Gene-modified microorganism for producing α-hydromuconic acid and / or adipic acid and method for producing said chemical product

Abstract

The present invention discloses a gene-modified microorganism capable of producing, with high productivity, an α-hydromuconic acid and / or an adipic acid. The gene-modified microorganism is achieved by using a microorganism which has the ability to produce an α-hydromuconic acid and / or an adipic acid, such that the activity of an enzyme which catalyzes a reaction from acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA to CoA is reinforced and such that the function of an organic phosphoric acid compound exporter is deleted or reduced.

Description

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

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

[0002] α-hydromuconic acid (IUPAC name: (E)-hex-2-enedioic acid) and adipic acid (IUPAC name: hexanedioic 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] As a document related to the production of a dicarboxylic acid having six carbon atoms using a microorganism, Patent Document 1 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 reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, and describes that the biosynthetic pathway for these substances goes through an enzymatic reaction that reduces 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.

[0004] Furthermore, in methods for producing chemical products using microorganisms, a method is known in which productivity of the chemical products is increased by deleting or reducing the function of exporters (transporters) possessed by the microorganisms and which excrete chemical products, which are metabolic products of the microorganisms, outside the cells. Patent Document 2 describes a method for producing 3-hydroxyadipic acid and / or α-hydromuconic acid using genetically modified microorganisms in which the function of dicarboxylic acid exporters such as YjjP, YjjB, YeeA, and YnfM is deleted or reduced, and describes that the productivity of these substances is improved by deleting or reducing the function of the dicarboxylic acid exporters.

[0005] WO2019 / 107516 WO2021 / 187533

[0006] An object of the present invention is to create a genetically modified microorganism capable of producing α-hydromuconic acid and / or adipic acid with high productivity, and a method for producing α-hydromuconic acid and / or adipic acid using the modified microorganism.

[0007] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they have found that, contrary to expectations based on the prior art, productivity of α-hydromuconic acid and / or adipic acid can be increased by deleting or reducing the function of an export carrier for organophosphate compounds possessed by a microorganism capable of producing α-hydromuconic acid and / or adipic acid, and have completed the present invention.

[0008] That is, the present invention provides the following (1) to (10): (1) A genetically modified microorganism capable of producing α-hydromuconic acid and / or adipic acid, in which the activity of an enzyme catalyzing the reaction of acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA and CoA is enhanced and the function of an organophosphate compound exporter is deleted or reduced. (2) The genetically modified microorganism according to (1), in which the deletion or reduction in the function of the organophosphate compound exporter is a deletion or reduction in the function of YhhS or a homolog thereof. (3) The genetically modified microorganism according to (2), in which the deletion or reduction in the function of YhhS or a homolog thereof is a deletion, loss-of-function mutation, or suppression of expression of the yhhS gene or a homolog thereof. (4) The genetically modified microorganism according to any one of (1) to (3), which has enhanced activity of an enzyme catalyzing the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA, the reaction of dehydrating 3-hydroxyadipyl-CoA to produce 2,3-dehydroadipyl-CoA, the reaction of reducing 2,3-dehydroadipyl-CoA to produce adipyl-CoA, the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA, and / or the reaction of producing adipic acid from adipyl-CoA. (5) The genetically modified microorganism according to any one of (1) to (4), wherein the microorganism belongs to the genus Escherichia. (6) A method for producing α-hydromuconic acid and / or adipic acid, which comprises a step of culturing the genetically modified microorganism according to (1). (7) A method for producing a genetically modified microorganism, the method comprising the steps of: enhancing the activity of an enzyme that catalyzes the reaction of acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA and CoA in a microorganism capable of producing α-hydromuconic acid and / or adipic acid through genetic modification; and deleting or reducing the function of an organophosphate compound exporter in the microorganism. (8) A method for producing adipic acid, the method comprising the steps of producing α-hydromuconic acid by the method described in (6) above; and reacting α-hydromuconic acid with hydrogen in the presence of a hydrogenation catalyst (hydrogenation step).(9) A method for producing a polyamide, comprising the steps of producing adipic acid by the method described in (6) or (8) and polycondensing the resulting adipic acid with a diamine. (10) The method for producing a polyamide described in (9), wherein the diamine is 1,4-butanediamine, 1,5-pentanediamine, and / or hexamethylenediamine. (11) α-hydromuconic acid and / or adipic acid obtained by culturing the genetically modified microorganism described in any one of (1) to (5). (12) Adipic acid obtained by a process comprising the steps of producing α-hydromuconic acid by the method described in (6) and reacting the resulting α-hydromuconic acid with hydrogen in the presence of a hydrogenation catalyst (hydrogenation process). (13) Polyamide obtained by a process comprising the steps of producing adipic acid by the method described in (6) or (8) and polycondensing the resulting adipic acid with a diamine. (14) The polyamide according to (13), wherein the diamine is 1,4-butanediamine, 1,5-pentanediamine, and / or hexamethylenediamine. (15) Fibers made from the polyamide according to (13).

[0009] The genetically modified microorganism can increase the productivity of α-hydromuconic acid and / or adipic acid compared to a parent strain of a microorganism in which the function of the organophosphate compound exporter is not modified.

[0010] Hereinafter, in this specification, α-hydromuconic acid may be abbreviated as HMA, and adipic acid may be abbreviated as ADA. Also, 3-oxoadipyl-CoA may be abbreviated as 3OA-CoA, 3-hydroxyadipyl-CoA may be abbreviated as 3HA-CoA, 2,3-dehydroadipyl-CoA may be abbreviated as HMA-CoA, and adipyl-CoA may be abbreviated as ADA-CoA. The enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA may be referred to as "3-oxoadipyl-CoA reductase." Furthermore, the protein encoded by the yhhS gene may be referred to as YhhS, the protein encoded by the mdtD gene as MdtD, and the protein encoded by the rhtB gene as RhtB. Furthermore, a nucleic acid encoding a functional polypeptide may be referred to as a gene.

[0011] The following Reaction Scheme 1 shows an example of a reaction pathway for a microorganism to produce α-hydromuconic acid and / or adipic acid. Here, Reaction A shows a reaction for producing 3-oxoadipyl-CoA and CoA from acetyl-CoA and succinyl-CoA. Reaction B shows a reaction for producing 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA. Reaction C shows a reaction for producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction D shows a reaction for producing adipyl-CoA from 2,3-dehydroadipyl-CoA. Reaction E shows a reaction for producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA. Reaction F shows a reaction for producing adipic acid from adipyl-CoA. Of these reaction pathways, microorganisms capable of producing α-hydromuconic acid and / or adipic acid are known to have an enzyme that catalyzes at least reaction A (see WO 2019 / 107516).

[0012]

[0013] The reaction for producing α-hydromuconic acid and / or adipic acid from 3-oxoadipyl-CoA preferably has the biosynthetic pathway shown in Reaction Scheme 1. In other words, 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).

[0014] Similarly, when the genetically modified microorganism of the present invention has the ability to produce adipic acid, the host microorganism for the genetically modified microorganism 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), the ability to produce adipyl-CoA from 2,3-dehydroadipyl-CoA (reaction D), and the ability to produce adipic acid from adipyl-CoA (reaction F).

[0015] The genetically modified microorganism of the present invention has a technical feature of improving the productivity of α-hydromuconic acid and / or adipic acid in a microorganism capable of producing α-hydromuconic acid and / or adipic acid by enhancing the activity of an enzyme in the reaction (reaction A) that produces 3-oxoadipyl-CoA and CoA from acetyl-CoA and succinyl-CoA, and by deleting or reducing the function of an organophosphate compound exporter. Note that "genetic modification" means artificially modifying a gene. Furthermore, "enhancing enzyme activity" and "deleting or reducing the function of an organophosphate compound exporter" mean doing these things artificially.

[0016] The genetically modified microorganism of the present invention has been genetically modified to eliminate or reduce the function of an organophosphate compound exporter. "Deleting or reducing the function of an organophosphate compound exporter" refers to deleting or reducing the organophosphate compound exporter activity. While the method for deleting or reducing the function of an organophosphate compound exporter is not particularly limited, a method that eliminates or reduces the expression of an organophosphate compound exporter is preferred. For example, this can be achieved by disrupting or deleting an organophosphate compound exporter gene or its homologous gene (hereinafter collectively referred to as "organophosphate compound exporter gene"), or the nucleotide sequence of the promoter or terminator region of the gene, by gene mutation treatment using a gene mutagen or ultraviolet irradiation, or by deleting part or all of the nucleotide sequence using site-directed mutagenesis, introducing a frameshift mutation into the nucleotide sequence, or 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 an organophosphate compound 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 a part or the whole of the base sequence of the organophosphate compound exporter gene is preferred.

[0017] Organophosphate compounds are compounds that have a structure in which phosphoric acid is bonded to organic compounds such as alcohols, carboxylic acids, and amino acids. Specific examples include phosphoserine, in which phosphoric acid is bonded to serine, an amino acid with a hydroxyl group, via an ester bond, and glyphosate, in which a phosphonomethyl group is substituted on the nitrogen atom of glycine.

[0018] Organophosphate compound exporters are membrane proteins that have the activity of exporting intracellular organophosphate compounds to the outside of cells. Specific examples of organophosphate compound exporters whose function is to be impaired or reduced in the present invention include phosphoserine exporters and / or glyphosate exporters.

[0019] Specific examples of phosphoserine exporters include the YhhS protein and homologs thereof, which are described in WO 2016 / 024771 as exporters of O-phosphoserine.

[0020] Specific examples of glyphosate excretion carriers include the YhhS protein and its homologs described in J. Ind. Microbiol Biotechnol. 2012 Apr; 39(4):641-7.

[0021] Organophosphate compounds such as phosphoserine and glyphosate are different from the substances on the reaction pathway to α-hydromuconic acid and adipic acid in Reaction Scheme 1. Therefore, the above-mentioned documents cited in the description of specific examples of phosphoserine efflux carriers and glyphosate efflux carriers do not describe or suggest the relationship between organophosphate compound efflux carriers such as YhhS and the productivity of α-hydromuconic acid or adipic acid by microorganisms. Therefore, it is an unexpected finding for those skilled in the art that the ability to produce α-hydromuconic acid and / or adipic acid, which are dicarboxylic acids having six carbon atoms, is improved by deleting or reducing the function of an organophosphate compound efflux carrier, as shown in the examples of this specification.

[0022] The organophosphate compound exporters whose functions are deleted or reduced in the present invention include YhhS, MdtD, RhtB, or homologs thereof, with YhhS or homologs thereof being preferred.

[0023] Hereinafter, YhhS and its homologues, which are suitable as organophosphate compound exporters whose functions are lost or reduced in the present invention, will be described in detail.

[0024] YhhS is a protein predicted to be a major facilitator superfamily (MFS)-type transporter, and a specific example thereof is YhhS derived from Escherichia coli str. K-12 substr. MG1655 (NCBI Protein ID: NP_417930, SEQ ID NO: 1). An example of a yhhS gene encoding the YhhS protein is yhhS derived from Escherichia coli str. K-12 substr. MG1655 (NCBI Gene ID: 947982, SEQ ID NO: 2). Furthermore, any protein can be used as YhhS if it consists of the amino acid sequence of SEQ ID NO: 1 with one or several amino acid substitutions, deletions, insertions, and / or additions, and has the function of improving the productivity of α-hydromuconic acid and / or adipic acid by deleting, deleting, and / or reducing the function of the amino acid sequence. Here, the range of "one or several" preferably means 10 or fewer, more preferably 5 or fewer, even more preferably 4 or fewer, and particularly preferably 1 or 2 or fewer. Furthermore, any protein can be used as YhhS if it consists of an amino acid sequence that has a sequence identity (defined below) of 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more to the amino acid sequence of SEQ ID NO: 1, and has the function of improving the productivity of α-hydromuconic acid and / or adipic acid by deleting or reducing the function of the amino acid sequence.

[0025] A YhhS homolog is a protein that has a high amino acid sequence identity with YhhS (usually 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more) and is predicted to have a function or structure similar to that of YhhS. Specific examples of such YhhS homologs include a YhhS homolog derived from Serratia grimesi NBRC13537 strain (SEQ ID NO: 31), a YhhS homolog derived from Salmonella enterica ATCC9150 strain (NCBI Protein ID: AAV79243, SEQ ID NO: 32), a YhhS homolog derived from Klebsiella pneumoniae MGH78578 strain (NCBI Protein ID: ABR79226, SEQ ID NO: 33), and a YhhS homolog derived from Pectobacterium carotovorum PC1 strain (NCBI Protein ID: ACT11150, SEQ ID NO: 34). Genes encoding these proteins include those derived from Serratia grimesi A yhhS homologue derived from the Salmonella enterica ATCC9150 strain (NCBI GeneID: CP000026, REGION: 3539049..3540266, SEQ ID NO: 36), a yhhS homologue derived from the Klebsiella pneumoniae MGH78578 strain (NCBI GeneID: CP000647, REGION: 4195189..4196409, SEQ ID NO: 37), a yhhS homologue derived from the Pectobacterium carotovorum PC1 strain (NCBI GeneID: CP000647, REGION: 4195189..4196409, SEQ ID NO: 37), a ...647, REGION: 4195189..4196409, SEQ ID NO: 37), a yhhS homologue derived from the Gene ID: CP001657, REGION: 113478. . 114677, SEQ ID NO: 38).

[0026] YhhS homologs can be easily obtained by, for example, using the amino acid sequence of YhhS as a query sequence, performing a BLAST (Basic Local Alignment Search Tool) search on public databases such as NCBI (National Center for Biotechnology Information) or KEGG (Kyoto Encyclopedia of Genes and Genomes), and referencing the gene encoding the corresponding sequence. Alternatively, they can be obtained by PCR using oligonucleotides prepared based on the nucleotide sequence of the gene encoding YhhS or its homolog as primers and the genomic DNA of another organism as a template.

[0027] As used herein, "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 start point) or base sequence (including the start codon) in the optimal alignment when two amino acid sequences or base sequences are aligned with or without gaps, and is calculated using formula (1). In formula (1), the shorter sequence length to be compared is 100 amino acids or 300 bases or more, and sequence identity is not defined for sequences less 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. According to formula (1), the sequence identities between the amino acid sequences of SEQ ID NOs: 32, 33, 34, and 31 relative to SEQ ID NO: 1 were calculated using the Genetyx function (% Identity Matrix) to yield 90.9, 84.4, 70.2, and 61.4%, respectively.

[0028] Sequence identity (%)=number of matches (gaps not counted) / length of shorter sequence (length not including gaps at both ends)×100 Formula (1).

[0029] The YhhS homologue whose function is to be deleted or reduced in the present invention preferably has the activity of effluxing organophosphate compounds. Whether the protein has the activity of effluxing organophosphate compounds can be determined by known methods. For example, the homologue gene can be introduced into Escherichia coli CA07-0012 strain (WO 2016 / 024771) capable of producing phosphoserine and verified to improve phosphoserine productivity, or by introducing the homologue gene into Escherichia coli BL21 DE3 strain using the method described in J. Ind. Microbiol Biotechnol. 2012 Apr;39(4):641-7 and verified to grow in the presence of glyphosate.

[0030] MdtD (also known as YegB) is a protein belonging to the major facilitator superfamily (MFS) known as putative multidrug resistance protein. Specific examples include MdtD derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Protein ID: NP_416581), and examples of genes encoding MdtD include mdtD derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Gene ID: 946601).

[0031] RhtB (also known as YigK) is a protein known as homoserine / homoserine lactone efflux protein. A specific example of RhtB is RhtB derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Protein ID: YP_026265). An example of a gene encoding RhtB is rhtB derived from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Gene ID: 948316).

[0032] Homologs of MdtD and RhtB are proteins that have a high degree of sequence identity between the amino acid sequences of the respective proteins and are predicted to have a similar function or structure to the respective proteins. MdtD (NCBI Protein ID: NP_416581) derived from Escherichia coli str. K-12 substr. MG1655 strain, RhtB (NCBI Protein ID: YP_026265) derived from Escherichia coli str. K-12 substr. MG1655 strain. Specifically, the sequence identity is 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, particularly preferably 95% or more, and has the function of improving the productivity of 3HA and / or 3OA by increasing the expression level. These homologs can be easily obtained by performing a BLAST (Basic Local Alignment Search Tool) search on public databases such as NCBI (National Center for Biotechnology Information) or KEGG (Kyoto Encyclopedia of Genes and Genomes) using the amino acid sequence of MdtD or RhtB as a query sequence and referencing the gene encoding the corresponding sequence. Alternatively, oligonucleotides prepared based on the base sequence of the gene encoding MdtD or RhtB, or a homolog thereof, can be used as primers for PCR using the genomic DNA of another organism as a template.

[0033] The three-dimensional structures of the proteins described in the present invention can be found using public databases such as Uniprot (https: / / www.uniprot.org / ) and AlphaFold Protein Structure Database (https: / / alphafold.ebi.ac.uk / ). For example, YhhS from Escherichia coli str. K-12 is registered as P37621 on Uniprot and has 12 transmembrane domains each consisting of an α-helical structure. These transmembrane domains are composed of regions of the amino acid sequence at positions 19 to 39, 47 to 67, 85 to 105, 107 to 127, 156 to 176, 178 to 198, 224 to 244, 252 to 272, 283 to 303, 309 to 329, 344 to 364, and 366 to 386. The transmembrane domain in an efflux carrier is involved in efflux activity, and when the amino acid sequence constituting this domain is highly conserved (when the sequence identity of the amino acid sequence is 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and particularly preferably 95% or more), it can be determined that the domain has efflux activity against the same compound.

[0034] The method for enhancing the enzyme activity that catalyzes the reaction (reaction A) producing 3-oxoadipyl-CoA and CoA from acetyl-CoA and succinyl-CoA is known per se, and can be preferably carried out according to the method described in WO 2017 / 209102, for example.

[0035] Enzymes that have the activity of catalyzing the reaction of producing 3-oxoadipyl-CoA and CoA from acetyl-CoA and succinyl-CoA include acetyl-CoA acetyltransferase, β-ketoacyl-CoA acyltransferase, 3-oxoadipyl-CoA acyltransferase, β-ketoadipyl-CoA acyltransferase, acetyl-CoA C-acetyltransferase, acetoacetyl-CoA thiolase, and beta-acetoacetyl coenzyme A. thiolase, 2-methylacetoacetyl-CoA thiolase, 3-oxothiolase, acetyl coenzyme A thiolase, acetyl-CoA acetyltransferase, acetyl-CoA: N-acetyltransferase, acetyl-CoA C-acyltransferase, beta-ketothiolase, 3-ketoacyl-CoA thiolase, beta-ketoacyl coenzyme A thiolase, beta-ketoacyl-CoA thiolase, beta-ketoadipyl coenzyme A thiolase, beta-ketoadipyl-CoA thiolase, 3-ketoacyl coenzyme A thiolase, 3-ketoacyl thiolase, 3-ketothiolase, 3-oxoacyl-CoA thiolase, 3-oxoacyl-coenzyme A thiolase, 6-oxoacyl-CoA thiolase, acetoacetyl-CoA beta-ketothiolase, acetyl-CoA acyltransferase, ketoacyl-CoA acyltransferase, ketoacyl-coenzyme A thiolase, long-chain 3-oxoacyl-CoA thiolase, oxoacyl-coenzyme A thiolase, pro-3-ketoacyl-CoA thiolase, 3-oxoadipyl-CoA thiolase, 3-oxo-5,Examples of the enzyme include 6-didehydrosuberyl-CoA thiolase. Although there is no particular limitation in classification based on the EC number, acyltransferases classified in EC 2.3.1.- are preferred, and specific examples include enzymes classified in EC 2.3.1.174, EC 2.3.1.9, EC 2.3.1.16, and EC 2.3.1.223.

[0036] Furthermore, whether a protein encoded by a gene sequence of unknown function corresponds to the above-mentioned enzyme can be estimated by performing a BLAST search on a website such as NCBI.

[0037] Preferred examples of these enzymes include those derived from Acinetobacter genus such as Acinetobacter baylyi and Acinetobacter radioresistens, Aerobacter genus such as Aerobacter cloacae, Alcaligenes genus such as Alcaligenes faecalis, Bacillus genus such as Bacillus badius, Bacillus megaterium, Bacillus roseus, Brevibacterium genus such as Brevibacterium iodinum, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, Corynebacterium Corynebacterium genus such as C. glutamicum, Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus numazuensis, Cupriavidus Cupriavidus genus such as Delftia oxalaticus, Delftia genus such as Delftia acidovorans, Escherichia coli, Escherichia species such as Escherichia fergusonii, Hafnia species such as Hafnia alvei, Microbacterium species such as Microbacterium ammoniaphilum, Nocardioides species such as Nocardioides albus, Planomicrobium species such as Planomicrobium okeanokoites, Pseudomonas azotoformans, Pseudomonas chlororaphis, Pseudomonas fluorescens, Pseudomonas fragii, Pseudomonas the genus Pseudomonas such as Pseudomonas putida, Pseudomonas reptilivora, and Pseudomonas taetrolens; the genus Rhizobium such as Rhizobium radiobacter; the genus Rhodosporidium such as Rhodosporidium toruloides;The genus Saccharomyces such as Saccharomyces cerevisiae, the genus Serratia such as Serratia entomophila, Serratia ficaria, Serratia fonticola, Serratia grimesi, Serratia nematodiphila, Serratia odorifera, and Serratia plymuthica, the genus Shimwellia such as Shimwellia blattae, Streptomyces vinaceus, Streptomyces karnatakensis, and Streptomyces Examples of suitable enzymes include enzymes derived from microorganisms of the genus Streptomyces, such as Streptomyces olivaceus and Streptomyces vinaceus, the genus Yarrowia, such as Yarrowia lipolytica, and the genus Yersinia, such as Yersinia ruckeri, and preferably those derived from Escherichia coli str. K-12 substr. Examples of such PcaF include PaaJ derived from the MG1655 strain (NCBI Protein ID: NP_415915), PcaF derived from the Pseudomonas putida KT2440 strain (NCBI Protein ID: NP_743536), and PcaF derived from the Corynebacterium glutamicum ATCC13032 strain (NCBI Protein ID: CAF21057).

[0038] Whether the above acyltransferase can produce 3-oxoadipyl-CoA using acetyl-CoA and succinyl-CoA as substrates can be examined by known methods, for example, the method described in WO 2019 / 107516. That is, the 3-oxoadipyl-CoA production reaction by the purified acyltransferase is combined with a reduction reaction by purified 3-oxoadipyl-CoA reductase using 3-oxoadipyl-CoA as a substrate, and the amount of NADH decreased due to the reduction of 3-oxoadipyl-CoA can be confirmed.

[0039] Furthermore, in the genetically modified microorganism of the present invention, in accordance with the descriptions in WO2017 / 209102 and WO2019 / 107516, it is preferable to enhance the activity of the enzyme catalyzing the reaction of producing 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA (reaction B), the reaction of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA (reaction C), the reaction of producing adipyl-CoA from 2,3-dehydroadipyl-CoA (reaction D), the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA (reaction E), and / or the reaction of producing adipic acid from adipyl-CoA (reaction F).

[0040] Examples of the enzyme that catalyzes the reaction (reaction B) of producing 3-hydroxyadipyl-CoA from 3-oxoadipyl-CoA include an enzyme classified as a 3-hydroxyacyl-CoA dehydrogenase in EC 1.1.1.35, and an enzyme classified as a 3-hydroxybutyryl-CoA dehydrogenase in EC 1.1.1.157. Specific examples include PaaH derived from Pseudomonas putida KT2440 strain (NCBI Protein ID: NP_745425.1), PaaH derived from Escherichia coli str. K-12 substr. Examples of usable enzymes include PaaH derived from the MG1655 strain (NCBI Protein ID: NP_415913.1), DcaH derived from the Acinetobacter baylyi ADP1 strain (NCBI Protein ID: CAG68533.1), PaaH derived from the Serratia plymuthica NBRC102599 strain (NCBI Protein ID: WP_063197120), and 3-oxoadipyl-CoA reductase derived from the Serratia marcescens ATCC13880 strain (NCBI Gene ID: JMPQ01000047.1).

[0041] Examples of enzymes that catalyze the reaction (reaction C) that produces 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA include enzymes classified as enoyl-CoA hydratases or 2,3-dehydroadipyl-CoA hydratases in EC 4.2.1.17. Specifically, PaaF derived from Escherichia coli MG1655 strain (NCBI-Protein ID: NP_415911) and PaaF derived from Pseudomonas putida KT2440 strain (NCBI-Protein ID: NP_745427) can be used.

[0042] Examples of the enzyme that catalyzes the reaction (reaction D) that produces adipyl-CoA from 2,3-dehydroadipyl-CoA include enzymes classified as trans-2-enoyl-CoA reductases in EC 1.3.1.44 and enzymes classified as acyl-CoA dehydrogenases in EC 1.3.8.7. Specifically, TER (UniProtKB: Q5EU90) derived from Euglena gracilis Z strain, Tfu_1647 (NCBI-Protein ID: AAZ55682) derived from Thermobifida fusca YX strain, and DcaA (NCBI-Protein ID: AAL09094.1) derived from Acinetobacter baylyi ADP1 strain can be used.

[0043] As the enzyme that catalyzes the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA (reaction E) and the reaction of producing adipic acid from adipyl-CoA (reaction F), for example, CoA transferase or acyl-CoA hydrolase can be used. Among these, CoA transferase is preferably used.

[0044] CoA transferase refers to an enzyme having catalytic activity (CoA transferase activity) for a reaction that produces carboxylic acid and succinyl-CoA using acyl-CoA and succinic acid as substrates. CoA transferases are not particularly limited in classification by EC number, but include those belonging to EC 2.8.3. -, preferably CoA transferases or acyl-CoA transferases classified in EC 2.8.3.6. Specific examples that can be used include PcaI (NCBI Protein ID: NP_746081) and PcaJ (NCBI Protein ID: NP_746082) derived from Pseudomonas putida KT2440 strain, and DcaI and DcaJ (NCBI Protein ID: CAG68538 and CAG68539) derived from Acinetobacter baylyi ADP1 strain.

[0045] The genes encoding the enzymes that catalyze reactions A to F may be genes that are conventionally possessed by microorganisms, or may be artificially introduced.

[0046] Methods for enhancing the enzyme activity are not particularly limited. Preferred methods include introducing genes encoding various enzymes or polypeptides having various functions into the host microorganism from outside the host microorganism, increasing the copy number of the genes, or modifying the promoter region or ribosome binding sequence upstream of the genes to increase the expression level of polypeptides having various enzyme activities or functions. These methods may be performed alone or in combination. The gene to be introduced may be artificially synthesized based on the amino acid sequence of the enzyme 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 to be introduced. The method for introducing nucleic acids is not particularly limited. Methods that can be used include incorporating the nucleic acid into an expression vector capable of autonomous replication within the microorganism and introducing it into the host microorganism, or integrating the nucleic acid into the genome of the microorganism.

[0047] The gene to be introduced may be one or more types, and gene introduction and enhancement of gene expression may be combined.

[0048] When a nucleic acid encoding a polypeptide 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 nucleic acid encoding the polypeptide to be expressed, and a transcription termination sequence. It may also contain a gene that controls promoter activity.

[0049] 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.

[0050] When an expression vector is used in the present invention to introduce a nucleic acid or enhance expression of a polypeptide, the vector is not particularly limited as long as it is capable of autonomous replication in the microorganism, and examples thereof include pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above-mentioned vectors.

[0051] In the present invention, when a nucleic acid for genome integration is used to introduce a nucleic acid or enhance the expression of a polypeptide, the nucleic acid can be introduced using site-specific homologous recombination. The method of site-specific homologous recombination is not particularly limited, and examples thereof 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).

[0052] 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 (N.M. Calvin, P.C. Hanawalt. J. Bacteriol. 170 (1988), pp. 2796-2801).

[0053] Microorganisms that are presumed to inherently have the ability to produce α-hydromuconic acid include the following: the genus Escherichia, such as Escherichia fergusonii and Escherichia coli, and the genus Serratia, such as Serratia grimesi, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila, and Serratia 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. The genus Acinetobacter, such as Acinetobacter baylyi and Acinetobacter radioresistens. The genus Alcaligenes, such as Alcaligenes faecalis. The genus Delftia, such as Delftia acidovorans. The genus Shimwellia, such as Shimwellia blattae.

[0054] Microorganisms that are presumed to inherently have the ability to produce adipic acid include microorganisms belonging to the genus Thermobifida, such as Thermobifida fusca.

[0055] Among the microorganisms that inherently have the ability to produce α-hydromuconic acid and / or adipic acid, microorganisms belonging to the genus Escherichia or Serratia are preferably used in the present invention.

[0056] If the genetically modified microorganism of the present invention does not originally have the ability to produce α-hydromuconic acid and / or adipic acid, these production abilities can be imparted by introducing into the microorganism an appropriate combination of nucleic acids encoding enzymes that catalyze reactions A to F, in accordance with the descriptions in WO 2017 / 209102 and WO 2019 / 107516.

[0057] The genetically modified microorganism of the present invention has a technical feature of deleting or reducing the function of an organophosphate compound exporter, and therefore a microorganism having an organophosphate exporter gene is used as a host for obtaining the genetically modified microorganism. The microorganism having an organophosphate exporter gene may be a microorganism that originally possesses the gene or a microorganism into which an organophosphate exporter gene has been introduced by genetic modification, but examples thereof include Escherichia, Salmonella, Shigella, Enterobacter, Cronobacter, Klebsiella, Raoultella, Citrobacter, Pluralibacter, Kosakonia, Kluyvera, and the like. Genus, Leclercia, Lelliotia, Buttiauxella, Symbiopectobacterium, Atlantibacter, Phytobacter, Izhakiella, Pseudoci trobacter, Siccibacter, Pseudescherichia, Superficieibacter, Trabulsiella, Yersinia, Serratia, Rahnella, Gibbs genus iella, genus Ewingella, genus Rouxiella, genus Chimaeribacter, genus Pectobacterium, genus Dickeya, genus Musicola, genus Brenneria, genus Prodigiosinella, S odalis, Erwinia, Duffyella, Pantoea, Mixta, Tatumella, Kalamiella, Pseudomonas, Celerinatantimonas, Burkholder Microorganisms belonging to the genera Escherichia, Klebsiella, Serratia, Pantoea, Pseudomonas, Burkholderia, and Sphingomonas are preferred, and microorganisms belonging to the genus Escherichia, Klebsiella, Serratia, Pantoea, Pseudomonas, Burkholderia, and Sphingomonas are more preferred, with microorganisms belonging to the genus Escherichia or Serratia being particularly preferred.

[0058] The genetically modified microorganisms of the present invention are cultured in a medium, preferably a liquid medium, containing a carbon source that can be utilized by conventional microorganisms as a fermentation raw material. In addition to the carbon source that can be utilized by the genetically modified microorganisms, a medium containing an appropriate amount of a nitrogen source, inorganic salts, and, as needed, organic trace nutrients such as amino acids and vitamins is used. Either a natural or synthetic medium can be used as long as it contains the above nutrient sources.

[0059] 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 intracellularly from another chemical compound into another chemical compound through an enzymatic reaction. Sugars are preferably used as carbon sources. Specific examples of sugars include monosaccharides such as glucose, fructose, galactose, mannose, xylose, and arabinose, disaccharides such as sucrose formed by combining these monosaccharides, polysaccharides, and starch saccharification solutions, molasses, and cellulose-containing biomass saccharification solutions containing these sugars.

[0060] 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.

[0061] Examples of nitrogen sources that can be used in culturing the genetically modified microorganism include 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.

[0062] 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.

[0063] The culture conditions for the genetically modified microorganism to produce α-hydromuconic acid and / or adipic 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.

[0064] 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.

[0065] There are no particular limitations on the range of aeration conditions during culture as long as α-hydromuconic acid and / or adipic acid can be produced. However, in order to grow the microbial mutant well, it is preferable that oxygen remain in the liquid phase or gas phase in the culture vessel at least at the start of culture.

[0066] 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.

[0067] After α-hydromuconic acid and / or adipic 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 bacterial cells can be separated by centrifugation, filtration, or the like, and then the product can be isolated from the culture 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.

[0068] Adipic acid can be produced by reacting the α-hydromuconic acid obtained in the present invention with hydrogen (hydrogenation) in the presence of a hydrogenation catalyst.

[0069] The hydrogenation catalyst preferably contains a transition metal element, and specifically, it preferably contains one or more elements selected from the group consisting of palladium, platinum, ruthenium, rhodium, rhenium, nickel, cobalt, iron, iridium, osmium, copper, and chromium, and more preferably contains one or more elements selected from the group consisting of palladium, platinum, nickel, cobalt, iron, copper, and chromium.

[0070] The hydrogenation catalyst is preferably used supported on a carrier from the viewpoints of saving the amount of metal used, increasing the active surface of the catalyst, etc. Supporting the hydrogenation catalyst on a carrier can be carried out by known methods such as impregnation, deposition / precipitation, and vapor phase support. Examples of the carrier include carbon, polymers, metal oxides, metal sulfides, zeolites, clays, heteropolyacids, solid phosphoric acid, and hydroxyapatite.

[0071] The hydrogen to be reacted with α-hydromuconic acid may be added to the reactor all at once or gradually. The partial pressure of hydrogen is not particularly limited, but if it is too low, the reaction time will be long, while if it is too high, it is undesirable from the viewpoint of equipment safety. Therefore, the partial pressure of hydrogen is preferably 0.1 MPa or more and 10 MPa or less (gauge pressure) at room temperature, more preferably 0.3 MPa or more and 5 MPa or less (gauge pressure), and even more preferably 0.5 MPa or more and 3 MPa or less (gauge pressure).

[0072] The reaction may be carried out in any reactor, such as a batch reactor, a semi-batch reactor, a continuous reactor, a continuous tubular reactor, or a trickle-bed tubular reactor. When a solid hydrogenation catalyst is used, the reaction may be carried out in any of a suspension bed, a fixed bed, a moving bed, or a fluidized bed.

[0073] The reaction temperature for hydrogenation is not particularly limited, but if it is too low, the reaction rate will be slow, and if it is too high, the amount of energy consumed will be large, which are undesirable. From these viewpoints, the reaction temperature is preferably 100 to 350°C, more preferably 120 to 300°C, even more preferably 130 to 280°C, still more preferably 140 to 250°C, even more preferably 150 to 230°C, and even more preferably 160 to 220°C.

[0074] In addition to hydrogen, the atmosphere in the reactor may contain inert gases such as nitrogen, helium, and argon, but the oxygen concentration is preferably 5% by volume or less because this leads to deterioration of the hydrogenation catalyst and the generation of detonation gas. Also, from the viewpoint of the stability of 3HA and / or 3OA and adipic acid, the amount of ammonia relative to 3HA and / or 3OA is preferably 5% by weight or less, more preferably 3% by weight or less, and even more preferably 0% by weight (i.e., reaction in the absence of ammonia).

[0075] The hydrogenation of α-hydromuconic acid is preferably carried out in the presence of a solvent.

[0076] Examples of solvents that can be used for the hydrogenation include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, pentane, hexane, cyclohexane, heptane, octane, decane, dimethyl ether, diethyl ether, 1,2-dimethoxyethane, diglyme, tetrahydrofuran, dioxane, methyl acetate, ethyl acetate, n-propyl acetate, n-butyl acetate, γ-butyrolactone, N-methylpyrrolidone, dimethyl sulfoxide, and aqueous solvents. A mixed solvent of two or more of these may also be used, but from the viewpoints of economy and environmental friendliness, it is preferable to use an aqueous solvent.

[0077] In the present invention, the aqueous solvent refers to a mixed solvent consisting mainly of water or water-miscible organic solvent. "Consisting mainly of water" means that the proportion of water in the mixed solvent is more than 50% by volume, preferably 70% by volume or more, and more preferably 90% by volume or more.

[0078] Examples of the water-miscible organic solvent include methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, 1,2-dimethoxyethane, diglyme, tetrahydrofuran, dioxane, γ-butyrolactone, N-methylpyrrolidone, dimethyl sulfoxide, dimethylformamide, dimethylacetamide, and acetone.

[0079] The pH of the aqueous solvent is not particularly limited, but in consideration of the prevention of catalyst deterioration, the prevention of by-product formation, corrosiveness to the reaction apparatus, etc., the pH is preferably 2 to 13, more preferably 3 to 11, and even more preferably 4 to 10.

[0080] When a solvent other than a primary alcohol or a secondary alcohol is used as the hydrogenation solvent, the carboxylic acid, carboxylate, and carboxylate ester of α-hydromuconic acid produce the corresponding adipic acid, adipate, and adipate, respectively. When a solvent containing a primary or secondary alcohol such as methanol, ethanol, n-propanol, isopropanol, n-butanol, or isobutanol is used as the hydrogenation solvent, a mixture of adipic acid, adipate, adipic acid monoester, and adipic acid diester is obtained after the reaction. In this specification, the carboxylic acid, carboxylate, and carboxylate ester of adipic acid, and mixtures thereof, are collectively referred to as "adipic acid."

[0081] The carboxylic acid of adipic acid obtained in the present invention can be further converted into an adipic acid ester by subjecting it to an esterification reaction. The esterification method is not particularly limited, and examples thereof include dehydration condensation of a carboxylic acid and an alcohol using an acid catalyst or a condensing agent, and a method using an alkylating reagent such as diazomethane or an alkyl halide.

[0082] The adipic acid obtained in the present invention can be separated and purified by ordinary unit operations such as centrifugation, filtration, membrane filtration, distillation, extraction, crystallization, and drying.

[0083] Adiponitrile can be produced from the adipic acid obtained by the present invention by a known method (for example, JP-B-61-24555). Hexamethylenediamine can be produced by hydrogenating the obtained adiponitrile by a known method (for example, JP-A-2000-508305).

[0084] Polyamides can be produced by polycondensing the adipic acid obtained by the present invention with diamines by a known method (see, for example, "Polyamide Resin Handbook," edited by Osamu Fukumoto, published by Nikkan Kogyo Shuppansha, January 1998). Specifically, by using 1,4-diaminobutane, 1,5-pentanediamine, or hexamethylenediamine as the diamine, polyamide 46, polyamide 56, and polyamide 66 can be produced, respectively.

[0085] Polyamide fibers can be produced by processing polyamides using known methods (e.g., WO 2019 / 208427). The polyamide fibers thus obtained can be used for clothing applications such as innerwear, sportswear, and casual wear, as well as industrial materials such as airbags and tire cords.

[0086] In addition, polyamide can be molded by a known method (for example, WO 2021 / 006257) to produce a polyamide molded product. The polyamide molded product thus obtained can be used for automotive parts, electrical parts, electronic parts, building materials, various containers, daily necessities, household goods, sanitary products, etc.

[0087] The present invention will be specifically described below with reference to examples.

[0088] Reference Example 1: Preparation of Plasmid 1 Expressing Enzymes Catalyzing the Reaction (Reaction A) of Producing 3OA-CoA and Coenzyme A from Acetyl-CoA and Succinyl-CoA, the Reaction (Reaction B) of Producing 3HA-CoA from 3OA-CoA, the Reaction (Reaction E) of Producing HMA from HMA-CoA, and the Reaction (Reaction F) of Producing ADA from ADA-CoA

[0089] Preparation of Plasmid Vector The plasmid vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166:175-176), which is capable of autonomous replication in E. coli, was cleaved with XhoI to obtain pBBR1MCS-2 / XhoI. In order to incorporate a constitutive expression promoter into this vector, primers were designed (SEQ ID NOs: 4 and 5) for PCR amplification of the 200 b upstream region (SEQ ID NO: 3) of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli (E. coli) str. K-12 substr. MG1655 as a template, and PCR reaction was carried out according to standard methods. The resulting DNA fragment (expression promoter: Pgap) and pBBR1MCS-2 / XhoI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli 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::Pgap. Subsequently, pBBR1MCS-2::Pgap was cleaved with ScaI to obtain pBBR1MCS-2::Pgap / ScaI.

[0090] Introduction of the Enzyme Gene Catalyzing Reaction A into a Plasmid Vector To amplify the gene encoding the enzyme that catalyzes Reaction A, primers were designed (SEQ ID NOs: 7 and 8) for PCR amplification of the full length of the acyltransferase gene pcaF (NCBI Gene ID: 1041755, SEQ ID NO: 6) 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" and introduced into 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 pBBR1MCS-2::AT.

[0091] Introduction of Enzyme Genes Catalyzing Reactions E and F into a Plasmid Vector pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT / HpaI. To amplify the genes encoding the enzymes catalyzing reactions E and F, primers were designed (SEQ ID NOS: 11 and 12) for PCR amplification of a continuous sequence including the full length of the CoA transferase genes pcaI (NCBI Gene ID: 1046613, SEQ ID NOS: 9) and pcaJ (NCBI Gene ID: 1046612, SEQ ID NOS: 10) 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::AT / HpaI were ligated using the "In-Fusion HD Cloning Kit" to generate E. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by a standard method. The resulting plasmid was designated pBBR1MCS-2::ATCT.

[0092] Introduction of the Enzyme Gene Catalyzing Reaction B into a Plasmid Vector pBBR1MCS-2::ATCT was cleaved with ScaI to obtain pBBR1MCS-2::ATCT / ScaI. To amplify the nucleic acid encoding 3-oxoadipyl-CoA reductase, which catalyzes Reaction B, primers for amplifying the nucleic acid set forth in SEQ ID NO: 13 were designed using the genomic DNA of Serratia marcescens ATCC13880 strain as a template (SEQ ID NOs: 14 and 15), and PCR was performed according to standard methods. The resulting fragment and pBBR1MCS-2::ATCT / ScaI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by a conventional method. The resulting plasmid was designated pBBR1MCS-2::ATCTOR.

[0093] Construction of Plasmid 1 Expressing Enzymes Catalyzing Reactions A, B, E, and F The kanamycin resistance gene of pBBR1MCS-2::ATCTOR was replaced with a chloramphenicol resistance gene. PCR was performed using pHSG396 (manufactured by Takara Bio Inc.) as a template and the nucleic acids of SEQ ID NOs: 16 and 17 as primers to amplify the chloramphenicol resistance gene. This PCR fragment and a fragment obtained by cleaving pBBR1MCS-2::ATCTOR with SapI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into Escherichia coli DH5α strain. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by standard methods. This plasmid was designated as Plasmid 1.

[0094] Reference Example 2 Preparation of Plasmid 3 for Expressing an Enzyme Catalyzing the Reaction (Reaction C) that Produces HMA-CoA from 3HA-CoA and an Enzyme Catalyzing the Reaction (Reaction D) that Produces ADA-CoA from HMA-CoA Preparation of Plasmid Vector A plasmid vector pMW119 (manufactured by Nippon Gene Co., Ltd.) capable of autonomous replication in Escherichia coli was cleaved with SacI to obtain pMW119 / SacI. In order to incorporate a constitutive expression promoter into this vector, primers were designed (SEQ ID NOs: 18 and 19) for PCR amplification of the upstream 200b (SEQ ID NO: 3) of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of E. coli str. K-12 substr. MG1655 as a template, and a PCR reaction was performed according to standard methods. The obtained fragment and pMW119 / SacI were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli DH5α. The plasmid was extracted from the obtained recombinant strain, and the nucleotide sequence was confirmed by a standard method. The plasmid was designated pMW119::Pgap.

[0095] Construction of Plasmid 2 Expressing an Enzyme Catalyzing Reaction C pMW119::Pgap was cleaved with SphI to obtain pMW119::Pgap / SphI. To amplify the gene encoding the enzyme catalyzing reaction C, primers were designed (SEQ ID NOS: 21 and 22) for PCR amplification of the full length of the enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 20) 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" (manufactured by Takara Bio Inc.) and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the base sequence was confirmed by a standard method. The resulting plasmid was designated as Plasmid 2.

[0096] Construction of Plasmid 3 Expressing Enzymes Catalyzing Reactions C and D Plasmid 2 was cleaved with HindIII to obtain plasmid 2 / HindIII. To amplify the gene encoding the enzyme that catalyzes reaction D, primers were designed (SEQ ID NOs: 24 and 25) for PCR amplification of the full length of dcaA (NCBI-Protein ID: AAL09094.1, SEQ ID NO: 23) derived from Acinetobacter baylyi ADP1 strain, and PCR was performed according to standard methods. The resulting fragment and plasmid 2 / HindIII were ligated using "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli DH5α. The plasmid was extracted from the resulting recombinant strain, and the nucleotide sequence was confirmed by standard methods. This plasmid was designated as plasmid 3.

[0097] Reference Example 3: Preparation of a mutant Escherichia microorganism lacking YhhS function To render Escherichia microorganisms lacking YhhS function, a mutant Escherichia microorganism lacking the gene encoding YhhS was prepared.

[0098] The target gene was deleted according to the method described in Proc Natl Acad Sci USA, 2000 Jun 6; 97(12):6640-6645.

[0099] PCR was performed using pKD4 as a template and oligo DNAs of SEQ ID NOs: 26 and 27 as primers to obtain a 1.6 kb PCR fragment for yhhS deletion. The FRT recombinase expression plasmid pKD46 was introduced into Escherichia coli str. K-12 substr. BW25113 to obtain an ampicillin-resistant strain. The resulting strain was inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and cultured with shaking at 37°C for 1 day. 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 with rotation at 37°C for 2 hours. The culture was ice-cooled 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 5 μL of the PCR fragment, and then ice-cooled in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene Pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF), followed by immediate addition of 1 mL of SOC medium and incubation at 37°C for 2 hours with shaking. The entire volume 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 deletion of the target gene and insertion of the kanamycin resistance gene were confirmed based on band lengths. Oligonucleotides of SEQ ID NOs: 28, 29, 29, and 30 were used as primers, respectively.

[0100] The kanamycin-resistant strain was then inoculated into 5 mL of LB medium and subcultured twice at 37°C to eliminate pKD46, yielding an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain, and an ampicillin-resistant strain was again obtained. The resulting strain was cultured at 40°C, followed by colony direct PCR, and the elimination of the kanamycin resistance gene was confirmed based on the band length. OligoDNAs of SEQ ID NOs: 28 and 29 were used as primers. The kanamycin-sensitive strain was inoculated into 5 mL of LB medium and subcultured twice at 37°C to eliminate pCP20. The resulting Escherichia microorganism mutant lacking YhhS function was designated EcΔyhhS.

[0101] Example 1: Preparation of an HMA-producing strain lacking YhhS function Plasmid 1 prepared in Reference Example 1 and plasmid 2 prepared in Reference Example 2 were introduced into EcΔyhhS prepared in Reference Example 3 to prepare an HMA-producing strain lacking YhhS function.

[0102] The EcΔyhhS strain was inoculated into 5 mL of LB medium and cultured with shaking at 37°C for 1 day. 0.5 mL of the culture was inoculated into 5 mL of LB medium and cultured with shaking at 37°C for 2 hours. The culture was ice-cooled 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 plasmid 1 and plasmid 2, and then ice-cooled in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene Pulser" (Bio-Rad) (3 kV, 200 Ω, 25 μF), after which 1 mL of SOC medium was immediately added and the cells were cultured with shaking at 37°C for 1 hour. 50 μL of the culture was spread on an LB agar medium containing 15 μg / mL of chloramphenicol and 100 μg / mL of ampicillin, and incubated for 1 day at 37° C. The resulting strain was designated EcΔyhhS / HMA.

[0103] Example 2: Preparation of an ADA-producing strain lacking YhhS function Plasmid 1 prepared in Reference Example 1 and plasmid 3 prepared in Reference Example 2 were introduced into EcΔyhhS in accordance with the method of Example 1 to obtain an ADA-producing strain lacking YhhS function (EcΔyhhS / ADA).

[0104] Comparative Example 1 Preparation of an HMA-producing strain and an ADA-producing strain in which the YhhS function is intact Plasmids 1 and 2, or plasmids 1 and 3, were introduced into Escherichia coli str. K-12 substr. BW25113 as a control strain by the method of Example 1 to obtain an HMA-producing strain in which the YhhS function is intact (EcWT / HMA) and an ADA-producing strain (EcWT / ADA), respectively.

[0105] Example 3 HMA and ADA Production Test Using an HMA-Producing Strain and an ADA-Producing Strain Deficient in YhhS Function EcΔyhhS / HMA and EcΔyhhS / ADA were each 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, chloramphenicol 15 μg / mL, ampicillin 100 μg / mL) (φ18 mm glass test tube, aluminum stopper) adjusted to pH 7, and cultured with shaking at 30°C and 120 min-1 for 24 hours. 0.6 mL of the culture solution was added to 12 mL of Medium II (16.5 mm diameter glass test tube, screw cap) (glucose 20 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, chloramphenicol 15 μg / mL, ampicillin 100 μg / mL) adjusted to pH 6.5, and the mixture was subjected to static culture at 30° C. for 24 hours.

[0106] The supernatant obtained by centrifuging the bacterial cells from the culture medium was passed through a membrane called "Millex-GV" (0.22 μm, PVDF, manufactured by Merck). The permeate was analyzed by LC-MS / MS under the following conditions to quantify the HMA and ADA accumulated in the culture supernatant, and the sugars remaining unused in the medium were quantified by HPLC under the following conditions.

[0107] Quantitative analysis of HMA or ADA 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: 0.3 mL / min Column temperature: 40° C. LC detector: 1260DAD VL+ (210 nm) MS / MS: Triple-Quad LC / MS (Agilent Technologies) Ionization method: ESI negative mode.

[0108] Quantitative analysis of sugars 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: 0.05 M aqueous sulfuric acid Flow rate: 0.6 mL / min Column temperature: 65° C. Detector: RID-10A (Shimadzu Corporation).

[0109] The quantitative results of HMA and ADA are shown in Table 1. Furthermore, the yields of HMA and ADA calculated by the following formula (2) based on the quantitative results of the sugars remaining unused in the medium are shown in Table 2.

[0110] Product yield (%) = Amount of product produced (mol) / Amount of sugar consumed (mol) × 100 Formula (2).

[0111] Comparative Example 2: HMA and ADA production test using HMA-producing strain and ADA-producing strain with intact YhhS function Using EcWT / HMA and EcWT / ADA from Comparative Example 1, HMA and ADA production tests were carried out in the same manner as in Example 3. The results are shown in Tables 1 and 2.

[0112] Comparing the results of Example 3 with those of Comparative Example 1, it was found that the productivity of HMA and / or ADA was improved by deleting the function of YhhS.

[0113] N. D. :Not Detected

[0114]

Claims

1. A genetically modified microorganism having the ability to produce α-hydromuconic acid and / or adipic acid, in which the activity of an enzyme that catalyzes the reaction from acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA and CoA is enhanced, and the function of an organophosphate compound exporter is deleted or reduced.

2. The genetically modified microorganism according to claim 1, wherein the deficiency or reduction in the function of the organophosphate compound exporter is a deficiency or reduction in the function of YhhS or a homolog thereof.

3. The genetically modified microorganism according to claim 2, wherein the deficiency or reduction in the function of YhhS or its homologue is a deletion, loss-of-function mutation or suppression of expression of the yhhS gene or its homologue gene.

4. The genetically modified microorganism according to claim 1, in which the activity of an enzyme that catalyzes the reaction of producing 3-hydroxyadipyl-CoA by reducing 3-oxoadipyl-CoA, the reaction of producing 2,3-dehydroadipyl-CoA by dehydrating 3-hydroxyadipyl-CoA, the reaction of producing adipyl-CoA by reducing 2,3-dehydroadipyl-CoA, the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA, and / or the reaction of producing adipic acid from adipyl-CoA is enhanced.

5. The genetically modified microorganism of claim 1, wherein the microorganism is a microorganism belonging to the genus Escherichia.

6. A method for producing α-hydromuconic acid and / or adipic acid, comprising a step of culturing the genetically modified microorganism according to claim 1.

7. A method for producing a genetically modified microorganism, comprising the steps of: enhancing the activity of an enzyme that catalyzes the reaction from acetyl-CoA and succinyl-CoA to 3-oxoadipyl-CoA and CoA in a microorganism capable of producing α-hydromuconic acid and / or adipic acid through genetic modification; and deleting or reducing the function of an organophosphate compound exporter in the microorganism.

8. A method for producing adipic acid, comprising the steps of producing α-hydromuconic acid by the method according to claim 6, and reacting the resulting α-hydromuconic acid with hydrogen in the presence of a hydrogenation catalyst (hydrogenation step).

9. A method for producing a polyamide, comprising the steps of producing adipic acid by the method according to claim 6 or 8, and polycondensing the adipic acid obtained with a diamine.

10. The method for producing a polyamide according to claim 9, wherein the diamine is 1,4-butanediamine, 1,5-pentanediamine and / or hexamethylenediamine.

11. α-hydromuconic acid and / or adipic acid obtained by culturing the genetically modified microorganism according to any one of claims 1 to 5.

12. Adipic acid obtained by a process comprising the steps of producing α-hydromuconic acid by the method according to claim 6 and reacting the obtained α-hydromuconic acid with hydrogen in the presence of a hydrogenation catalyst (hydrogenation process).

13. A polyamide obtained by a process comprising the steps of producing adipic acid by the method according to claim 6 or 8, and polycondensing the adipic acid obtained with a diamine.

14. The polyamide of claim 13, wherein the diamine is 1,4-butanediamine, 1,5-pentanediamine and / or hexamethylenediamine.

15. Fibers made from the polyamide according to claim 13.

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