA (reaction E) Vector pBBR1MCS-2 capable of autonomous replication in E. coli (ME Kovach, (1995), Gene 166:175-176) was digested with XhoI to obtain pBBR1MCS-2/XhoI. In order to incorporate a constitutive expression promoter into the vector, Escherichia coli str. K-12 substr. Using the genomic DNA of MG1655 as a template, primers for PCR amplification of the upstream region 200b (SEQ ID NO: 8) of gapA (NCBI-GeneID: NC_000913.3) were designed (SEQ ID NOs: 9 and 10), and a PCR reaction was performed according to a conventional method. went. The obtained fragment and pBBR1MCS-2/XhoI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant E. coli strain, and the nucleotide sequence was confirmed by a conventional method, and the plasmid was designated as pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was cut with ScaI to obtain pBBR1MCS-2::Pgap/ScaI. In order to amplify the gene encoding the enzyme that catalyzes reaction A, primers were designed to PCR amplify the full length of the acyltransferase gene pcaF (NCBI-Gene ID: 1041755) using the genomic DNA of Pseudomonas putida strain KT2440 as a template ( SEQ ID NO: 11, 12), PCR reaction was performed according to a conventional method. The obtained fragment and pBBR1MCS-2::Pgap/ScaI were ligated using "In-Fusion HD Cloning Kit" and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the nucleotide sequence was confirmed by a conventional method, and the plasmid was designated as pBBR1MCS-2::AT. Subsequently, pBBR1MCS-2::AT was cut with HpaI to obtain pBBR1MCS-2::AT/HpaI. In order to amplify the genes encoding the enzymes that catalyze reactions D and E, we used the genomic DNA of Pseudomonas putida strain KT2440 as a template to amplify continuous sequences containing the full length of the CoA transferase genes pcaI and pcaJ (NCBI-Gene ID: 1046613, 1046612). Primers for PCR amplification of the sequence were designed (SEQ ID NOs: 13 and 14), and a PCR reaction was performed according to a conventional method. The obtained fragment and pBBR1MCS-2::AT/HpaI were ligated using "In-Fusion HD Cloning Kit" and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the plasmid whose base sequence was confirmed by a conventional method was designated as pBBR1MCS-2::ATCT.

pBBR1MCS-2::ATCT was cut with ScaI to obtain pBBR1MCS-2::ATCT/ScaI. Using the genomic DNA of Serratia marcescens ATCC13880 strain as a template, primers for amplifying the nucleic acid set forth in SEQ ID NO: 15 were designed (SEQ ID NOs: 16 and 17), and a PCR reaction was performed according to a conventional method. The obtained fragment and pBBR1MCS-2::ATCT/ScaI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and the nucleotide sequence was confirmed by a conventional method, and the plasmid was designated as pBBR1MCS-2::ATCTOR.

(Reference Example 2) Preparation of Escherichia coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E Escherichia coli str. K-12 substr. MG1655 was inoculated into 5 mL of LB medium and cultured with shaking at 30°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium, and cultured with shaking at 30° C. for 2 hours. After cooling the culture solution on ice for 20 minutes, the bacterial 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 150 ng of pBBR1MCS-2::ATCTOR, and then cooled on ice for 10 minutes in an electroporation cuvette. After performing electroporation (3 kV, 200 Ω, 25 μF) using Gene pulser (manufactured by Bio-rad), 1 mL of SOC medium was immediately added and cultured with shaking at 30° C. for 1 hour. 50 μL was applied to an LB agar medium containing 25 μg/mL of kanamycin and incubated at 30° C. for 1 day. The obtained strain was named Ec/3HA.

(Reference Example 3) Production of E. coli with deletion of the pyruvate dehydrogenase complex transcription repressor gene (pdhR) Deletion of pdhR (NCBI-Gene ID: 944827), a gene encoding the pyruvate dehydrogenase complex transcription repressor gene of Escherichia coli E. coli in which the expression level of PDHc was increased was produced by doing this.

PCR was performed using pKD4 as a template and oligo DNAs of SEQ ID NOs: 18 and 19 as primers to obtain a PCR fragment for pdhR deficiency. Escherichia coli str. K-12 substr. After introducing pKD46 into MG1655, a PCR fragment for pdhR deletion was introduced. Colony direct PCR was performed using the obtained kanamycin-resistant strain, and deletion of the target gene and insertion of the kanamycin-resistant gene were confirmed from the band length. As primers, oligo DNAs having SEQ ID NOs: 20 and 22 were used.

Subsequently, pKD46 was eliminated to obtain an ampicillin-sensitive strain. pCP20 was introduced into an ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony direct PCR was performed using the obtained strain, and the loss of the kanamycin resistance gene was confirmed from the band length. As primers, oligo DNAs of SEQ ID NOs: 21 and 22 were used. pCP20 was shed from the kanamycin-sensitive strain. The obtained strain was designated as EcΔR.

(Reference Example 4) Production of pdhR-deficient E. coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E The EcΔR strain produced in Reference Example 3 was treated in the same manner as in Reference Example 2. The plasmid pBBR1MCS-2::ATCTOR prepared in Example 1 was introduced. The obtained strain was named EcΔR/3HA.

(Reference Example 5) Preparation of gapA promoter introduction plasmid (Preparation of pCDF::Pgap)
Expression vector pCDF-1b (manufactured by Merck Millipore) capable of autonomous replication in E. coli was cut with BamHI to obtain pCDF/BamHI. In order to incorporate a constitutive expression promoter into the vector, Escherichia coli str. K-12 substr. Using the genomic DNA of MG1655 as a template, primers for PCR amplification of the upstream region 200b (SEQ ID NO: 8) of gapA (NCBI-GeneID: NC_000913.3) were designed (SEQ ID NOs: 23 and 24), and a PCR reaction was performed according to a conventional method. went. The obtained fragment and pCDF/BamHI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant E. coli strain, and the base sequence was confirmed by a conventional method, and the plasmid was designated as pCDF::Pgap.

(Preparation of pMW119::Pgap)
Expression vector pMW119 capable of autonomous replication in E. coli was cut with SacI to obtain pMW119/SacI. In order to incorporate a constitutive expression promoter into the vector, Escherichia coli str. K-12 substr. Using MG1655 genomic DNA as a template, primers for PCR amplification of the upstream region 200b (SEQ ID NO: 8) of gapA (NCBI-GeneID: NC_000913.3) were designed (SEQ ID NOs: 25 and 26), and a PCR reaction was performed according to a conventional method. went. The obtained fragment and pMW119/SacI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant E. coli strain, and the base sequence was confirmed by a conventional method, and the plasmid was designated as pMW119::Pgap.

(Reference Example 6) Preparation of NADH-producing formate dehydrogenase (FDH1) expression plasmid The nucleotide sequence encoding FDH1 was introduced into the gapA promoter introduction plasmids pCDF::Pgap and pMW119::Pgap prepared in Reference Example 5, and the gapA promoter FDH1 expression plasmids pCDF::FDH1 and pMW119::FDH1 were created, which were designed to express FDH1 under control.

(Preparation of pCDF::FDH1)
pCDF::Pgap prepared in Reference Example 5 was cleaved with BamHI to obtain pCDF::Pgap/BamHI. A nucleotide sequence (SEQ ID NO: 27) encoding the NADH-producing formate dehydrogenase (FDH1) of Candida boidinii was gene synthesized, primers for PCR amplification of the region were designed (SEQ ID NOs: 28, 29), and PCR was performed according to a conventional method. The reaction was carried out. The obtained fragment and pCDF::Pgap/BamHI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) to create plasmid pCDF::FDH1. The base sequence of the plasmid was confirmed by a conventional method.

(Preparation of pMW119::FDH1)
In order to incorporate the gapA promoter and FDH1-containing region sequence of pCDF::FDH1 into the expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.) capable of autonomous replication in E. coli, pCDF::FDH1 and pMW119 were cut with XbaI and SacI. The obtained fragments were ligated together to create plasmid pMW119::FDH1. The base sequence of the plasmid was confirmed by a conventional method.

(Comparative Example 1) Production of E. coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E and a gapA promoter-introducing plasmid Ec/3HA was inoculated into 5 mL of LB medium containing 25 μg/mL of kanamycin, It was cultured with shaking at 30°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium containing 25 μg/mL of kanamycin, and cultured with shaking at 30° C. for 2 hours. After cooling the culture solution on ice for 20 minutes, the bacterial 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 150 ng of pMW119::Pgap, and then cooled on ice for 10 minutes in an electroporation cuvette. After performing electroporation (3 kV, 200 Ω, 25 μF) using Gene pulser (manufactured by Bio-rad), 1 mL of SOC medium was immediately added and cultured with shaking at 30° C. for 1 hour. 50 μL was applied to an LB agar medium containing 25 μg/mL of kanamycin and 100 μg/mL of ampicillin, and incubated at 30° C. for 1 day. The obtained strain was named Ec/3HA_pMW.

(Comparative Example 2) Production test of 3-hydroxyadipic acid in Escherichia coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E and a gapA promoter-introduced plasmid Medium I (Bacto tryptone (Difco Laboratories) adjusted to pH 7) Bacto yeast extract (manufactured by Difco Laboratories) 5 g/L, sodium chloride 5 g/L, kanamycin 25 μg/mL, ampicillin 100 μg/mL) 5 mL (φ18 mm glass test tube, aluminum stopper), comparative example One platinum loop of Ec/3HA_pMW prepared in 1 was inoculated and cultured with shaking at 30° C. and 120 min ⁻¹ for 24 hours. 0.25 mL of the culture solution was adjusted to pH 6.5 in 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 .7mg/L, calcium chloride 0.33mg/L, sodium chloride 1.25g/L, Bacto tryptone 2.5g/L, Bacto yeast extract 1.25g/L, kanamycin 25μg/mL, ampicillin 100μg/mL) 5mL ( The cells were added to a φ18 mm glass test tube, aluminum stopper) and cultured with shaking at 30°C. The concentrations of 3-hydroxyadipic acid accumulated in the culture supernatant are shown in Table 1.

(Comparative Example 3) Production of pdhR-deficient E. coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E and a gapA promoter-introduced plasmid Using the same method as in Comparative Example 1 for EcΔR/3HA Plasmid pMW119::Pgap produced in Example 5 was introduced to produce EcΔR/3HA_pMW.

(Comparative Example 4) 3-Hydroxyadipic acid production test using pdhR-deficient E. coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E and a gapA promoter-introduced plasmid EcΔR/3HA_pMW produced in Comparative Example 3 , and cultured in the same manner as in Comparative Example 2. The concentrations of 3-hydroxyadipic acid accumulated in the culture supernatant are shown in Table 1.

(Example 1) Production of pdhR-deficient E. coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E and FDH1 expression plasmid. The FDH1 expression plasmid pMW119::FDH1 produced in Example 6 was introduced to produce EcΔR/3HA_FDH1.

(Example 2) Production test of 3-hydroxyadipic acid using pdhR-deficient E. coli into which plasmids expressing enzymes that catalyze reactions A, B, D, and E and FDH1 expression plasmid were introduced EcΔR prepared in Example 1 /3HA_FDH1 was cultured in the same manner as in Comparative Example 2. The concentration of 3-hydroxyadipic acid accumulated in the culture supernatant is shown in Table 1. In Escherichia coli, which has the ability to produce 3-hydroxyadipic acid, increasing the expression level of PDHc due to deletion of pdhR By strengthening the reaction that generates acetyl-CoA from pyruvate and increasing the expression level of FDH1, the accumulated concentration of 3-hydroxyadipic acid is significantly increased by strengthening the reaction that generates carbon dioxide from formic acid. I understand.

(Reference Example 7) Preparation of a plasmid expressing PDHc subunit (Lpd) Expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.) capable of autonomous replication in E. coli was cut with SacI and KpnI to obtain pMW119/SacI and KpnI.

In order to amplify the gene encoding Lpd from Escherichia coli, Escherichia coli str. K-12 substr. Using MG1655 genomic DNA as a template, primers were designed to PCR amplify the full-length lpd (NCBI-GeneID: 944854) and a region including 0.5 kb on the 5' side and 0.1 kb on the 3' side (SEQ ID NOs: 30 and 31). ), PCR reaction was performed according to a conventional method. The obtained fragment, pMW119/SacI, and KpnI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) to produce pMW119::LPD. The base sequence of the plasmid was confirmed by a conventional method.

(Reference Example 8) Preparation of a plasmid expressing pyruvate formate lyase (PFL) A nucleotide sequence encoding PFL was introduced into the gapA promoter introduction plasmid pMW119::Pgap prepared in Reference Example 5, and PFL was produced under the control of the gapA promoter. A PFL expression plasmid pMW119::PFL was created, which was designed to express pMW119::PFL.

pMW119::Pgap prepared in Reference Example 5 was cleaved with SphI to obtain pMW119::Pgap/SphI. To amplify the gene encoding PFL, Escherichia coli str. K-12 substr. Using MG1655 genomic DNA as a template, primers were designed to PCR amplify the region containing the full lengths of pflB (NCBI-Gene ID: 945514) and pflA (NCBI-Gene ID: 945517) (SEQ ID NOs: 32 and 33), and PCR was carried out according to a conventional method. The reaction was carried out. The obtained fragment and pMW119::Pgap/SphI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) to create a plasmid pMW119::PFL. The base sequence of the plasmid was confirmed by a conventional method.

(Comparative Example 5) Preparation of E. coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E and two types of gapA promoter-introduced plasmids. The cells were inoculated into 5 mL of LB medium containing 100 μg/mL, and cultured with shaking at 30° C. for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium containing 25 μg/mL of kanamycin and 100 μg/mL of ampicillin, and cultured with shaking at 30° C. for 2 hours. After cooling the culture solution on ice for 20 minutes, the bacterial 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 150 ng of pCDF::Pgap, and then cooled on ice for 10 minutes in an electroporation cuvette. After performing electroporation (3 kV, 200 Ω, 25 μF) using Gene pulser (manufactured by Bio-rad), 1 mL of SOC medium was immediately added and cultured with shaking at 30° C. for 1 hour. 50 μL was applied to an LB agar medium containing 25 μg/mL of kanamycin, 100 μg/mL of ampicillin, and 50 μg/mL of streptomycin, and incubated at 30° C. for 1 day. The obtained strain was named Ec/3HA_pMWp_CDF.

(Comparative Example 6) Production test of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E and two types of gapA promoter-introduced plasmids Comparative Example 5 The prepared Ec/3HA_pMW_pCDF was cultured in the same manner as in Comparative Example 2 except that the medium contained 50 μg/mL of streptomycin. Table 2 shows the concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant.

(Comparative Example 7) Preparation of E. coli cells containing plasmids expressing enzymes that catalyze reactions A, B, D, and E, Lpd expression plasmid, and gapA promoter introduction plasmid In the same manner as in Comparative Example 1, Ec/3HA was Ec/3HA_LPD was prepared by introducing the plasmid pMW119::LPD prepared in Reference Example 7 to increase the expression level of Lpd, thereby increasing the expression level of PDHc. Subsequently, the plasmid pCDF::Pgap produced in Reference Example 5 was introduced into Ec/3HA_LPD in the same manner as in Comparative Example 5 to produce Ec/3HA_LPD_pCDF.

(Comparative Example 8) Production test of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E, an Lpd expression plasmid, and a gapA promoter introduction plasmid Comparative Example 7 The Ec/3HA_LPD_pCDF prepared in was cultured in the same manner as in Comparative Example 6. Table 2 shows the concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant.

(Example 3) Production of E. coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E, Lpd expression plasmid, and FDH1 expression plasmid Using the same method as Comparative Example 5, Ec/3HA_LPD was added to Reference Example The plasmid pCDF::FDH1 prepared in step 6 was introduced to produce Ec/3HA_LPD_FDH1 in which the expression levels of PDHc and FDH1 were increased.

(Example 4) Production test of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E, Lpd expression plasmid, and FDH1 expression plasmid In Example 3 The prepared Ec/3HA_LPD_FDH1 was cultured in the same manner as in Comparative Example 6. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant are shown in Table 2. It was found that by increasing the expression level of 3-hydroxyadipic acid and α-hydromuconic acid, the accumulated concentrations of 3-hydroxyadipic acid and α-hydromuconic acid were significantly improved.

(Comparative Example 9) Production of E. coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E, a PFL expression plasmid, and a gapA promoter introduction plasmid. Using the same method as in Comparative Example 1, use Ec/3HA as a reference. The plasmid pMW119::PFL prepared in Example 8 was introduced to create Ec/3HA_PFL with increased expression level of PFL. Subsequently, the plasmid pCDF::Pgap produced in Reference Example 5 was introduced into Ec/3HA_PFL in the same manner as in Comparative Example 5 to produce Ec/3HA_PFL_pCDF.

(Comparative Example 10) Production test of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E, a PFL expression plasmid, and a gapA promoter introduction plasmid Comparative Example 9 The Ec/3HA_PFL_pCDF prepared in was cultured in the same manner as in Comparative Example 6. Table 2 shows the concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant.

(Example 5) Production of E. coli having a plasmid expressing enzymes that catalyze reactions A, B, D, and E, a PFL expression plasmid, and an FDH1 expression plasmid In the same manner as in Comparative Example 5, Ec/3HA_PFL was added to the reference example. The plasmid pCDF::FDH1 prepared in step 6 was introduced to produce Ec/3HA_PFL_FDH1.

(Example 6) Production test of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia coli having plasmids expressing enzymes that catalyze reactions A, B, D, and E, PFL expression plasmid, and FDH1 expression plasmid In Example 5 The prepared Ec/3HA_PFL_FDH1 was cultured in the same manner as in Comparative Example 6. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant are shown in Table 2. By increasing the expression level of FDH1, the reaction to generate acetyl-CoA from pyruvate is strengthened, and by increasing the expression level of FDH1, the reaction to generate carbon dioxide from formic acid is strengthened. It was found that the accumulated concentrations of acid and α-hydromuconic acid were significantly improved.

Claims (12)

A gene that enhances the reaction of producing acetyl-CoA from pyruvic acid and the reaction of producing carbon dioxide from formic acid in a microorganism capable of producing 3-hydroxyadipic acid and/or α-hydromuconic acid. Modified microorganisms. The genetic modification according to claim 1, wherein the enhancement of the reaction for producing acetyl-CoA from pyruvate is enhancement of the reaction catalyzed by a pyruvate dehydrogenase complex and/or the enhancement of the reaction catalyzed by pyruvate formate lyase. Microorganisms. The genetically modified microorganism according to claim 2, wherein the reaction catalyzed by the pyruvate dehydrogenase complex is enhanced by increasing the expression level of the pyruvate dehydrogenase complex and/or enhancing the activity of the pyruvate dehydrogenase complex. 4. The genetically modified microorganism according to claim 3, wherein the expression level of the pyruvate dehydrogenase complex is increased by reducing the function of a pyruvate dehydrogenase complex transcription repressor. 3. The genetically modified microorganism according to claim 2, wherein the reaction catalyzed by pyruvate formate lyase is enhanced by increasing the expression level of pyruvate formate lyase. The genetically modified microorganism according to any one of claims 1 to 5, wherein the enhancement of the reaction for producing carbon dioxide from formic acid is an increase in the expression level of an enzyme that catalyzes the reaction for producing carbon dioxide from formic acid. The genetically modified microorganism according to any one of claims 1 to 6, wherein the enhancement of the reaction for producing carbon dioxide from formic acid is enhancement of the reaction catalyzed by NADH-producing formate dehydrogenase. The genetically modified microorganism according to any one of claims 1 to 7, which further enhances the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. The genetically modified microorganism according to any one of claims 1 to 8, which is a microorganism that does not metabolize sugar through the phosphoketolase pathway. A method for producing 3-hydroxyadipic acid and/or α-hydromuconic acid, comprising the step of culturing the genetically modified microorganism according to any one of claims 1 to 9. A step of genetically modifying a microorganism capable of producing 3-hydroxyadipic acid and/or α-hydromuconic acid to enhance the reaction of producing acetyl-CoA from pyruvic acid, and the reaction of producing carbon dioxide from formic acid. A method for producing a genetically modified microorganism, including a step of strengthening. 12. The method for producing a genetically modified microorganism according to claim 11, further comprising the step of enhancing the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA through genetic modification.