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

Abstract

The objective is to improve the productivity of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid while reducing the by-product of acetic acid in a method for producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid using microorganisms. [Solution] A genetically modified microorganism capable of producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, in which the function of isocitrate lyase is enhanced.

Description

Technical Field

[0001] The present invention relates to a genetically modified microorganism that highly produces 3-hydroxyadipic acid, α-hydroxymuconic acid, and / or adipic acid, and a method for producing 3-hydroxyadipic acid, α-hydroxymuconic acid, and / or adipic acid using the genetically modified microorganism.

Background Art

[0002] 3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid), α-hydroxymuconic acid (IUPAC name: (E)-hex-2-enedioic acid), and adipic acid (IUPAC name: hexanedioic acid) are dicarboxylic acids having 6 carbon atoms. These can be used as a polyester by polymerizing with a polyhydric alcohol, and as a raw material for a polyamide by polymerizing with a polyvalent amine. Also, compounds obtained by adding ammonia to the terminals thereof and subjecting them to lactamization or diamination can be used as a raw material for a polyamide.

[0003] As literature related to the production of 3-hydroxyadipic acid, α-hydroxymuconic acid, and / or adipic acid using a microorganism, Patent Document 1 describes a method for producing 3-hydroxyadipic acid, α-hydroxymuconic acid, and / or adipic acid using a polypeptide showing excellent activity in catalyzing the reduction reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, and describes that as a biosynthetic pathway of these substances, it passes through an enzymatic reaction of reducing 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.

[0004] Patent Document 2 describes a method for producing 3-hydroxyadipic acid, α-hydroxymuconic acid, and / or adipic acid using a polypeptide showing excellent activity in catalyzing the reduction reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA and a genetically modified microorganism having a defective function of pyruvate kinase.

[0005] Patent Document 3 describes a method for producing 3-hydroxyadipic acid and / or α-hydromuconic acid using genetically modified microorganisms in which the reaction for producing acetyl-CoA from pyruvate is enhanced and the function of pyruvate kinase and / or phosphotransferase enzymes is reduced.

[0006] Incidentally, in the production of chemicals using microorganisms, suppressing by-products and improving the productivity of the target substance is a universal technical challenge. In the production of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid using microorganisms, as shown in Patent Document 2, acetic acid is the main by-product. Therefore, if the by-production of acetic acid by microorganisms capable of producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid can be reduced, an improvement in the productivity of those microorganisms for 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid can be expected.

[0007] However, in the production of chemicals using microorganisms, it is known that blocking the acetic acid production pathway to reduce the by-product of acetic acid also reduces the productivity of the target substance. Specifically, one method of blocking the acetic acid production pathway is to inactivate or delete the genes encoding enzymes that catalyze the acetic acid production reaction. The main enzymes that catalyze the acetic acid production reaction are known to be phosphate acetyltransferase (EC 2.3.1.8), which catalyzes the reaction that produces acetyl phosphate and CoA from acetyl-CoA and phosphate; acetic acid kinase (EC 2.7.2.1), which catalyzes the reaction that produces acetic acid from acetyl phosphate; and pyruvate dehydrogenase (EC 1.2.5.1), which catalyzes the reaction that produces acetic acid and carbon dioxide from pyruvate. However, as exemplified in Non-Patent Literature 1, in industrially widely used E. coli, it is known that the deficiency of ackA, pta, and poxB, which are genes encoding enzymes that catalyze the acetic acid production reaction, reduces the growth of microorganisms, and consequently, the productivity of the target substance also decreases. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] WO2019 / 107516 [Patent Document 2] WO2020 / 230718 issue [Patent Document 3] WO2022 / 102635 issue [Non-patent literature]

[0009] [Non-Patent Document 1] Front. Microbiol. 2020, 11, 233. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a genetically modified microorganism capable of producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, which improves the productivity of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid while reducing the by-production of acetic acid, by modifying the metabolic pathway of the microorganism without blocking the acetic acid production pathway. [Means for solving the problem]

[0011] As a result of diligent research to achieve the above objective, the inventors have discovered that by enhancing the function of isocitrate lyase in microorganisms capable of producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, the productivity of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid is improved and the by-production of acetic acid is reduced, thus completing the present invention.

[0012] Specifically, the present invention consists of the following (1) to (22). (1) A genetically modified microorganism having the ability to produce 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, in which the function of isocitrate lyase is enhanced. (2) The genetically modified microorganism described in (1), wherein the enhancement of the function of isocitrate lyase is achieved by increasing the expression level of the isocitrate lyase gene. (3) The genetically modified microorganism described in (1), wherein the enhancement of the function of isocitrate lyase is achieved by increasing the copy number of the isocitrate lyase gene. (4) The genetically modified microorganism described in (1) further having enhanced function of malate synthase and / or isocitrate dehydrogenase / phosphatase. (5) The genetically modified microorganism described in (4), wherein the enhancement of the function of malate synthase and / or isocitrate dehydrogenase / phosphatase is achieved by increasing the expression level of the malate synthase gene and / or isocitrate dehydrogenase / phosphatase gene. (6) A genetically modified microorganism as described in (1), which does not impair the function of isocitrate lyase repressor, or maintains said function. (7) A genetically modified microorganism as described in (6), which has not been genetically modified to suppress the expression of the isocitrate lyase repressor gene. (8) The genetically modified microorganism described in (1), wherein the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA is further enhanced. A method for producing 3-hydroxyadipic acid, α-hydromuconic acid and / or adipic acid, comprising the step of culturing a genetically modified microorganism described in any of (1) to (8). A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid and / or α-hydromuconic acid by the method described in (10)(9), and reacting 3-hydroxyadipic acid and / or α-hydromuconic acid with hydrogen in the presence of a catalyst. A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid and / or α-hydromuconic acid by the method described in (11)(9), and reacting 3-hydroxyadipic acid and / or α-hydromuconic acid with ammonia and hydrogen in the presence of a catalyst. A method for producing 3-hydroxyadipic acid-3,6-lactone, comprising the steps of producing 3-hydroxyadipic acid by the method described in (12)(9), and condensing 3-hydroxyadipic acid to produce 3-hydroxyadipic acid-3,6-lactone. A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in (13)(12), and reacting 3-hydroxyadipic acid-3,6-lactone with hydrogen in the presence of a catalyst. A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in (14)(12), and reacting 3-hydroxyadipic acid-3,6-lactone with ammonia and hydrogen in the presence of a catalyst. A method for producing ε-caprolactam, comprising the steps of producing adipic acid by the method described in (15)(9), (10), or (13), and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst. A method for producing hexamethylenediamine, comprising the steps of producing adipic acid by the method described in (16)(9), (10), or (13), and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst. A method for producing a polyamide, comprising the steps of producing adipic acid by the method described in (17)(9), (10), or (13), and polycondensing adipic acid and a diamine. (18) The method for producing a polyamide according to (17), wherein the diamine is a diamine containing 1,4-butanediamine, 1,5-pentanediamine, or hexamethylenediamine. A method for producing a polyamide, comprising a step of producing hexamethylenediamine by the method described in (19)(16), and a step of polycondensing hexamethylenediamine and a dicarboxylic acid. (20) The method for producing a polyamide according to (19), wherein the dicarboxylic acid is adipic acid or sebacic acid. (21) A method for producing polyamide 6,6, comprising a step of producing adipic acid by the method described in (9), (10), or (13), a step of producing hexamethylenediamine by the method described in (16), and a step of polycondensing adipic acid and hexamethylenediamine. (22) A method for producing polyamide 6, comprising a step of producing ε-caprolactam by the method described in (11), (14), or (15), and a step of polycondensing ε-caprolactam. [[Effect of the Invention]]

[0013] The genetically modified microorganism according to the present invention can enhance the productivity of 3-hydroxyadipic acid, α-hydroxymuconic acid and / or adipic acid, and further reduce the productivity of acetic acid, as compared with the microorganism of the parent strain that has not been modified with the gene. That is, the production amounts of 3-hydroxyadipic acid, α-hydroxymuconic acid and / or adipic acid can be increased, and further, the production ratios of 3-hydroxyadipic acid, α-hydroxymuconic acid and / or adipic acid to acetic acid can be increased. [[Modes for Carrying Out the Invention]]

[0014] Hereinafter, the present invention will be described in more detail. However, the present invention is not limited to the following embodiments, and various modifications can be made and implemented within the scope of the gist of the present invention.

[0015] Hereinafter, in this specification, 3-hydroxyadipic acid may be abbreviated as "3HA", α-hydroxymuconic acid as "HMA", and adipic acid as "ADA". Also, 3-oxoadipyl-CoA may be abbreviated as "3OA-CoA", 3-hydroxyadipyl-CoA as "3HA-CoA", 2,3-dehydroadipyl-CoA as "HMA-CoA", and adipyl-CoA as "ADA-CoA". Further, the enzyme that catalyzes the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA may be described as "3-oxoadipyl-CoA reductase". Also, isocitrate lyase may be described as "AceA", malate synthase as "AceB", and isocitrate dehydrogenase kinase / phosphatase as "AceK". Also, a nucleic acid encoding a polypeptide having a function may sometimes be described as a gene.

[0016] [Genetically modified microorganism] The genetically modified microorganism of the present invention can biosynthesize 3HA, HMA, and / or ADA using acetyl-CoA and succinyl-CoA as intermediates as shown in the following metabolic pathway. The metabolic pathway from glucose to acetyl-CoA is the glycolysis pathway, the metabolic pathway to succinyl-CoA is the TCA cycle, the glyoxylate cycle, and a metabolic pathway consisting of a replenishment pathway that generates oxaloacetate from PEP and a reductive TCA cycle, which is well-known.

[0017] [Chemical formula]

[0018] Microorganisms capable of producing 3HA, HMA, and / or ADA are not particularly limited as long as they have a metabolic pathway capable of producing these substances, but it is preferable that they have the ability to produce 3HA, HMA, and / or ADA 3-hydroxyadipic acid through some or all of the metabolic pathways shown in reaction scheme 2 below. Specifically, microorganisms capable of producing 3HA are preferably capable of producing 3HA through reactions A to C of the reaction scheme shown below, more preferably that the activity of the enzymes catalyzing reactions A, B, and / or C is enhanced, even more preferably that the activity of at least the enzymes catalyzing reactions A and / or B is enhanced, and particularly preferably that the activity of at least the enzyme catalyzing reaction B is enhanced. Microorganisms capable of producing HMA are preferably capable of producing HMA by reactions A to C and F of the reaction scheme shown below, more preferably with enhanced activity of the enzymes catalyzing reactions A, B, C and / or F, even more preferably with enhanced activity of at least the enzymes catalyzing reaction A and / or B, and particularly preferably with enhanced activity of at least the enzyme catalyzing reaction B. Microorganisms capable of producing ADA are preferably capable of producing ADA by reactions A to D and G, preferably with enhanced activity of the enzymes catalyzing reactions A, B, C, D and / or G, even more preferably with enhanced activity of at least the enzymes catalyzing reaction A and / or B, and particularly preferably with enhanced activity of at least the enzyme catalyzing reaction B.

[0019] Here, reaction A shows the reaction of producing 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA. Reaction B shows the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. Reaction C shows the reaction of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction D shows the reaction of producing adipyl-CoA from 2,3-dehydroadipyl-CoA. Reaction E shows the reaction of producing 3-hydroxyadipyl-CoA to produce 3-hydroxyadipic acid. Reaction F shows the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA. Reaction G shows the reaction of producing adipic acid from adipyl-CoA. The enzymes that catalyze each reaction in the following metabolic pathways, as well as methods for creating microorganisms capable of producing 3HA, HMA, and / or ADA using these metabolic pathways, are described in detail in WO2019 / 107516. Furthermore, enhancing the activity of the enzymes that catalyze each reaction can be achieved, for example, by increasing the expression level of the enzymes. Methods for increasing expression levels include, for example, introducing the enzyme gene into the host microorganism from outside the cell, increasing the copy number of the gene, or modifying the promoter region upstream of the coding region or the ribosome binding sequence of the gene. These methods may be performed individually or in combination, but it is preferable to introduce each enzyme gene into the host microorganism from outside the cell using the method described in WO2019 / 107516.

[0020] [ka]

[0021] The present invention is characterized by enhancing the function of isocitrate lyase in microorganisms capable of producing 3HA, HMA, and / or ADA.

[0022] Isocitrate lyase is an enzyme (EC 4.1.3.1) that reversibly catalyzes the reaction that produces succinic acid and glyoxylic acid from isocitrate. A specific example of isocitrate lyase is AceA (NCBI-ProteinID:NP_418439, SEQ ID NO: 1) from Escherichia coli str.K-12 substr.MG1655 strain, and a specific example of the aceA gene encoding AceA is the nucleic acid sequence of SEQ ID NO: 2.

[0023] Methods to enhance the function of isocitrate lyase include, for example, introducing mutations that lead to the deletion, substitution, and / or insertion of some amino acids in the polypeptide having the catalytic activity of the enzyme, thereby expressing an enzyme that exhibits higher catalytic activity compared to before the mutation was introduced, or replacing the enzyme gene originally possessed by the microorganism used in the present invention with a foreign enzyme gene having higher catalytic activity. In addition, the function of the enzyme can be enhanced by increasing the expression level of isocitrate lyase, for example, by increasing the copy number of the gene or by modifying the promoter region upstream of the coding region of the gene or the ribosome binding sequence. Methods for increasing the copy number of the gene may include artificially introducing the gene originally possessed by the host gene or introducing a foreign gene. Methods for enhancing the function of the enzyme may be performed individually or in combination, but enhancement by increasing the expression level of the enzyme is preferred, and enhancement by increasing the copy number of the gene encoding the enzyme is more preferred.

[0024] Isocitric acid lyase is known to catalyze a reaction that forms the glyoxylate cycle, bypassing part of the TCA cycle, together with malate synthase and isocitrate dehydrogenase kinase / phosphatase. Furthermore, the aceA gene encoding isocitrate lyase, the aceB gene encoding malate synthase, and the aceK gene encoding isocitrate dehydrogenase kinase / phosphatase are known to form the aceBAK operon. Thus, since isocitrate lyase functions in conjunction with malate synthase and / or isocitrate dehydrogenase kinase / phosphatase, it is preferable in the present invention to enhance the function of isocitrate lyase as well as the function of malate synthase and / or isocitrate dehydrogenase kinase / phosphatase.

[0025] Malate synthase is an enzyme (EC 2.3.3.9) that catalyzes the reaction that produces malate and acetyl-CoA from glyoxylic acid and acetyl-CoA. A specific example of malate synthase is AceB (NCBI-ProteinID:NP_418438, SEQ ID NO: 3) from Escherichia coli str.K-12 substr.MG1655 strain, and a specific example of the aceB gene encoding AceB is the nucleic acid sequence of SEQ ID NO: 4.

[0026] Isocitrate dehydrogenase kinase / phosphatase is an enzyme (EC 2.7.11.5) that catalyzes the phosphorylation of isocitrate dehydrogenase, which is an enzyme that catalyzes the reaction in the TCA cycle that produces α-ketoglutarate and carbon dioxide from isocitrate. It is known that the catalytic activity of isocitrate dehydrogenase decreases upon phosphorylation. A specific example of isocitrate dehydrogenase kinase / phosphatase is AceK (NCBI-ProteinID:NP_418440, SEQ ID NO: 5) from Escherichia coli str.K-12 substr.MG1655 strain, and a specific example of the aceK gene encoding AceK is the nucleic acid gene SEQ ID NO: 6.

[0027] Furthermore, as a method to enhance the function of isocitrate lyase, one could consider reducing the function of isocitrate lyase repressor (IclR), specifically by deleting, losing-of-function, or suppressing the expression of the iclR gene encoding isocitrate lyase repressor. However, in the present invention, it is preferable to enhance the function of isocitrate lyase without reducing the function of IclR, or even maintaining the function of IclR, preferably by not suppressing the expression of the iclR gene through gene modification.

[0028] Methods for enhancing the function of malate synthase and / or isocitrate dehydrogenase kinase / phosphatase may be the same as those described above for enhancing the function of isocitrate lyase, and enhancement by increasing the expression level of the enzyme is preferred, while enhancement by increasing the copy number of the gene encoding the enzyme is more preferred.

[0029] In addition to acetic acid, lactic acid and ethanol are among the main by-products obtained by culturing microorganisms capable of producing 3HA, HMA, and / or ADA. In this invention, modifications may be made to the microorganisms capable of producing 3HA, HMA, and / or ADA to reduce the lactic acid or ethanol production reaction.

[0030] Lactic acid is produced by the reduction of pyruvate. A specific example of an enzyme that catalyzes the reduction of pyruvate to lactic acid is lactate dehydrogenase (EC 1.1.1.27, 1.1.1.28). A specific example of lactate dehydrogenase is LdhA (NCBI-ProteinID:NP_415898) derived from Escherichia coli str.K-12 substr.MG1655 strain.

[0031] Ethanol is produced by the reduction of acetyl-CoA. A specific example of an enzyme that catalyzes the reduction of acetyl-CoA to ethanol is alcohol dehydrogenase (EC 1.2.1.10, 1.1.1.1). A specific example of alcohol dehydrogenase is AdhE (NCBI-ProteinID:NP_415757) derived from Escherichia coli str.K-12 substr.MG1655 strain.

[0032] The method for reducing the lactic acid or ethanol production reaction in the present invention is not particularly limited, but examples include reducing the function of the aforementioned lactate dehydrogenase or alcohol dehydrogenase. The method for reducing the function of lactate dehydrogenase or alcohol dehydrogenase is not particularly limited, but examples include deletion, loss-of-function mutation, or suppression of the gene encoding the enzyme, with deletion or suppression of the gene encoding the enzyme being preferred. The ldhA gene is preferred as the gene encoding lactate dehydrogenase whose function is reduced, and the adhE gene is preferred as the gene encoding alcohol dehydrogenase whose function is reduced.

[0033] In the present invention, the method for gene deletion or loss-of-function mutation is not particularly limited, but can be performed by, for example, gene mutation treatment using a gene mutagenetic agent or ultraviolet irradiation, deletion of part or all of the nucleic acid sequence by site-directed mutagenesis, introduction of frameshift mutations into the nucleic acid sequence, insertion of stop codons into the base sequence, etc. Alternatively, the function can be lost by removing all or part of the nucleic acid sequence or by replacing it with another nucleic acid sequence using genetic recombination technology. Among these, a method of deleting part or all of the nucleic acid sequence is preferred.

[0034] Furthermore, in the present invention, it is particularly preferable to enhance the reaction (reaction B) that reduces 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA, as described above. Enhancing the reaction that reduces 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA can be achieved, for example, by increasing the expression level of the enzyme that catalyzes this reaction. Methods for increasing the expression level include, for example, introducing the enzyme gene into the cell of a host microorganism from outside the cell, increasing the copy number of the gene, or modifying the promoter region upstream of the coding region or the ribosome binding sequence of the gene. These methods may be performed individually or in combination, but it is preferable to introduce the enzyme gene into the cell of a host microorganism from outside the cell using the method described in WO2019 / 107516.

[0035] Specific examples of 3-oxoadipyl-CoA reductases that catalyze the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA include enzymes classified as 3-hydroxyacyl-CoA dehydrogenases under EC1.1.1.35, enzymes classified as 3-hydroxybutyryl-CoA dehydrogenases under EC1.1.1.157, and enzymes that have 70% or more, preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 97% or more, and more preferably 99% or more sequence identity with any of the amino acid sequences of SEQ ID NOs. 1-6 and 213 described in WO2019 / 107516, and that also have 3-oxoadipyl-CoA reductase activity. Specific examples include PaaH derived from Pseudomonas putida strain KT2440 (NCBI-ProteinID:NP_745425.1), PaaH derived from Escherichia coli str.K-12 substr.MG1655 strain (NCBI-ProteinID:NP_415913.1), DcaH derived from Acinetobacter baylyi strain ADP1 (NCBI-ProteinID:CAG68533.1), PaaH derived from Serratia plymuthica strain NBRC102599 (NCBI-ProteinID:WP_063197120), and polypeptide derived from Serratia marcescens strain ATCC13880 (NCBI-ProteinID:KFD11732.1). The amino acid sequences and encoding base sequences of the polypeptides of sequence numbers 1-6 and 213 described in WO2019 / 107516 are shown in sequence numbers 7-13 and 14-20, respectively.

[0036] In this invention, "sequence identity" refers to the percentage of identical amino acids or bases relative to the total overlapping amino acid sequence (including the amino acid that serves as the translation start site) or base sequence (including the start codon) when two amino acid sequences or base sequences are aligned with or without introducing gaps, and is calculated by formula (1). In formula (1), the shorter sequence being compared must be 400 amino acids or longer; if it is less than 400 amino acids, sequence identity is not defined. Sequence identity can be easily checked using BLAST (Basic Local Alignment Search Tool), a commonly used algorithm in this field. For example, BLAST is available to anyone from websites such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes), and sequence identity can be easily checked using default parameters. Sequence identity can also be checked using similar functions included in software such as Genetyx.

[0037] Sequence identity (%) = Number of matches (gaps are not counted) / Length of the shorter sequence (length excluding gaps at both ends) × 100 ... Formula (1).

[0038] The following microorganisms are examples of microorganisms that originally possess the ability to produce 3-hydroxyadipic acid, which can serve as the parent strain of the genetically modified microorganism of the present invention.

[0039] The genus Escherichia includes species such as Escherichia fergusonii and Escherichia coli.

[0040] Serratia grimesii、Serratia ficaria、Serratia fonticola、Serratia odorifera、Serratia plymuthica、Serratia entomophila or Serratia nematodiphila.

[0041] Pseudomonas chlororaphis、Pseudomonas putida、Pseudomonas azotoformans、Pseudomonas chlororaphis subsp.aureofaciens。

[0042] Hafnia, etc.

[0043] Corynebacterium genus, such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, and Corynebacterium glutamicum.

[0044] Bacillus genus, such as Bacillus badius, Bacillus megaterium, and Bacillus roseus.

[0045] Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus, etc.

[0046] Cupriavidus metaldurans, Cupriavidus necator, Cupriavidus oxalaticus etc.

[0047] The genus Acinetobacter includes species such as Acinetobacter baylyi and Acinetobacter radioresistens.

[0048] The genus Alcaligenes, such as Alcaligenes faecalis.

[0049] The genus Nocardioides, including Nocardioides albus.

[0050] Brevibacterium genus, such as Brevibacterium iodinum.

[0051] Delftia species such as Delftia acidovorans.

[0052] The genus Shimwellia, including Shimwellia blattae.

[0053] The genus Aerobacter, including Aerobacter cloacae.

[0054] Rhizobium species, such as Rhizobium radiobacter.

[0055] Among microorganisms that inherently possess the ability to produce 3-hydroxyadipic acid, the present invention prefers microorganisms that do not undergo sugar metabolism via the phosphoketolase pathway, such as those belonging to the genera Escherichia, Serratia, Hafnia, Corynebacterium, Brevibacterium, Shimwellia, and Aerobacter, and more preferably microorganisms belonging to the genera Escherichia or Serratia.

[0056] The following microorganisms are presumed to inherently possess the ability to produce α-hydromuconic acid, which can serve as the parent strain of the genetically modified microorganism of the present invention.

[0057] The genus Escherichia includes species such as Escherichia fergusonii and Escherichia coli.

[0058] Genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia nematodiphila.

[0059] The genus Pseudomonas includes species such as Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens.

[0060] Hafnia species such as Hafnia alvei.

[0061] The genus Bacillus, including Bacillus badius.

[0062] Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus numazuensis, Cupriavidus oxalaticus.

[0063] The genus Acinetobacter includes species such as Acinetobacter baylyi and Acinetobacter radioresistens.

[0064] The genus Alcaligenes, such as Alcaligenes faecalis.

[0065] Delftia species such as Delftia acidovorans.

[0066] The genus Shimwellia, including Shimwellia blattae.

[0067] Microorganisms that are presumed to inherently possess the ability to produce adipic acid, which can serve as the parent strain of the genetically modified microorganism of the present invention, include the genus Thermobifida, such as Thermobifida fusca.

[0068] If the genetically modified microorganism of the present invention does not originally possess the ability to produce 3-hydroxyadipic acid, this ability can be conferred to the microorganism by introducing a suitable combination of nucleic acids encoding enzymes that catalyze reactions A, B, and E into the microorganism. If the microorganism does not originally possess the ability to produce α-hydromuconic acid, this ability can be conferred to the microorganism by introducing a suitable combination of nucleic acids encoding enzymes that catalyze reactions A, B, C, and F into the microorganism. Furthermore, if the microorganism does not originally possess the ability to produce adipic acid, this ability can be conferred to the microorganism by introducing a suitable combination of nucleic acids encoding enzymes that catalyze reactions A, B, C, D, and G into the microorganism.

[0069] The microorganisms that can be used as hosts to obtain genetically modified microorganisms in this invention are not particularly limited as long as they are capable of genetic modification, and may be microorganisms that have the ability to produce 3-hydroxyadipic acid, α-hydromuconic acid and / or adipic acid or not, but may belong to the genera Escherichia, Serratia, Hafnia, Pseudomonas, Corynebacterium, Bacillus, Streptomyces, Cupriavidus, Acinetobacter, Alcaligene Microorganisms belonging to the genera s, Brevibacterium, Delftia, Shimwellia, Aerobacter, Rhizobium, Thermobifida, Clostridium, Schizosaccharomyces, Kluyveromyces, Pichia, and Candida are preferred, microorganisms belonging to the genera Escherichia, Serratia, Hafnia, and Pseudomonas are more preferred, and microorganisms belonging to the genera Escherichia or Serratia are particularly preferred.

[0070] The genetically modified microorganisms of the present invention preferably maintain a reaction pathway that produces acetic acid from acetyl-CoA or pyruvate. This can be achieved, for example, by possessing a gene encoding one of the enzymes that catalyzes the acetic acid production reaction and maintaining the function of said enzyme. Examples of enzymes that catalyze the acetic acid production reaction include phosphate acetyltransferase (EC 2.3.1.8), which catalyzes the reaction that produces acetyl phosphate and CoA from acetyl-CoA and phosphate; acetate kinase (EC 2.7.2.1), which catalyzes the reaction that produces acetic acid from acetyl phosphate; and pyruvate dehydrogenase (EC 1.2.5.1), which catalyzes the reaction that produces acetic acid and carbon dioxide from pyruvate.

[0071] The method for introducing a gene to create the genetically modified microorganism of the present invention is not particularly limited. Methods such as incorporating the gene into an expression vector capable of autonomous replication within the microorganism and introducing it into a host microorganism, or incorporating the gene into the genome of the microorganism, can be used.

[0072] One or more genes may be introduced. Furthermore, gene introduction and gene expression enhancement may be combined.

[0073] When incorporating a gene to be expressed or a nucleic acid fragment used to repress gene expression in the present invention into an expression vector or host microbial genome, it is preferable that the expression vector or nucleic acid for genome integration consists of a promoter, a ribosome binding sequence, the gene to be expressed or the nucleic acid fragment, and a transcription termination sequence. It may also include a gene that controls promoter activity.

[0074] The promoter used in this invention is not particularly limited as long as it can express a gene within the host microorganism, but examples include the gap promoter, trp promoter, lac promoter, tac promoter, T7 promoter, and λ promoter (PL or PR).

[0075] In the present invention, when using an expression vector to introduce a gene or enhance gene expression, there are no particular limitations as long as it can autonomously replicate within the microorganism, but examples include the pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above-mentioned vectors.

[0076] In this invention, when introducing genes or enhancing gene expression using nucleic acids for genome integration, site-directed homologous recombination is used. The method of site-directed homologous recombination is not particularly limited, but examples include a method using λ Red recombinase and FLP recombinase (Proc. Natl. Acad. Sci. USA. 2000, 97(12), 6640-6645.) and a method using λ Red recombinase and the sacB gene (Biosci. Biotechnol. Biochem. 2007, 71(12), 2905-2911.).

[0077] The method for introducing expression vectors or nucleic acids 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 (J.Mol.Biol.1970,53,159-162.) and the electroporation method (J.Bacteriol,1988,170,2796-2801.).

[0078] In the method for producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid using genetically modified microorganisms of the present invention, the production ratio of 3-hydroxyadipic acid to acetic acid is calculated according to formula (2). The production ratio of α-hydromuconic acid to acetic acid or the production ratio of adipic acid to acetic acid is calculated by replacing 3HA in formula (2) with HMA or ADA.

[0079] The production ratio of 3HA to acetic acid = 3HA production (g / L) / acetic acid production (g / L) ... Equation (2).

[0080] The genetically modified microorganisms of the present invention are cultured in a culture medium, preferably a liquid medium, that contains a carbon source available to ordinary microorganisms as a fermentation raw material. In addition to the carbon source available to the genetically modified microorganisms, the culture medium also contains a nitrogen source, inorganic salts, and, if necessary, organic micronutrients such as amino acids and vitamins in appropriate amounts. Any culture medium, whether natural or synthetic, can be used as long as it contains the above nutrients.

[0081] Fermentation raw materials are raw materials that the genetically modified microorganism can metabolize. "Metabolism" refers to the process by which a chemical compound taken in from outside the cell by a microorganism, or produced from another chemical compound within the cell, is converted into another chemical compound by an enzymatic reaction. Sugars can preferably be used as a carbon source. Specific examples of sugars include monosaccharides such as glucose, fructose, galactose, mannose, xylose, and arabinose; disaccharides such as sucrose, which are formed by the combination of these monosaccharides; polysaccharides; and starch saccharification solutions, molasses, and cellulose-containing biomass saccharification solutions containing these.

[0082] The carbon sources listed above may be used individually or in combination, but it is particularly preferable to culture in a medium containing glucose. When adding the 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.

[0083] For example, nitrogen sources used in culturing the genetically modified microorganisms include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oilseed meal, soybean hydrolysate, casein hydrolysates, other amino acids, vitamins, corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermentation cells and their hydrolysates. The concentration of the nitrogen source in the culture medium is not particularly limited, but is preferably 0.1 to 50 g / L.

[0084] Inorganic salts used in culturing the genetically modified microorganisms may include, for example, phosphates, magnesium salts, calcium salts, iron salts, and manganese salts, which can be added as appropriate.

[0085] The culture conditions for genetically modified microorganisms to produce 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid are set by appropriately adjusting or selecting the culture medium with the above-mentioned component composition, culture temperature, stirring speed, pH, aeration rate, inoculation rate, etc., according to the type of genetically modified microorganism and external conditions.

[0086] The pH range during cultivation is not particularly limited as long as the genetically modified microorganism can grow, but it is preferably pH 5 to 8, more preferably pH 5.5 to 7.0.

[0087] The range of aeration conditions in culture is not particularly limited as long as 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid can be produced. However, in order to grow the microbial mutant well, it is preferable that oxygen remains in the gas phase and / or liquid phase of the culture vessel at least at the start of culture.

[0088] If foaming occurs during liquid culture, defoaming agents such as mineral oil, silicone oil, and surfactants can be appropriately added to the culture medium.

[0089] After 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid have been produced in the culture of the aforementioned microorganism to a recoverable amount, the produced products can be recovered. Recovery of the produced products, for example isolation, can be carried out in accordance with the general method for collecting fermentation products from the culture after stopping the culture when the accumulated amount has reached a suitable level. Specifically, after separating the microbial cells by centrifugation, filtration, etc., the products can be isolated from the culture by column chromatography, ion exchange chromatography, activated carbon treatment, crystallization, membrane separation, distillation, etc. More specifically, possible and not limited to methods include, but are limited to, methods such as adding an acid component to the salt of the product to recover the precipitate, methods of increasing the concentration of the product by removing water from the culture using a reverse osmosis membrane or evaporator, then precipitating the product and / or the salt of the product by cooling crystallization or adiabatic crystallization, and obtaining the crystals of the product and / or the salt of the product by centrifugation or filtration, and methods of adding alcohol to the culture to esterify the product, recovering the ester of the product by distillation, and then obtaining the product by hydrolysis. Furthermore, these recovery methods can be appropriately selected and optimized depending on the physical properties of the product.

[0090] [Production of adipic acid] Adipic acid can be produced by reacting (hydrogenating) the 3-hydroxyadipic acid and / or α-hydromuconic acid obtained in the present invention with hydrogen in the presence of a hydrogenation catalyst. Furthermore, adipic acid can be produced by reacting (hydrogenating) the 3-hydroxyadipic acid-3,6-lactone obtained by condensing the 3-hydroxyadipic acid obtained in the present invention with hydrogen in the presence of a hydrogenation catalyst. Methods for producing 3-hydroxyadipic acid-3,6-lactone by condensing 3-hydroxyadipic acid, and methods for producing adipic acid from 3-hydroxyadipic acid-3,6-lactone, are described in detail in WO2021 / 060335. The condensation reaction of 3-hydroxyadipic acid can be carried out by dissolving 3-hydroxyadipic acid in water and adjusting the pH to 4 or less.

[0091] [ka]

[0092] The hydrogenation catalyst preferably contains a transition metal element, more preferably one or more selected from the group consisting of palladium, platinum, ruthenium, rhodium, rhenium, nickel, cobalt, iron, iridium, osmium, copper, and chromium, and more preferably one or more selected from the group consisting of palladium, platinum, nickel, cobalt, iron, copper, and chromium.

[0093] Hydrogenation catalysts are preferably used supported on a carrier, as this allows for savings in the amount of metal used and increases the active surface area of ​​the catalyst. Hydrogenation catalysts can be supported on a carrier by known methods such as impregnation, precipitation, and gas-phase support. Examples of carriers include carbon, polymers, metal oxides, metal sulfides, zeolites, clays, heteropoly acids, solid phosphoric acid, and hydroxyapatite.

[0094] The hydrogen to be reacted with 3-hydroxyadipic acid, α-hydromuconic acid, and / or 3-hydroxyadipic acid-3,6-lactone may be added to the reactor all at once or sequentially. The partial pressure of hydrogen is not particularly limited, but if it is too low the reaction time will be long, and if it is too high the partial pressure of hydrogen is undesirable from a safety standpoint for the equipment. Therefore, it is preferably 0.1 MPa to 10 MPa (gauge pressure) at room temperature, more preferably 0.3 MPa to 5 MPa (gauge pressure), and even more preferably 0.5 MPa to 3 MPa (gauge pressure).

[0095] The reaction can be carried out using any of the following reactor types: batch-type tank reactor, semi-batch-type tank reactor, continuous-type tank reactor, continuous-type tubular reactor, or trickle-bed tubular reactor. When using a solid hydrogenation catalyst, the reaction can be carried out using any of the following methods: suspension-bed, fixed-bed, moving-bed, or fluidized-bed.

[0096] 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 reaction temperature will be high, which is undesirable. From this viewpoint, the reaction temperature is preferably 100 to 350°C, more preferably 120 to 300°C, even more preferably 130 to 280°C, even more preferably 140 to 250°C, even more preferably 150 to 230°C, and even more preferably 160 to 220°C.

[0097] The atmosphere in the reactor may contain inert gases such as nitrogen, helium, and argon in addition to hydrogen, but the oxygen concentration is preferably 5% by volume or less, as this can lead to degradation of the hydrogenation catalyst and the generation of roaring gases. Furthermore, from the viewpoint of the stability of α-hydromuconic acid and / or 3-hydroxyadipic acid-3,6-lactone and adipic acid, the amount of ammonia relative to the α-hydromuconic acid and / or 3-hydroxyadipic acid-3,6-lactone raw materials 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).

[0098] The hydrogenation of 3-hydroxyadipic acid, α-hydromuconic acid, and / or 3-hydroxyadipic acid-3,6-lactone is preferably carried out in the presence of a solvent.

[0099] Suitable solvents for 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 mixture of two or more of these solvents may be used, but aqueous solvents are preferred from the viewpoint of economy and environmental friendliness.

[0100] In the present invention, an aqueous solvent means water or a mixed solvent in which water is the main component and a water-miscible organic solvent is mixed. "Mainly 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.

[0101] Examples of water-miscible organic solvents 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.

[0102] The pH of the aqueous solvent is not particularly limited, but considering the suppression of catalyst degradation, suppression of by-product formation, and corrosion to the reaction apparatus, it is preferably pH 2 to 13, more preferably pH 3 to 11, and even more preferably pH 4 to 10.

[0103] When a solvent other than primary and secondary alcohols is used as the solvent for hydrogenation, the carboxylic acid, carboxylate salt, and carboxylic acid ester of α-hydromuconic acid and / or 3-hydroxyadipic acid-3,6-lactone are converted to the corresponding adipic acid, adipic acid salt, and adipic acid ester, 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 solvent for hydrogenation, a mixture of adipic acid, adipic acid salt, adipic acid monoester, and adipic acid diester is obtained after the reaction. In this specification, the carboxylic acid, carboxylate salt, carboxylic acid ester, and mixtures thereof of adipic acid are collectively referred to as "adipic acid."

[0104] The adipic acid carboxylic acid obtained in this invention can be further converted to an adipic acid ester by subjecting it to an esterification reaction. The esterification method is not particularly limited, but examples include dehydration condensation of the carboxylic acid and alcohol using an acid catalyst or a condensing agent, and methods using alkylating reagents such as diazomethane or alkyl halides.

[0105] The adipic acid obtained in this invention can be separated and purified by conventional operations such as centrifugation, filtration, membrane filtration, distillation, extraction, crystallization, and drying.

[0106] [Manufacturing of ε-caprolactam] ε-caprolactam can be produced from 3-hydroxyadipic acid, α-hydromuconic acid, and / or 3-hydroxyadipic acid-3,6-lactone obtained in the present invention by known methods (see, for example, WO2016 / 068108). Specifically, ε-caprolactam can be produced by reacting 3-hydroxyadipic acid, α-hydromuconic acid, and / or 3-hydroxyadipic acid-3,6-lactone with hydrogen and ammonia in the presence of a catalyst.

[0107] Furthermore, ε-caprolactam can be produced from the adipic acid obtained in the present invention by known methods (see, for example, US 8946411 and WO 2012 / 141997). Specifically, ε-caprolactam can be produced by reacting the adipic acid of the present invention with hydrogen and ammonia using a catalyst containing metals such as Ru, Pt, and Pd. The resulting ε-caprolactam can be purified by known methods such as distillation and can be suitably used as a raw material for polyamide 6.

[0108] [Manufacturing of hexamethylenediamine] Hexamethylenediamine can be produced from the adipic acid obtained in the present invention by known methods (see, for example, Organic Synthesis Chemistry, Vol. 35, No. 6, 59-66 (1977)). Specifically, hexamethylenediamine can be obtained by reacting adipic acid with ammonia and hydrogen in the presence of a catalyst. In this process, it is preferable to react adipic acid with ammonia, dehydrate it with a dehydrating catalyst to produce adiponitrile, and then hydrogenate the adiponitrile to produce hexamethylenediamine.

[0109] One method for producing adiponitrile from adipic acid is to heat the adipic acid of the present invention in the presence of ammonia using a dehydration catalyst such as phosphoric acid or silica gel. The reaction may be a liquid-phase reaction using a solvent or a gas-phase reaction in which the adipic acid is vaporized and reacted. The resulting adiponitrile can be purified by known methods such as distillation and used in a subsequent hydrogenation reaction.

[0110] One method for producing hexamethylenediamine from adiponitrile is to heat adiponitrile in the presence of hydrogen using a common hydrogenation catalyst such as nickel or cobalt. The produced hexamethylenediamine can be purified by known methods such as distillation and can be suitably used as a raw material for polyamides.

[0111] [Production of polyamides] The adipic acid obtained in this invention can be used to produce polyamides by polycondensation with a diamine using a known method (see, for example, Osamu Fukumoto (ed.), "Polyamide Resin Handbook," Nikkan Kogyo Shuppansha (January 1998)). Specifically, by using 1,4-diaminobutane, 1,5-pentanediamine, and hexamethylenediamine as the diamine, polyamide 46, polyamide 56, and polyamide 66 can be produced, respectively.

[0112] The hexamethylenediamine obtained in this invention can be used to produce polyamides by polycondensation with adipic acid using a known method (for example, see Fukumoto, Osamu (ed.), "Polyamide Resin Handbook," Nikkan Kogyo Shuppansha (January 1998)). Specifically, by using adipic acid and sebacic acid as the dicarboxylic acid, polyamide 6,6 and polyamide 6,10 can be produced, respectively.

[0113] Furthermore, by polycondensing the adipic acid obtained in this invention with the hexamethylenediamine obtained in this invention, polyamide 6,6 in which all monomers are derived from biomass resources can be produced.

[0114] As a method for polymerizing the ε-caprolactam obtained in the present invention to produce polyamide 6, known methods for ring-opening polymerization of ε-caprolactam can be applied (see Fukumoto, Osamu (ed.), "Polyamide Resin Handbook," Nikkan Kogyo Shuppansha (January 1998)).

[0115] Polyamide can be processed by known methods (e.g., WO2019 / 208427) to produce polyamide fibers. The resulting polyamide fibers can be used in clothing applications such as innerwear, sportswear, and casual wear, as well as in industrial materials such as airbags and tire cords.

[0116] Furthermore, polyamide can be molded using known methods (e.g., WO2021 / 006257) to produce polyamide molded articles. The resulting polyamide molded articles can be used in automotive parts, electrical components, electronic components, building materials, various containers, daily necessities, household goods, and sanitary products, etc. [Examples]

[0117] (Reference Example 1) Construction of plasmids expressing enzymes that catalyze the following reactions: the reaction to produce 3OA-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (reaction A); the reaction to produce 3HA-CoA from 3OA-CoA (reaction B); the reaction to produce 3-hydroxyadipic acid from 3HA-CoA (reaction E); the reaction to produce α-hydromuconic acid from HMA-CoA (reaction F); and the reaction to produce adipic acid from ADA-CoA (reaction G). The autologous replication vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166:175-176) in Escherichia coli was cleaved with XhoI to obtain pBBR1MCS-2 / XhoI. To incorporate a constitutive expression promoter into this vector, primers were designed (sequence numbers 22, 23) for PCR amplification of the upstream region 200b (sequence number 21) of gapA (NCBI-GeneID: NC_000913.3) using the genomic DNA of Escherichia coli str.K-12 substr.MG1655 as a template, and PCR reactions were performed according to standard procedures. The obtained fragments and pBBR1MCS-2 / XhoI were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into Escherichia coli strain DH5α. The plasmid was extracted from the obtained recombinant E. coli strain, and its sequence was confirmed by a standard method to obtain pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was cleaved with ScaI to obtain pBBR1MCS-2::Pgap / ScaI. To amplify the gene encoding the enzyme that catalyzes reaction A, primers were designed (sequence numbers 25, 26) for PCR amplification of the full-length acyltransferase gene pcaF (NCBI-GeneID: 1041755, SEQ ID NO: 24) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and the PCR reaction was performed according to a standard method. The obtained fragments and pBBR1MCS-2::Pgap / ScaI were ligated using the "In-Fusion HD Cloning Kit" and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and its nucleotide sequence was confirmed by a standard method. This plasmid was designated as pBBR1MCS-2::AT.

[0118] Next, pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT / HpaI. To amplify the genes encoding the enzymes that catalyze reactions D and E, primers were designed (sequence numbers 29, 30) for PCR amplification of continuous sequences containing the full-length CoA transferase genes pcaI and pcaJ (NCBI-GeneID: 1046613, 1046612, SEQ ID NOs. 27, 28) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and PCR reactions were performed according to standard procedures. The obtained fragments and pBBR1MCS-2::AT / HpaI were ligated using the "In-Fusion HD Cloning Kit" and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pBBR1MCS-2::ATCT.

[0119] pBBR1MCS-2::ATCT was cleaved with ScaI to obtain pBBR1MCS-2::ATCT / ScaI. To amplify the gene encoding the enzyme that catalyzes reaction B, primers for amplifying the nucleic acid described in SEQ ID NO: 31 were designed using the genomic DNA of Serratia marcescens strain ATCC13880 as a template (SEQ ID NOs: 32, 33), and PCR was performed according to standard procedures. The obtained fragments and pBBR1MCS-2::ATCT / ScaI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pBBR1MCS-2::ATCTOR.

[0120] (Reference Example 2) Creation of a template plasmid for gene expression repression For gene expression repression, the method described in Nat. Biotechnol. 2013, 31(2), 170-176. was used. As a template plasmid for gene expression repression, a nucleic acid fragment (SEQ ID NO: 34) with an NcoI restriction enzyme-treated site was introduced to insert a 24-nucleotide nucleic acid that forms the complementary strand of the gene, and this fragment was synthesized. The autoclavable vector pCDF-1b in E. coli was cleaved with XbaI and BamHI to obtain pCDF-1b / XbaI,BamHI. To incorporate the nucleic acid fragment for gene repression into this vector, primers for PCR amplification of the nucleic acid fragment were designed (SEQ ID NOs: 35, 36), and the PCR reaction was performed according to standard procedures. The obtained fragments and pCDF-1b / XbaI,BamHI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by standard procedures was designated as pCDF-1b::temp.

[0121] (Reference Example 3) Creation of a plasmid for suppressing iclR gene expression pCDF-1b::temp was cleaved with NcoI and then dephosphorylated with bacterial alcohol phosphate to obtain pCDF-1b::temp / NcoI,BAP. To incorporate a nucleic acid fragment for suppressing iclR gene expression into this vector, a nucleic acid fragment (SEQ ID NO: 37) containing a 24-base complementary strand including the start codon of the iclR gene was synthesized. This fragment and pCDF-1b::temp / NcoI,BAP were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a standard method was designated as pCDF-1b::iclRi.

[0122] (Reference Example 4) Creation of a plasmid for aceB gene expression The expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.), which can autonomously replicate in E. coli, was cleaved with SacI to obtain pMW119 / SacI. To incorporate a constitutive expression promoter into this vector, primers were designed (sequence numbers 38 and 39) for PCR amplification of the upstream region 200b (sequence number 21) of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli K-12 MG1655 as a template, and the PCR reaction was performed according to standard procedures. The obtained fragments and pMW119 / SacI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pMW119::Pgap.

[0123] Next, pMW119::Pgap was cleaved with KpnI to obtain pMW119::Pgap / KpnI. To amplify the aceB gene encoding malate synthase, primers were designed (sequence numbers 40 and 41) for PCR amplification of the full-length aceB (sequence number 4) from Escherichia coli MG1655 strain, and the PCR reaction was performed according to standard procedures. The obtained fragments and pMW119::Pgap / KpnI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pMW119::aceB.

[0124] (Reference Example 5) Creation of a plasmid for aceA gene expression pMW119::Pgap was cleaved with KpnI to obtain pMW119::Pgap / KpnI. To amplify the aceA gene encoding isocitrate lyase, primers were designed (sequence numbers 42 and 43) for PCR amplification of the full-length aceA (sequence number 2) from Escherichia coli strain MG1655, and PCR was performed according to standard procedures. The obtained fragments and pMW119::Pgap / KpnI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pMW119::aceA.

[0125] (Reference Example 6) Creation of a plasmid for aceK gene expression pMW119::Pgap was cleaved with KpnI to obtain pMW119::Pgap / KpnI. To amplify the aceK gene encoding isocitrate dehydrogenase kinase / phosphatase, primers were designed (sequence numbers 44, 45) for PCR amplification of the full-length aceK (sequence number 6) from Escherichia coli strain MG1655, and PCR was performed according to standard procedures. The obtained fragments and pMW119::Pgap / KpnI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pMW119::aceK.

[0126] (Reference Example 7) Preparation of a plasmid for aceBAK gene expression pMW119::Pgap was cleaved with KpnI to obtain pMW119::Pgap / KpnI. To amplify the aceBAK operon encoding malate synthase, isocitrate lyase, and isocitrate dehydrogenase kinase / phosphatase, a PCR reaction was performed using primers (sequence numbers 40 and 45) for PCR amplification of the full-length aceBAK (sequence number 46) from Escherichia coli strain MG1655, according to a standard procedure. The obtained fragments and pMW119::Pgap / KpnI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by a standard procedure was designated as pMW119::aceBAK.

[0127] (Reference Example 8) Plasmid for expressing an enzyme that catalyzes the reaction (reaction C) that produces HMA-CoA from 3HA-CoA. pMW119::Pgap was cleaved with SphI to obtain pMW119::Pgap / SphI. To amplify the gene encoding the enzyme that catalyzes reaction C, primers were designed (sequence numbers 48, 49) for PCR amplification of the full-length enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 47) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and PCR was performed according to standard procedures. The obtained fragments and pMW119::Pgap / SphI were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and its nucleotide sequence was confirmed by standard procedures. The obtained plasmid was designated pMW119::EH.

[0128] (Reference Example 9) Plasmid for expressing enzymes that catalyze the reaction to produce HMA-CoA from 3HA-CoA (Reaction C) and the reaction to produce ADA-CoA from HMA-CoA (Reaction D) pMW119::EH was cleaved with HindIII to obtain pMW119::EH / HindIII. To amplify the gene encoding the enzyme that catalyzes reaction D, primers were designed (sequence numbers 51, 52) for PCR amplification of the full-length dcaA (NCBI-ProteinID: AAL09094.1, SEQ ID NO: 50) from Acinetobacter baylyi ADP1 strain, and the PCR reaction was performed according to standard procedures. The obtained fragments and pMW119::EH / HindIII were ligated using the “In-Fusion HD Cloning Kit” (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pMW119::EHER.

[0129] (Reference Example 10) Plasmid for expressing the enzyme that catalyzes the reaction (reaction C) that produces HMA-CoA from 3HA-CoA. Primers (SEQ ID NOs. 53, 54) were designed to amplify the region containing the replication start site and chloramphenicol resistance gene of the expression vector pACYCDutet-1 (Novagen), which can autonomously replicate in E. coli. Primers (SEQ ID NOs. 55, 56) were also designed to amplify the upstream region 200b of gapA and the region containing the paaF gene of pMW119::EH. PCR reactions were performed according to standard procedures. The resulting fragments were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pACYC::EH.

[0130] (Reference Example 11) Plasmid preparation for expressing enzymes that catalyze the reaction to produce HMA-CoA from 3HA-CoA (reaction C) and the reaction to produce ADA-CoA from HMA-CoA (reaction D) 2 Primers (SEQ ID NOs. 57, 54) were designed to amplify the region containing the replication start site and chloramphenicol resistance gene of the expression vector pACYCDutet-1 (Novagen), which can autonomously replicate in E. coli. Primers (SEQ ID NOs. 55, 58) were designed to amplify the upstream region 200b of gapA, the paaF gene, and the dcaA gene of pMW119::EHER. PCR reactions were performed according to standard procedures. The resulting fragments were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pACYC::EHER.

[0131] (Reference Example 12) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid. The pBBR1MCS-2::ATCTOR plasmid was introduced into Escherichia coli str.K-12 substr.MG1655 strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin. The resulting strain was then introduced into the pCDF-1b::temp plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin and 50 μg / mL streptomycin.

[0132] The strain was inoculated with a platinum loop into 5 mL of LB medium (containing 25 μg / mL kanamycin and 50 μg / mL streptomycin, adjusted to pH 7, with 10 g / L Bacto tryptone (Difco Laboratories), 5 g / L Bacto yeast extract (Difco Laboratories), and 5 g / L sodium chloride) and cultured with shaking at 30°C for 24 hours at 120 min-1. 0.05 mL of the culture medium was added to 5 mL of Medium I (glucose 10 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) in a test tube, which had been adjusted to pH 6.5, and incubated with shaking at 30°C for 120 min-1 for 24 hours.

[0133] The supernatant obtained by centrifugation of bacterial cells from the culture medium was treated with a Millex-GV membrane (0.22 μm, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS / MS. Quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed, and the production ratio of 3HA to acetic acid, calculated using formula (2), is shown in Table 1.

[0134] [Conditions for quantitative analysis of acetic acid by HPLC] HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation) Column: Shodex Sugar SH1011 (manufactured by Showa Denko Corporation), length 300 mm, inner diameter 8 mm, particle size 6 μm Mobile phase: 0.05M sulfuric acid aqueous solution Flow rate: 0.6mL / min Column temperature: 65℃ Detector: RI.

[0135] [Conditions for quantitative analysis of 3HA by LC-MS / MS] HPLC: 1290 Infinity (manufactured by Agilent Technologies) Column: Synergi hydro-RP (Phenomenex), 100 mm length, 3 mm inner diameter, 2.5 μm particle size Mobile phase: 0.1% formic acid aqueous solution / methanol = 70 / 30 Flow rate: 0.3mL / min Column temperature: 40℃ LC detector: 1260DAD VL+ (210nm) MS / MS: Triple-Quad LC / MS (manufactured by Agilent Technologies) Ionization method: ESI negative mode.

[0136] (Comparative Example 1) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid. To create a strain with suppressed iclR gene expression, the pCDF-1b::iclRi plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and streptomycin 50 μg / mL.

[0137] After culturing the strain in question using the same method as in Reference Example 12, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0138] (Reference Example 13) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid. The pBBR1MCS-2::ATCTOR plasmid was introduced into Escherichia coli str.K-12 substr.W3110 strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin. The resulting strain was then introduced into the pCDF-1b::temp plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin and 50 μg / mL streptomycin.

[0139] After culturing the strain in question using the same method as in Reference Example 12, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0140] (Comparative Example 2) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid. To create a strain with suppressed iclR gene expression, the pCDF-1b::iclRi plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.W3110 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and streptomycin 50 μg / mL.

[0141] After culturing the strain in question using the same method as in Reference Example 12, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0142] (Reference Example 14) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid. The pMW119::Pgap plasmid was introduced into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid, by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0143] The strain was cultured in the same manner as in Reference Example 12, except that 100 μg / mL of ampicillin was used instead of streptomycin. Then, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0144] (Comparative Example 3) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid. To create a strain with enhanced aceB gene expression, the pMW119::aceB plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0145] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0146] (Comparative Example 4) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid. To create a strain with enhanced aceK gene expression, the pMW119::aceK plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0147] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0148] (Example 1) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. To create a strain with enhanced aceA gene expression, the pMW119::aceA plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0149] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0150] (Example 2) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceBAK plasmid. To create strains with enhanced aceBAK operon expression, the pMW119::aceBAK plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0151] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0152] (Reference Example 15) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid. Escherichia coli str.K-12 substr.W3110 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid, was introduced with the pMW119::Pgap plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0153] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0154] (Comparative Example 5) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid. To create a strain with enhanced aceB gene expression, the pMW119::aceB plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.W3110 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0155] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0156] (Comparative Example 6) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid. To create a strain with enhanced aceK gene expression, the pMW119::aceK plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.W3110 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0157] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0158] (Example 3) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. To create a strain with enhanced aceA gene expression, the pMW119::aceA plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.W3110 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0159] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0160] (Example 4) Preparation and culture of Escherichia coli str.K-12 substr.W3110 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceBAK plasmid. To create strains with enhanced aceBAK operon expression, the pMW119::aceBAK plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.W3110 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0161] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0162] (Comparative Example 7) Preparation and culture of ldhA gene-deficient Escherichia coli str.K-12 substr.MG1655 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::Pgap plasmid. First, we created the Escherichia coli str.K-12 substr.MG1655 strain, which lacked the full-length deletion of the ldhA gene encoding lactate dehydrogenase. The gene deletion method followed the procedure described in Proc.Natl.Acad.Sci.USA.2000,97(12):6640-6645. The pKD46 plasmid, necessary for expressing λ Red recombinase, was introduced into the Escherichia coli str.K-12 substr.MG1655 strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 50 μg / mL ampicillin to obtain the E. coli MG1655 / pKD46 strain.

[0163] Next, a nucleic acid fragment for ldhA gene deletion was synthesized. This nucleic acid fragment contains the upstream region 500b of the ldhA gene, the sacB gene, the kanamycin resistance gene, and the downstream region 500b of the ldhA gene on the genome of E. coli MG1655 strain (SEQ ID NO: 59). Primers for PCR amplification of the nucleic acid fragment obtained by gene synthesis were designed (SEQ ID NOs: 60, 61), and PCR reactions were performed according to standard procedures. The obtained fragments were purified by agarose gel electrophoresis and nucleic acid column purification kits and used to create ldhA gene deletion strains. The nucleic acid fragment for ldhA gene deletion was introduced into E. coli MG1655 / pKD46 strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 25 μg / mL of kanamycin. The resulting recombinant strain is E. coli MG1655 / ldhA-sacB-kan, in which the entire ldhA gene sequence is replaced with a sequence containing the full lengths of the sacB gene and the kanamycin resistance gene (sacB-kan sequence).

[0164] Next, a nucleic acid fragment for removing the sacB-kan sequence was synthesized. This fragment contains the upstream and downstream 500b regions of the ldhA gene on the genome of E. coli MG1655 strain (SEQ ID NO: 62). To PCR amplify the nucleic acid fragment obtained by gene synthesis, PCR reactions were performed according to standard procedures using primers SEQ ID NOs: 60 and 61. The obtained fragments were purified by agarose gel electrophoresis and nucleic acid column purification kits and used for sacB-kan sequence removal. The nucleic acid fragment for sacB-kan sequence removal was introduced into the E. coli MG1655 / ldhA-sacB-kan strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 50 g / L sucrose. From the obtained colonies, they were cultured at 30°C on LB agar medium and LB agar medium containing 25 μg / mL kanamycin to select strains that were not resistant to kanamycin. The resulting strain is an E. coli MG1655 / ldhA gene deletion strain, in which the ldhA gene is deleted from the genome.

[0165] The pBBR1MCS-2::ATCTOR plasmid was introduced into E. coli MG1655 / ldhA gene deletion strains by electroporation. After introduction, the strains were cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin. The resulting strains were then introduced into the pMW119::Pgap plasmid by electroporation. After introduction, the strains were cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin.

[0166] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0167] (Example 5) Preparation and culture of ldhA gene-deficient Escherichia coli str.K-12 substr.MG1655 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceA plasmid. To create a strain with enhanced aceA gene expression, the pMW119::aceA plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains lacking the ldhA gene, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0168] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0169] (Example 6) Preparation and culture of ldhA gene-deficient Escherichia coli str.K-12 substr.MG1655 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceBAK plasmid. To create strains with enhanced aceBAK operon expression, the pMW119::aceBAK plasmid was introduced by electroporation into ldhA gene-deleted Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0170] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0171] (Comparative Example 8) Preparation and culture of adhE gene-deficient Escherichia coli str.K-12 substr.W3110 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::Pgap plasmid. First, we created the Escherichia coli str.K-12 substr.W3110 strain, which lacked the full-length adhE gene encoding alcohol dehydrogenase. The gene deletion method followed the procedure described in Proc.Natl.Acad.Sci.USA.2000,97(12):6640-6645. The pKD46 plasmid, necessary for expressing λ Red recombinase, was introduced into the Escherichia coli str.K-12 substr.W3110 strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 50 μg / mL ampicillin to obtain the E. coli W3110 / pKD46 strain.

[0172] Next, a nucleic acid fragment for adhE gene deletion was synthesized. This nucleic acid fragment contains the upstream region 500b of the adhE gene, the sacB gene, the kanamycin resistance gene, and the downstream region 500b of the adhE gene on the genome of E. coli W3110 strain (SEQ ID NO: 63). Primers for PCR amplification of the nucleic acid fragment obtained by gene synthesis were designed (SEQ ID NOs: 64, 65), and PCR reactions were performed according to standard procedures. The obtained fragments were purified by agarose gel electrophoresis and nucleic acid column purification kits and used to create adhE gene deletion strains. The nucleic acid fragment for adhE gene deletion was introduced into E. coli W3110 / pKD46 strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 25 μg / mL of kanamycin. The resulting recombinant strain is E. coli W3110 / adhE-sacB-kan, in which the entire adhE gene sequence is replaced with a sequence containing the full-length sacB gene and kanamycin resistance gene (sacB-kan sequence).

[0173] Next, a nucleic acid fragment for removing the sacB-kan sequence was synthesized. This fragment contains the upstream and downstream 500b regions of the adhE gene on the genome of E. coli W3110 strain (SEQ ID NO: 66). To PCR amplify the nucleic acid fragment obtained by gene synthesis, PCR reactions were performed according to standard procedures using primers SEQ ID NOs: 64 and 65. The obtained fragments were purified by agarose gel electrophoresis and nucleic acid column purification kits and used for sacB-kan sequence removal. The nucleic acid fragment for sacB-kan sequence removal was introduced into the E. coli W3110 / adhE-sacB-kan strain by electroporation. After introduction, the strain was cultured at 30°C on LB agar medium containing 50 g / L sucrose. From the obtained colonies, they were cultured at 30°C on LB agar medium and LB agar medium containing 25 μg / mL kanamycin to select strains that were not resistant to kanamycin. The resulting strain is an E. coli W3110 / adhE gene deletion strain, in which the adhE gene is deleted from the genome.

[0174] The pBBR1MCS-2::ATCTOR plasmid was introduced into E. coli W3110 / adhE gene deletion strains by electroporation. After introduction, the strains were cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin. The resulting strains were then introduced into the pMW119::Pgap plasmid by electroporation. After introduction, the strains were cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin.

[0175] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0176] (Example 7) Preparation and culture of adhE gene-deficient Escherichia coli str.K-12 substr.W3110 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceA plasmid. To create a strain with enhanced aceA gene expression, the pMW119::aceA plasmid was introduced by electroporation into an adhE gene-deficient Escherichia coli str.K-12 substr.W3110 strain that had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0177] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0178] (Example 8) Preparation and culture of adhE gene-deficient Escherichia coli str.K-12 substr.W3110 strain by introducing the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceBAK plasmid. To create a strain with enhanced aceBAK operon expression, the pMW119::aceBAK plasmid was introduced by electroporation into adhE gene-deleted Escherichia coli str.K-12 substr.W3110 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL.

[0179] After culturing the strain in question using the same method as in Reference Example 14, quantitative analysis of acetic acid and 3HA accumulated in the culture supernatant was performed using the same method as in Reference Example 12, and the production ratio of 3HA to acetic acid calculated using formula (2) is shown in Table 1.

[0180] [Table 1]

[0181] The results from Reference Examples 12 and 13 and Comparative Examples 1 and 2 revealed that in strains with suppressed iclR gene expression, 3HA production decreased, and the 3HA production ratio to acetic acid decreased.

[0182] The results from Reference Examples 14 and 15 and Comparative Examples 3-6 revealed that in strains with increased expression levels of the aceB gene or aceK gene, 3HA production was equivalent to or decreased, and the ratio of 3HA production to acetic acid decreased.

[0183] From the results of Reference Examples 14 and 15 and Examples 1-4, it was revealed that strains with increased expression levels of the aceA gene or aceBAK operon produced increased 3HA and improved the 3HA production ratio to acetic acid.

[0184] The results from Comparative Example 7 and Examples 5 and 6 revealed that strains lacking the ldhA gene and exhibiting increased expression of the aceA gene or aceBAK operon showed increased 3HA production and an improved 3HA production ratio to acetic acid.

[0185] The results from Comparative Example 8 and Examples 7 and 8 revealed that strains lacking the adhE gene and exhibiting increased expression of the aceA gene or aceBAK operon showed increased 3HA production and an improved 3HA production ratio to acetic acid.

[0186] (Reference Example 16) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid, and pMW119::EH plasmid. Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::temp plasmid, was then introduced with the pMW119::EH plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, streptomycin 50 μg / mL, and ampicillin 100 μg / mL.

[0187] The strain was inoculated with a platinum loop into 5 mL of LB medium (containing 10 g / L Bacto tryptone (Difco Laboratories), 5 g / L Bacto yeast extract (Difco Laboratories), and 5 g / L sodium chloride) adjusted to pH 7, and cultured with shaking at 30°C for 120 min-1 for 24 hours. 0.05 mL of the culture medium was added to 5 mL of Medium I (glucose 10 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) in a test tube, which had been adjusted to pH 6.5, and cultured with shaking at 30°C for 120 min-1 for 24 hours.

[0188] The supernatant obtained by centrifugation of bacterial cells from the culture medium was treated with a Millex-GV membrane (0.22 μm, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS / MS. Quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed, and the production ratio of HMA to acetic acid, calculated by replacing 3HA with HMA in equation (2), is shown in Table 2.

[0189] [Conditions for quantitative analysis of acetic acid by HPLC] The analysis was carried out under the same conditions as the quantitative analysis of acetic acid by HPLC described in Reference Example 12.

[0190] [Conditions for quantitative analysis of HMA by LC-MS / MS] The analysis was carried out under the same conditions as the quantitative analysis of 3HA by LC-MS / MS described in Reference Example 12.

[0191] (Comparative Example 9) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::iclRi plasmid, and pMW119::EH plasmid. To create a strain with suppressed iclR gene expression, pMW119::EH plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::iclRi plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, streptomycin 50 μg / mL, and ampicillin 100 μg / mL.

[0192] After culturing the strain in question using the same method as in Reference Example 16, quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed using the same method as in Reference Example 12. The production ratio of HMA to acetic acid, calculated by replacing 3HA with HMA in formula (2), is shown in Table 2.

[0193] (Reference Example 17) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid, pMW119::Pgap plasmid, and pACYC::EH plasmid have been introduced. Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::Pgap plasmid, was introduced with the pACYC::EH plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0194] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of HMA to acetic acid calculated using formula (2) is shown in Table 2.

[0195] (Comparative Example 10) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain into which pBBR1MCS-2::ATCTOR plasmid, pMW119::aceB plasmid, and pACYC::EH plasmid have been introduced. To create strains with enhanced aceB gene expression, pACYC::EH plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceB plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0196] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of HMA to acetic acid calculated using formula (2) is shown in Table 2.

[0197] (Comparative Example 11) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceK plasmid, and pACYC::EH plasmid. To create strains with enhanced aceK gene expression, pACYC::EH plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0198] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of HMA to acetic acid calculated using formula (2) is shown in Table 2.

[0199] (Example 9) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pMW119::aceA plasmid, and pACYC::EH plasmid. To create strains with enhanced aceA gene expression, pACYC::EH plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceA plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0200] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of HMA to acetic acid calculated using formula (2) is shown in Table 2.

[0201] (Example 10) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pMW119::aceBAK plasmid, and pACYC::EH plasmid. To create strains with enhanced aceBAK operon expression, pACYC::EH plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceBAK plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0202] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetic acid and HMA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of HMA to acetic acid calculated using formula (2) is shown in Table 2.

[0203] [Table 2]

[0204] The results from Reference Example 16 and Comparative Example 9 revealed that in strains with suppressed iclR gene expression, HMA production decreased, and the HMA production ratio to acetic acid decreased.

[0205] The results from Reference Example 17 and Comparative Examples 10 and 11 revealed that in strains with increased expression levels of the aceB gene or aceK gene, HMA production was equivalent to or decreased, and the HMA production ratio to acetic acid decreased.

[0206] The results from Reference Example 17 and Examples 9 and 10 revealed that in strains with increased expression levels of the aceA gene or aceBAK operon, HMA production increased and the HMA production ratio to acetic acid improved.

[0207] (Reference Example 18) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid, and pMW119::EHER plasmid. Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pCDF-1b::temp plasmid, was then introduced with the pMW119::EHER plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, streptomycin 50 μg / mL, and ampicillin 100 μg / mL.

[0208] The strain was inoculated with a platinum loop into 5 mL of LB medium (containing 10 g / L Bacto tryptone (Difco Laboratories), 5 g / L Bacto yeast extract (Difco Laboratories), and 5 g / L sodium chloride) adjusted to pH 7, and cultured with shaking at 30°C for 120 min-1 for 24 hours. 0.05 mL of the culture medium was added to 5 mL of Medium I (glucose 10 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) in a test tube, which had been adjusted to pH 6.5, and cultured with shaking at 30°C for 120 min-1 for 24 hours.

[0209] The supernatant obtained by centrifugation of bacterial cells from the culture medium was treated with a Millex-GV membrane (0.22 μm, PVDF, Merck), and the permeate was analyzed by HPLC and LC-MS / MS. Quantitative analysis of acetic acid and ADA accumulated in the culture supernatant was performed, and the production ratio of ADA to acetic acid, calculated by replacing 3HA with ADA in equation (2), is shown in Table 3.

[0210] [Conditions for quantitative analysis of acetic acid by HPLC] The analysis was carried out under the same conditions as the quantitative analysis of acetic acid by HPLC described in Reference Example 12.

[0211] [Conditions for quantitative analysis of ADA by LC-MS / MS] The analysis was carried out under the same conditions as the quantitative analysis of 3HA by LC-MS / MS described in Reference Example 12.

[0212] (Comparative Example 12) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::iclRi plasmid, and pMW119::EHER plasmid. To create a strain with suppressed iclR gene expression, pMW119::EHER plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, streptomycin 50 μg / mL, and ampicillin 100 μg / mL.

[0213] After culturing the strain in question using the same method as in Reference Example 16, quantitative analysis of acetic acid and ADA accumulated in the culture supernatant was performed using the same method as in Reference Example 12. The production ratio of ADA to acetic acid, calculated by replacing 3HA with ADA in formula (2), is shown in Table 3.

[0214] (Reference Example 19) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pMW119::Pgap plasmid, and pACYC::EHER plasmid. Escherichia coli str.K-12 substr.MG1655 strains, which had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::Pgap plasmid, were then introduced with the pACYC::EHER plasmid by electroporation. After introduction, the strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0215] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetate and ADA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of ADA to acetate calculated using formula (2) is shown in Table 3.

[0216] (Comparative Example 13) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceB plasmid, and pACYC::EHER plasmid. To create strains with enhanced aceB gene expression, pACYC::EHER plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceB plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0217] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetate and ADA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of ADA to acetate calculated using formula (2) is shown in Table 3.

[0218] (Comparative Example 14) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceK plasmid, and pACYC::EHER plasmid. To create strains with enhanced aceK gene expression, pACYC::EHER plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0219] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetate and ADA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of ADA to acetate calculated using formula (2) is shown in Table 3.

[0220] (Example 11) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pMW119::aceA plasmid, and pACYC::EHER plasmid. To create strains with enhanced aceA gene expression, pACYC::EHER plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0221] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetate and ADA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of ADA to acetate calculated using formula (2) is shown in Table 3.

[0222] (Example 12) Preparation and culture of Escherichia coli str.K-12 substr.MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid, pMW119::aceBAK plasmid, and pACYC::EHER plasmid. To create strains with enhanced aceBAK operon expression, pACYC::EHER plasmid was introduced by electroporation into Escherichia coli str.K-12 substr.MG1655 strains that had been introduced with the pBBR1MCS-2::ATCTOR plasmid and the pMW119::aceBAK plasmid. After introduction, these strains were cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL, ampicillin 100 μg / mL, and chloramphenicol 15 μg / mL.

[0223] The strain was cultured in the same manner as in Reference Example 16, except that chloramphenicol 15 μg / mL was used instead of streptomycin. Then, quantitative analysis of acetate and ADA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12, and the production ratio of ADA to acetate calculated using formula (2) is shown in Table 3.

[0224] [Table 3]

[0225] The results from Reference Example 18 and Comparative Example 12 revealed that strains in which iclR gene expression was suppressed produced the same amount of ADA and the same ratio of ADA production to acetate.

[0226] The results from Reference Example 19 and Comparative Examples 13 and 14 revealed that in strains with increased expression levels of the aceB gene or aceK gene, ADA production decreased, and the ADA production ratio to acetic acid remained the same or decreased.

[0227] The results from Reference Example 19 and Examples 11 and 12 revealed that strains with increased expression levels of the aceA gene or aceBAK operon produced increased ADA and improved the ADA production ratio to acetic acid.

Claims

1. A genetically modified microorganism capable of producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, in which the function of isocitrate lyase is enhanced.

2. The genetically modified microorganism according to claim 1, wherein the enhancement of isocitrate lyase function is achieved by increasing the expression level of the isocitrate lyase gene.

3. The genetically modified microorganism according to claim 1, wherein the enhancement of the function of isocitrate lyase is achieved by increasing the copy number of the isocitrate lyase gene.

4. Furthermore, the genetically modified microorganism according to claim 1, wherein the function of malate synthase and / or isocitrate dehydrogenase / phosphatase is enhanced.

5. The genetically modified microorganism according to claim 4, wherein the enhancement of the function of malate synthase and / or isocitrate dehydrogenase / phosphatase is achieved by increasing the expression level of the malate synthase gene and / or isocitrate dehydrogenase / phosphatase gene.

6. The genetically modified microorganism according to claim 1, wherein the function of the isocitrate lyase repressor is not impaired, or the function of said isocitrate lyase repressor is maintained.

7. The genetically modified microorganism according to claim 6, which is a microorganism that has not undergone genetic modification to suppress the expression of the isocitrate lyase repressor gene.

8. The genetically modified microorganism according to claim 1, further comprising an enhanced reaction that reduces 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA.

9. A method for producing 3-hydroxyadipic acid, α-hydromuconic acid and / or adipic acid, comprising the step of culturing a genetically modified microorganism according to any one of claims 1 to 8.

10. A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid and / or α-hydromuconic acid by the method of claim 9, and reacting 3-hydroxyadipic acid and / or α-hydromuconic acid with hydrogen in the presence of a catalyst.

11. A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid and / or α-hydromuconic acid by the method described in claim 9, and reacting 3-hydroxyadipic acid and / or α-hydromuconic acid with ammonia and hydrogen in the presence of a catalyst.

12. A method for producing 3-hydroxyadipic acid-3,6-lactone, comprising the steps of producing 3-hydroxyadipic acid by the method described in claim 9, and condensing 3-hydroxyadipic acid to produce 3-hydroxyadipic acid-3,6-lactone.

13. A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in claim 12, and reacting 3-hydroxyadipic acid-3,6-lactone with hydrogen in the presence of a catalyst.

14. A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in claim 12, and reacting 3-hydroxyadipic acid-3,6-lactone with ammonia and hydrogen in the presence of a catalyst.

15. A method for producing ε-caprolactam, comprising the steps of producing adipic acid by the method of claim 9, 10, or 13, and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst.

16. A method for producing hexamethylenediamine, comprising the steps of producing adipic acid by the method of claim 9, 10, or 13, and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst.

17. A method for producing a polyamide, comprising the steps of producing adipic acid by the method of claim 9, 10, or 13, and polycondensing adipic acid and a diamine.

18. The method for producing a polyamide according to claim 17, wherein the diamine is a diamine containing 1,4-butanediamine, 1,5-pentanediamine, or hexamethylenediamine.

19. A method for producing a polyamide, comprising the steps of producing hexamethylenediamine by the method described in claim 16, and polycondensing hexamethylenediamine with a dicarboxylic acid.

20. The method for producing a polyamide according to claim 19, wherein the dicarboxylic acid is adipic acid or sebacic acid.

21. A method for producing polyamide 6,6, comprising the steps of producing adipic acid by the method of claim 9, 10, or 13, producing hexamethylenediamine by the method of claim 16, and polycondensing adipic acid and hexamethylenediamine.

22. A method for producing polyamide 6, comprising the steps of producing ε-caprolactam by the method of claim 11, 14, or 15, and polycondensing the ε-caprolactam.

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