Genetically modified microorganism for producing 3-hydroxyhexanedioic acid, (e)-hex-2-enedioic acid, and / or hexanedioic acid, and method for producing said chemicals

Abstract

The present invention makes it possible to improve the production yield of 3-hydroxyhexanedioic acid, (E)-hex-2-enedioic acid, and / or hexanedioic acid while reducing the by-production of acetic acid by enhancing the function of isocitrate lyase in a microorganism capable of producing 3-hydroxyhexanedioic acid, (E)-hex-2-enedioic acid, and / or hexanedioic acid. The present invention further makes it possible to improve the production yield of 3-hydroxyhexanedioic acid by increasing the expression level of YnfM or a homolog thereof in a microorganism capable of producing 3-hydroxyhexanedioic acid.

Description

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

[0001] The present invention relates to genetically modified microorganisms that highly produce 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 said genetically modified microorganisms.

[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 polymerization with a polyhydric alcohol, and as a raw material for a polyamide by polymerization with a polyvalent amine. Further, compounds obtained by adding ammonia to the ends thereof and lactamizing or diamining them can also be used as a raw material for a polyamide.

[0003] As a document related to the production of 3-hydroxyadipic acid, α-hydroxymuconic acid and / or adipic acid using microorganisms, 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 lacking the 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; 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. 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.

[0008] Furthermore, in methods for producing chemicals using microorganisms, transporters are used to expel the target chemical from cells. Patent Document 4 discloses that organophosphate efflux carriers also function as efflux carriers for 3-hydroxyadipic acid, and that the productivity of 3-hydroxyadipic acid can be improved by using genetically modified microorganisms with increased expression levels of organophosphate efflux carriers. Patent Document 5 also discloses that the productivity of 3-hydroxyadipic acid by microorganisms capable of producing 3-hydroxyadipic acid can be improved by deleting or reducing the function of YnfM or its homolog, which is known to function as a dicarboxylic acid efflux carrier.

[0009] WO2019 / 107516 WO2020 / 230718 WO2022 / 102635 WO2023 / 182322 WO2021 / 187533

[0010] Front. Microbiol. 2020, 11, 233.

[0011] 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.

[0012] Furthermore, the present invention aims to create a genetically modified microorganism capable of producing 3-hydroxyadipic acid with high productivity, and a method for producing 3-hydroxyadipic acid using the genetically modified microorganism.

[0013] As a result of diligent research to achieve the above objective, the present inventors have found that in microorganisms capable of producing 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, enhancing the function of isocitrate lyase improves the productivity of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, while decreasing the productivity of acetic acid.

[0014] Furthermore, the inventors have found that the productivity of 3-hydroxyadipic acid can be improved in microorganisms capable of producing 3-hydroxyadipic acid by increasing the expression level of YnfM or its homolog.

[0015] In other words, the present invention comprises the following (1) to (32): (1) A genetically modified microorganism having the ability to produce 3-hydroxyadipic acid, α-hydromuconic acid and / or adipic acid, wherein the function of isocitrate lyase is enhanced. (2) The genetically modified microorganism according to (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 according to (1) or (2), 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 according to any one of (1) to (3), 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) The genetically modified microorganism described in any of (1) to (5), wherein the function of the isocitrate lyase repressor is not reduced or the function is maintained. (7) The genetically modified microorganism described in (6), wherein the microorganism has not undergone genetic modification to suppress the expression of the isocitrate lyase repressor gene. (8) The genetically modified microorganism described in any of (1) to (7), wherein the microorganism belongs to the genus Escherichia or Serratia. (9) A method for producing 3-hydroxyadipic acid, α-hydromuconic acid and / or adipic acid, comprising the step of culturing the 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.(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 (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 (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 (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 (12), and reacting 3-hydroxyadipic acid-3,6-lactone with ammonia and hydrogen in the presence of a catalyst. (15) A genetically modified microorganism according to any one of (1) to (8), wherein the expression level of YnfM or its homolog is further increased. (16) A genetically modified microorganism having the ability to produce 3-hydroxyadipic acid, wherein the expression level of YnfM or its homolog is increased. (17) A genetically modified microorganism according to (16), wherein the expression level of YnfM or its homolog is increased by introducing a gene encoding one of the polypeptides (A) to (C) below. (A) A polypeptide comprising the amino acid sequence of SEQ ID NO: 1. (B) A polypeptide having 60% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 and having a function to improve the productivity of 3-hydroxyadipic acid. (C) A polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having a function to improve the productivity of 3-hydroxyadipic acid. (18) The genetically modified microorganism according to (16) or (17), wherein the microorganism is a microorganism belonging to the genus Escherichia or Serratia.(19) A method for producing 3-hydroxyadipic acid, comprising the step of culturing a genetically modified microorganism described in any of (16) to (18). (20) A method for producing adipic acid, comprising the step of producing 3-hydroxyadipic acid by the method described in (19), and the step of reacting 3-hydroxyadipic acid with hydrogen in the presence of a catalyst. (21) A method for producing ε-caprolactam, comprising the step of producing 3-hydroxyadipic acid by the method described in (19), and the step of reacting 3-hydroxyadipic acid with ammonia and hydrogen in the presence of a catalyst. (22) A method for producing 3-hydroxyadipic acid-3,6-lactone, comprising the step of producing 3-hydroxyadipic acid by the method described in (19), and the step of condensing 3-hydroxyadipic acid to produce 3-hydroxyadipic acid-3,6-lactone. (23) A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in (22), and reacting 3-hydroxyadipic acid-3,6-lactone with hydrogen in the presence of a catalyst. (24) A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in (22), and reacting 3-hydroxyadipic acid-3,6-lactone with ammonia and hydrogen in the presence of a catalyst. (25) A method for producing ε-caprolactam, comprising the steps of producing adipic acid by the method described in (9), (10), (13), (20), or (23), and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst. (26) A method for producing hexamethylenediamine, comprising the steps of producing adipic acid by the method described in (9), (10), (13), (20), or (23), and reacting the adipic acid with ammonia and hydrogen in the presence of a catalyst. (27) A method for producing polyamide, comprising the steps of producing adipic acid by the method described in (9), (10), (13), (20), or (23), and polycondensing the adipic acid and the diamine. (28) The method for producing polyamide according to (27), wherein the diamine is a diamine containing 1,4-butanediamine, 1,5-pentanediamine, or hexamethylenediamine.(29) A method for producing a polyamide, comprising the steps of producing hexamethylenediamine by the method described in (26), and polycondensing hexamethylenediamine with a dicarboxylic acid. (30) The method for producing a polyamide according to (29), wherein the dicarboxylic acid is adipic acid or sebacic acid. (31) A method for producing polyamide 6, 6, comprising the steps of producing adipic acid by the method described in (9), (10), (13), (20), or (23), producing hexamethylenediamine by the method described in (26), and polycondensing adipic acid and hexamethylenediamine. (32) A method for producing polyamide 6, comprising the steps of producing ε-caprolactam by the method described in (11), (14), (21), (24), or (25), and polycondensing ε-caprolactam.

[0016] The genetically modified microorganism according to the present invention can increase the productivity of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid, and further reduce the productivity of acetic acid, compared to the parental microorganism that has not been genetically modified. In other words, the production of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid can be increased, and the production ratio of 3-hydroxyadipic acid, α-hydromuconic acid, and / or adipic acid to acetic acid can be increased.

[0017] Furthermore, the genetically modified microorganisms of the present invention can produce 3-hydroxyadipic acid with higher productivity compared to parental microorganisms that do not have increased expression levels of YnfM or its homolog.

[0018] The present invention will be described in more detail below, but the present invention is not limited to the following embodiments and can be implemented with various modifications within the scope of the gist of the present invention.

[0019] Hereinafter, in this specification, 3-hydroxyadipic acid may be abbreviated as "3HA", α-hydromuconic acid as "HMA", and adipic acid as "ADA". Furthermore, coenzyme A may be abbreviated as "CoA", 3-oxoadipyl-CoA as "3OA-CoA", 3-hydroxyadipyl-CoA as "3HA-CoA", 2,3-dehydroadipyl-CoA as "HMA-CoA", and adipyl-CoA as "ADA-CoA". In addition, the enzyme that catalyzes the reaction to reduce 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA may be described as "3-oxoadipyl-CoA reductase". Furthermore, isocitrate lyase is sometimes referred to as "AceA," malate synthase as "AceB," and isocitrate dehydrogenase kinase / phosphatase as "AceK." Also, nucleic acids that code for functional polypeptides are sometimes referred to as genes. Additionally, the name of a protein coded by a gene may be expressed by changing the first letter of the gene name from lowercase to uppercase.

[0020] [Genetically Modified Microorganisms] The genetically modified microorganisms 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 pathways. The metabolic pathway from glucose to acetyl-CoA is known as glycolysis, and the metabolic pathway to succinyl-CoA is known as a metabolic pathway consisting of the TCA cycle, the glyoxylate cycle, and a supplementation pathway and a reductive TCA cycle that produce oxaloacetate from PEP. Note that "genetic modification" means artificially altering genes.

[0021]

[0022] Microorganisms capable of producing 3HA, HMA, and / or ADA are not particularly limited as long as they have metabolic pathways capable of producing these substances, but it is preferable that they have the ability to produce 3HA, HMA, and / or ADA through some or all of the metabolic pathways in the reaction scheme shown below. Specifically, it is preferable that microorganisms capable of producing 3HA have the ability to produce 3HA through reactions A to B and E in the reaction scheme shown below, more preferably that the activity of the enzymes catalyzing reactions A, B, and / or E 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 preferably have the ability to produce HMA by reactions A to C and F of the reaction scheme shown below, more preferably have enhanced activity of the enzymes catalyzing reactions A, B, C and / or F, even more preferably have enhanced activity of at least the enzymes catalyzing reaction A and / or B, and particularly preferably have enhanced activity of at least the enzyme catalyzing reaction B. Microorganisms capable of producing ADA preferably have the ability to produce ADA by reactions A to D and G, preferably have enhanced activity of the enzymes catalyzing reactions A, B, C, D and / or G, even more preferably have enhanced activity of at least the enzymes catalyzing reaction A and / or B, and particularly preferably have enhanced activity of at least the enzyme catalyzing reaction B.

[0023] Here, reaction A represents the reaction of producing 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA. Reaction B represents the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. Reaction C represents the reaction of producing 2,3-dehydroadipyl-CoA from 3-hydroxyadipyl-CoA. Reaction D represents the reaction of producing adipyl-CoA from 2,3-dehydroadipyl-CoA. Reaction E represents the reaction of producing 3-hydroxyadipic acid from 3-hydroxyadipyl-CoA. Reaction F represents the reaction of producing α-hydromuconic acid from 2,3-dehydroadipyl-CoA. Reaction G represents the reaction of producing adipic acid from adipyl-CoA. The enzymes that catalyze each reaction in the following metabolic pathways, and 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 that catalyze each reaction. Methods for increasing expression levels 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 each enzyme gene into the cell of a host microorganism from outside the cell using the method described in WO2019 / 107516.

[0024]

[0025] The present invention is characterized by enhancing the function of isocitrate lyase in microorganisms capable of producing 3HA, HMA, and / or ADA.

[0026] 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-Protein ID: NP_418439, SEQ ID NO: 35) derived 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: 36.

[0027] 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.

[0028] 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 and the function of malate synthase and / or isocitrate dehydrogenase kinase / phosphatase.

[0029] Malate synthase is an enzyme (EC 2.3.3.9) that catalyzes the reaction that produces malate and CoA from glyoxylic acid and acetyl-CoA. A specific example of malate synthase is AceB (NCBI-Protein ID: NP_418438, SEQ ID NO: 37) derived 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: 38.

[0030] Isocitrate dehydrogenase kinase / phosphatase is an enzyme (EC 2.7.11.5) that catalyzes the phosphorylation of isocitrate dehydrogenase, 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: 39) derived from Escherichia coli str. K-12 substr. MG1655 strain, and a specific example of the aceK gene encoding AceK is the nucleic acid gene of SEQ ID NO: 40.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Furthermore, the genetically modified microorganism of the present invention is technically characterized by increasing the expression level of YnfM or its homolog in microorganisms capable of producing 3-hydroxyadipic acid, thereby improving the productivity of 3-hydroxyadipic acid in microorganisms capable of producing 3-hydroxyadipic acid.

[0039] YnfM is known to function as an efflux carrier for succinic acid and malic acid (see, for example, Japanese Patent Publication No. 2017-216881), and also as an efflux carrier for glutaric acid (see, for example, US Patent No. 2022 / 0205000). On the other hand, YnfM is not known to have efflux activity for hydroxydicarboxylic acids having 5 or more carbon atoms, and Appl. Microbiol. Biotechnol. 2023 (Kataoka et al.) states that YnfM does not have efflux activity for 2-hydroxyglutaric acid. Furthermore, WO2021 / 187533 discloses that the productivity of 3-hydroxyadipic acid by a microorganism capable of producing 3-hydroxyadipic acid can be improved by deleting or reducing the function of YnfM or its homolog, which is known to function as a dicarboxylic acid efflux carrier. Therefore, it is a discovery for those skilled in the art that increasing the expression level of YnfM or its homolog improves the productivity of 3-hydroxyadipic acid, a C6 hydroxydicarboxylic acid, as shown in the examples herein.

[0040] The following describes in detail YnfM and its homologs, which increase the expression level in the present invention.

[0041] YnfM is a protein known as a putative membrane transport protein. A specific example of YnfM is YnfM derived from Escherichia coli, specifically YnfM from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Protein ID: NP_416113, SEQ ID NO: 1). The gene encoding this protein is YnfM from Escherichia coli str. K-12 substr. MG1655 strain (NCBI Gene ID: 946138, SEQ ID NO: 2). Furthermore, any protein that has an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added to the amino acid sequence of SEQ ID NO: 1, and that has the effect of improving the productivity of 3-hydroxyadipic acid by increasing its expression level, can be used as YnfM. Here, the range of "one or several" is preferably 10 or less, more preferably 5 or less, even more preferably 4 or less, and particularly preferably 1 or 2 or less. In addition, any protein that has an amino acid sequence with sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more to the amino acid sequence of SEQ ID NO: 1, and that has the effect of improving the productivity of 3-hydroxyadipic acid by increasing its expression level, can also be used as YnfM.

[0042] A YnfM homolog is a protein that has a high degree of amino acid sequence identity with YnfM (usually 60% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and especially preferably 95% or more) and is presumed to have a similar function or structure to YnfM.

[0043] Homologs of YnfM can be easily obtained, for example, by using the amino acid sequence of YnfM as a query sequence to perform a BLAST search on public databases such as NCBI and KEGG, and referring to the gene encoding the matching sequence. Also, using an oligonucleotide prepared based on the nucleotide sequence of the gene encoding YnfM or its homolog as a primer, it can be obtained by PCR using the genomic DNA of other organisms as a template.

[0044] In the present invention, "sequence identity" means the percentage of identical amino acids or bases with respect to the total overlapping amino acid sequence (including the amino acid that is the translation start point) or nucleotide sequence (including the start codon) in the optimal alignment when two amino acid sequences or nucleotide sequences are aligned with or without introducing gaps, and is calculated by formula (1). In formula (1), the shorter sequence length for comparing YnfM or its homolog is 100 amino acids or 300 bases or more, and when it is less than 100 amino acids or 300 bases, sequence identity is not defined. Sequence identity can be easily examined using BLAST (Basic Local Alignment Search Tool), an algorithm widely used 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 examined using the default parameters. Also, sequence identity can be examined using a similar function incorporated in software such as Genetyx.

[0045] Sequence identity (%) = number of matches (gaps are not counted) / shorter sequence length (length not including the gaps at both ends) × 100... formula (1).

[0046] Specific examples of YnfM homologs include, for instance, MFS transporter (NCBI-ProteinID: USJ84179) derived from Shigella sp. PIB strain, and ynfM derived from Escherichia albertii.membrane protein (NCBI-ProteinID: AHE62265), MFS transporter from Escherichia ruysiae (NCBI-ProteinID: WGM53076), MFS transporter from Citrobacter enshiensis (NCBI-ProteinID: WET38817), Arabinose efflux permease from Enterobacter hormaechei (NCBI-ProteinID: CBK85064), membrane protein from Salmonella bongori protein (NCBI-ProteinID: AID24813), MFS transporter derived from Trabulsiella odontotermitis (NCBI-ProteinID: WHP29441), MFS transporter derived from Yokenella regensburgei (NCBI-ProteinID: QIU88899), Leclercia sp. MFS transporter derived from strain J807 (NCBI-Protein ID: QGU10738), hyperthermic protein derived from Kosakonia radicincitans (NCBI-Protein ID: APG17921), Klebsiella electrica (NCBI-Protein ID: QDI08490), Raoultella sp. MFS transporter derived from strain X13 (NCBI-Protein ID: AXC30147), MFS transporter derived from Franconibacter pulveris (NCBI-Protein ID: XES82270), MFS transporter derived from Buttiauxella ferragutiae (NCBI-Protein ID: UNK59519), hypothetical protein derived from Plaralibacter gergoviae (NCBI-Protein ID: AIR01365), and ynfM derived from Cronobacter sakazakii;Examples include putative MFS superfamily transporter (NCBI-ProteinID: AFJ99489).;

[0047] Examples of microorganisms that originally have the ability to produce 3-hydroxyadipic acid and can be used as parent strains of the genetically modified microorganisms of the present invention include the following microorganisms.

[0048] Genus Escherichia such as Escherichia fergusonii and Escherichia coli.

[0049] Genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia nematodiphila.

[0050] Genus Pseudomonas such as Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotofomans, Pseudomonas chlororaphis subsp. aureofaciens.

[0051] Genus Hafnia such as Hafnia alvei.

[0052] Genus Corynebacterium such as Corynebacterium acetophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, Corynebacterium glutamicum.

[0053] Genus Bacillus such as Bacillus badius, Bacillus megaterium, Bacillus roseus.

[0054] Streptomyces genus such as Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus.

[0055] Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus oxalaticus.

[0056] The genus Acinetobacter includes species such as Acinetobacter baylyi and Acinetobacter radioresistens.

[0057] The genus Alcaligenes, such as Alcaligenes faecalis.

[0058] The genus Nocardioides, such as Nocardioides albus.

[0059] Brevibacterium species, such as Brevibacterium iodinum.

[0060] The genus Delftia, such as Delftia acidovorans.

[0061] The genus Simwellia, including Simwellia blattae.

[0062] Aerobacter species such as Aerobacter cloacae.

[0063] The genus Rhizobium includes species such as Rhizobium radiobacter.

[0064] 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.

[0065] 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.

[0066] The genus Escherichia includes species such as Escherichia fergusonii and Escherichia coli.

[0067] Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia Genus Serratia, such as Entomophila or Serratia nematodiphila.

[0068] The genus Pseudomonas includes species such as Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens.

[0069] Hafnia species such as Hafnia alvei.

[0070] The genus Bacillus, such as Bacillus badius.

[0071] Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus numazuensis, Cupriavidus oxalaticus.

[0072] The genus Acinetobacter includes species such as Acinetobacter baylyi and Acinetobacter radioresistens.

[0073] The genus Alcaligenes, such as Alcaligenes faecalis.

[0074] The genus Delftia, such as Delftia acidovorans.

[0075] The genus Simwellia, including Simwellia blattae.

[0076] 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 members of the genus Thermobifida, such as Thermobifida fusca.

[0077] 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.

[0078] 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 genetically modifiable microorganisms, and may be microorganisms that have the ability to produce 3-hydroxyadipic acid, α-hydromuconic acid and / or adipic acid, or not, but may include genera such as Escherichia, Serratia, Hafnia, Pseudomonas, Corynebacterium, Bacillus, Streptomyces, Cupriavidus, Acinetobacter, and 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, Serratiia, Hafnia, and Pseudomonas are more preferred, and microorganisms belonging to the genera Escherichia or Serratiia are particularly preferred.

[0079] As a method to increase the expression level of YnfM or its homolog, or to enhance the enzyme activity of any of reactions A to G, as described above, one can increase the copy number of the gene encoding the polypeptide having these functions by introducing it into the host microorganism from outside the cell to inside the cell, or by modifying the promoter region or ribosome binding sequence upstream of the gene to increase the expression level of various polypeptides. These methods may be performed individually or in combination. The gene to be introduced may be artificially synthesized based on the amino acid sequence of the enzyme that exists in a database, or it may be isolated from nature. When artificially synthesized, the frequency of use of codons corresponding to each amino acid may be changed to suit the host microorganism to which it is introduced. The method of introducing nucleic acids is not particularly limited, and methods such as incorporating the nucleic acid into an expression vector that can autonomously replicate within the microorganism and introducing it into the host microorganism, or incorporating the nucleic acid into the genome of the microorganism can be used.

[0080] The genetically modified microorganism of the present invention may be introduced with one or more genes. Furthermore, the introduction of genes and the enhancement of gene expression may be combined. It is preferable that the genetically modified microorganism of the present invention maintains 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.

[0081] The method for introducing a gene to create the genetically modified microorganism of the present invention is not particularly limited. Methods include 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.

[0082] One or more genes may be introduced. Furthermore, gene introduction and gene expression enhancement may be combined.

[0083] 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.

[0084] The promoter used in the present 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).

[0085] In the present invention, when an expression vector is used to introduce a gene or enhance gene expression, there are no particular limitations as long as it can autonomously replicate in 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.

[0086] In the present invention, when introducing genes or enhancing gene expression using nucleic acids for genome integration, site-directed homologous recombination is used for introduction. 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.).

[0087] 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.).

[0088] 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.

[0089] The production ratio of 3HA to acetic acid = 3HA production (g / L) / acetic acid production (g / L) ... Equation (2).

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] If foaming occurs during liquid culture, defoaming agents such as mineral oil, silicone oil, and surfactants can be appropriately added to the culture medium.

[0099] 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.

[0100] It is known that 3-hydroxyadipic acid exists as 3-hydroxyadipic acid-3,6-lactone under acidic conditions through carboxyl group condensation (see WO2020 / 196459). By appropriately combining the above methods, 3-hydroxyadipic acid-3,6-lactone can also be recovered from cultures containing 3-hydroxyadipic acid. The form of 3-hydroxyadipic acid-3,6-lactone is not particularly limited as long as it is obtained from the 3-hydroxyadipic acid of the present invention. For example, it may be a carboxylic acid, a carboxylate salt, a carboxylic acid ester, or a mixture thereof. In the present invention, these are collectively referred to as "3-hydroxyadipic acid-3,6-lactone".

[0101] [Production of Adipic Acid] Adipic acid can be produced by reacting (hydrogenating) 3-hydroxyadipic acid and / or α-hydromuconic acid obtained in the present invention with hydrogen in the presence of a hydrogenation catalyst. Alternatively, adipic acid can be produced by reacting (hydrogenating) 3-hydroxyadipic acid-3,6-lactone, obtained by condensing 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, and 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.

[0102]

[0103] The hydrogenation catalyst preferably contains a transition metal element, and more preferably contains one or more elements selected from the group consisting of palladium, platinum, ruthenium, rhodium, rhenium, nickel, cobalt, iron, iridium, osmium, copper, and chromium, and more preferably contains one or more elements selected from the group consisting of palladium, platinum, nickel, cobalt, iron, copper, and chromium.

[0104] 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.

[0105] 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, at room temperature, it is preferably 0.1 MPa to 10 MPa (gauge pressure), more preferably 0.3 MPa to 5 MPa (gauge pressure), and even more preferably 0.5 MPa to 3 MPa (gauge pressure).

[0106] 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.

[0107] 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.

[0108] 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 deterioration 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 material 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).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] The pH of the aqueous solvent is not particularly limited, but considering the suppression of catalyst degradation, the suppression of by-product formation, and corrosion to the reactor, it is preferably pH 2 to 13, more preferably pH 3 to 11, and even more preferably pH 4 to 10.

[0114] 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."

[0115] 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.

[0116] The adipic acid obtained in this invention can be separated and purified by conventional unit operations such as centrifugation, filtration, membrane filtration, distillation, extraction, crystallization, and drying.

[0117] [Production 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.

[0118] 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 a metal catalyst such as Ru, Pt, or Pd in ​​the presence of hydrogen and ammonia. 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.

[0119] [Production 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.

[0120] 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.

[0121] 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.

[0122] [Production of Polyamides] The adipic acid obtained in the present invention can be polycondensed with a diamine by a known method (see, for example, Fukumoto Osamu (ed.), "Polyamide Resin Handbook," Nikkan Kogyo Shuppansha (January 1998)) to produce polyamides. 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.

[0123] Polyamides can be produced by polycondensation of the hexamethylenediamine obtained in the present invention with adipic acid using a known method (for example, see Fukumoto, Osamu (ed.), "Polyamide Resin Handbook," Nikkan Kogyo Shuppansha (January 1998)). Specifically, polyamides 6,6 and 6,10 can be produced by using adipic acid and sebacic acid as the dicarboxylic acid, respectively.

[0124] Furthermore, by polycondensing the adipic acid obtained in the present invention and the hexamethylenediamine obtained in the present invention, polyamide 6,6 in which all monomers are derived from biomass resources can be produced.

[0125] 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)).

[0126] 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.

[0127] Furthermore, polyamide can be molded using known methods (for example, 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 hygiene products.

[0128] (Reference Example 1) Construction of plasmids expressing enzymes that catalyze the reactions of producing 3OA-CoA and coenzyme A from acetyl-CoA and succinyl-CoA (Reaction A), producing 3HA-CoA from 3OA-CoA (Reaction B), producing 3-hydroxyadipic acid from 3HA-CoA (Reaction E), producing α-hydromuconic acid from HMA-CoA (Reaction F), and producing adipic acid from ADA-CoA (Reaction G). The vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166:175-176), which can autonomously replicate in E. coli, was cleaved with XhoI to obtain pBBR1MCS-2 / XhoI. In order to incorporate a constitutive expression promoter into the vector, Escherichia coli str. K-12 substr. Using the genomic DNA of MG1655 as a template, primers were designed (sequence numbers 56, 57) for PCR amplification of the upstream region 200b (sequence number 55) of gapA (NCBI-GeneID: NC_000913.3), 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 E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the plasmid whose base sequence was confirmed by standard procedures was designated as pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was cleaved with ScanI to obtain pBBR1MCS-2::Pgap / ScaI. To amplify the gene encoding the enzyme that catalyzes reaction A, primers were designed (sequence numbers 59, 60) for PCR amplification of the full-length acyltransferase gene pcaF (NCBI-GeneID: 1041755, SEQ ID NO: 58) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and PCR was performed according to standard procedures. The resulting fragment 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 resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pBBR1MCS-2::AT.

[0129] Next, pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT / HpaI. To amplify the genes encoding the enzymes that catalyze reactions E, F, and G, primers were designed (sequence numbers 63, 64) for PCR amplification of continuous sequences containing the full-length CoA transferase genes pcaI and pcaJ (NCBI-GeneID: 1046613, 1046612, SEQ ID NOs: 61, 62) 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 obtained recombinant strain, and its nucleotide sequence was confirmed by a standard method. This plasmid was designated as pBBR1MCS-2::ATCT.

[0130] pBBR1MCS-2::ATCT was cleaved with CaI 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: 65 were designed using the genomic DNA of Serratia marcescens strain ATCC13880 as a template (SEQ ID NOs: 66, 67), and PCR was performed according to a standard procedure. 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 base sequence was confirmed by a standard procedure was designated as pBBR1MCS-2::ATCTOR.

[0131] (Reference Example 2) Construction of a template plasmid for gene expression repression The method described in Nat. Biotechnol. 2013, 31(2), 170-176. was used for gene expression repression. As a template plasmid for gene expression repression, a nucleic acid fragment (SEQ ID NO: 68) into which an NcoI restriction enzyme-treated site was introduced was synthesized to insert a 24-base nucleic acid that forms the complementary strand of the gene. The vector pCDF-1b, which can autonomously replicate in E. coli, was cleaved with XbaI and BamHI to obtain pCDF-1b / XbaI,BamHI. In order to incorporate the nucleic acid fragment for gene repression into this vector, primers for PCR amplification of the nucleic acid fragment for gene repression were designed (SEQ ID NOs: 69, 70), and the PCR reaction was performed according to a standard method. The obtained fragments and pCDF-1b / XbaI,BamHI 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 strain, and the plasmid whose nucleotide sequence was confirmed by a standard method was designated as pCDF-1b::temp.

[0132] (Reference Example 3) Preparation of Plasmid for IclR Gene Expression Repression pCDF-1b::temp was cleaved with NcoI and then dephosphorylated with Bacterial Alkaline Phosphate to obtain pCDF-1b::temp / NcoI,BAP. To incorporate a nucleic acid fragment for IclR gene expression repression into this vector, a nucleic acid fragment (SEQ ID NO: 71) 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" (manufactured by 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.

[0133] (Reference Example 4) Production of a plasmid for aceB gene expression The expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.), which can autonomously replicate in Escherichia coli, was cut with SacI to obtain pMW119 / SacI. In order to incorporate a constitutive expression promoter into this vector, primers were designed (sequence number 72, 73) for PCR amplification of the upstream region 200b (sequence number 55) of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli K-12 MG1655 as a template, and a PCR reaction was performed according to the standard procedure. The obtained fragments and pMW119 / SacI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into the Escherichia coli strain DH5α. The plasmid was extracted from the obtained recombinant E. coli strain, and its nucleotide sequence was confirmed by a standard method. The plasmid was designated pMW119::Pgap.

[0134] 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 74, 75) for PCR amplification of the full-length aceB (sequence number 38) from Escherichia coli MG1655 strain, and the PCR reaction was performed 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::aceB.

[0135] (Reference Example 5) Preparation of 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 (SEQ ID NOs: 76, 77) for PCR amplification of the full-length aceA (SEQ ID NO: 36) from Escherichia coli MG1655 strain, and a PCR reaction was performed according to a standard procedure. The obtained fragments and pMW119::Pgap / KpnI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a standard procedure was designated as pMW119::aceA.

[0136] (Reference Example 6) Preparation of 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 (SEQ ID NOs: 78, 79) for PCR amplification of the full-length aceK (SEQ ID NO: 40) from Escherichia coli MG1655 strain, and a PCR reaction was performed according to a standard procedure. The obtained fragments and pMW119::Pgap / KpnI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into Escherichia coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a standard procedure was designated as pMW119::aceK.

[0137] (Reference Example 7) Preparation of aceBAK gene expression plasmid 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 according to a standard procedure using primers (sequence numbers 74, 79) for PCR amplification of the full-length aceBAK (sequence number 80) from Escherichia coli MG1655 strain. The obtained fragments and pMW119::Pgap / KpnI were ligated using the "In-Fusion HD Cloning Kit" (manufactured by Takara Bio Inc.) and introduced into Escherichia 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 pMW119::aceBAK.

[0138] (Reference Example 8) Preparation of a plasmid for expressing an enzyme that catalyzes the reaction (reaction C) to produce 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 82, 83) for PCR amplification of the full length of the enoyl-CoA hydratase gene paaF (NCBI Gene ID: 1046932, SEQ ID NO: 81) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and the PCR reaction was performed according to the standard procedure. 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 a standard method. The obtained plasmid was designated pMW119::EH.

[0139] (Reference Example 9) Preparation of plasmids 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 85, 86) for PCR amplification of the full length of dcaA (NCBI-Protein ID: AAL09094.1, SEQ ID NO: 84) from Acinetobacter baylyi ADP1 strain, and PCR reactions were performed according to the standard procedure. The obtained fragments and pMW119::EH / HindIII 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 strain, and the plasmid whose nucleotide sequence was confirmed by a standard method was designated as pMW119::EHER.

[0140] (Reference Example 10) Plasmid for expressing an enzyme that catalyzes the reaction (reaction C) to produce HMA-CoA from 3HA-CoA 2 Primers (SEQ ID NOs. 87, 88) for amplifying 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, and primers (SEQ ID NOs. 89, 90) for amplifying the upstream region 200b of gapA and the region containing the paaF gene of pMW119::EH were designed, and PCR reactions were performed according to standard procedures. The obtained 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 obtained recombinant strain, and the plasmid whose base sequence was confirmed by standard procedures was designated as pACYC::EH.

[0141] (Reference Example 11) Plasmid production 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. 91, 88) for amplifying 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, and primers (SEQ ID NOs. 89, 92) for amplifying the upstream region 200b of gapA in pMW119::EHER, the paaF gene, and the dcaA gene were designed, and PCR reactions were performed according to the standard method. The obtained 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 obtained recombinant strain, and its nucleotide sequence was confirmed by a standard method. The plasmid was designated as pACYC::EHER.

[0142] (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 kanamycin 25 μg / mL. The pCDF-1b::temp plasmid was introduced into the resulting strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and streptomycin 50 μg / mL.

[0143] 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) which was pH 7 and contained 25 μg / mL kanamycin and 50 μg / mL streptomycin, and then cultured with shaking at 30°C and 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 and 120 min⁻¹ for 24 hours.

[0144] The supernatant obtained by centrifugation of bacterial cells from the culture medium was treated with Millex-GV (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.

[0145] [Conditions for quantitative analysis of acetic acid by HPLC] HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation) Column: Shodex Sugar SH1011 (manufactured by Showa Denko K.K.), length 300 mm, inner diameter 8 mm, particle size 6 μm Mobile phase: 0.05 M Sulfuric acid aqueous solution flow rate: 0.6 mL / min Column temperature: 65 °C Detector: RI.

[0146] [Conditions for quantitative analysis of 3HA by LC-MS / MS] HPLC: 1290 Infinity (Agilent Technologies) Column: Synergi hydro-RP (Phenomenex), length 100 mm, inner diameter 3 mm, particle size 2.5 μm Mobile phase: 0.1% formic acid aqueous solution / methanol = 70 / 30 Flow rate: 0.3 mL / min Column temperature: 40°C LC detector: 1260 DAD VL+ (210 nm) MS / MS: Triple-Quad LC / MS (Agilent Technologies) Ionization method: ESI negative mode.

[0147] (Comparative Example 1) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid In order to prepare a strain with suppressed iclR gene expression, pCDF-1b::iclRi plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with 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 streptomycin 50 μg / mL.

[0148] After culturing the strain in the same manner as in Reference Example 12, 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.

[0149] (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 kanamycin 25 μg / mL. The pCDF-1b::temp plasmid was introduced into the resulting strain by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing kanamycin 25 μg / mL and streptomycin 50 μg / mL.

[0150] After culturing the strain in the same manner as in Reference Example 12, 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.

[0151] (Comparative Example 2) Preparation and Culture of Escherichia coli str. K-12 substr. W3110 strain with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::iclRi plasmid In order to prepare a strain with suppressed iclR gene expression, pCDF-1b::iclRi plasmid was introduced into Escherichia coli str. K-12 substr. W3110 strain with 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 streptomycin 50 μg / mL.

[0152] After culturing the strain in the same manner as in Reference Example 12, 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.

[0153] (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. Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid, was introduced with 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.

[0154] 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.

[0155] (Comparative Example 3) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid To prepare a strain with enhanced aceB gene expression, pMW119::aceB plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with 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.

[0156] 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.

[0157] (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 prepare a strain with enhanced aceK gene expression, pMW119::aceK plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with 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.

[0158] 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.

[0159] (Example 1) Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. In order to prepare a strain with enhanced aceA gene expression, pMW119::aceA plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with 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.

[0160] 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.

[0161] (Example 2) Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceBAK plasmid. In order to prepare a strain with enhanced aceBAK operon expression, pMW119::aceBAK plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with 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.

[0162] 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.

[0163] (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 introduced with pBBR1MCS-2::ATCTOR plasmid was introduced with 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.

[0164] 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.

[0165] (Comparative Example 5) Preparation and Culture of Escherichia coli str. K-12 substr. W3110 strain with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB plasmid To prepare a strain with enhanced aceB gene expression, pMW119::aceB plasmid was introduced into Escherichia coli str. K-12 substr. W3110 strain with 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.

[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] (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 prepare a strain with enhanced aceK gene expression, pMW119::aceK plasmid was introduced into Escherichia coli str. K-12 substr. W3110 strain, which had been introduced with 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.

[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 3) Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. In order to prepare a strain with enhanced aceA gene expression, pMW119::aceA plasmid was introduced into Escherichia coli str. K-12 substr. W3110 strain, which had been introduced with 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.

[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] (Example 4) Preparation and culture of Escherichia coli str. K-12 substr. W3110 strain with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceBAK plasmid. In order to prepare a strain with enhanced aceBAK operon expression, pMW119::aceBAK plasmid was introduced into Escherichia coli str. K-12 substr. W3110 strain with 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.

[0172] 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.

[0173] (Comparative Example 7) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain with ldhA gene deletion by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid First, Escherichia coli str. K-12 substr. MG1655 strain was prepared by deleting the entire length of the ldhA gene encoding lactate dehydrogenase. The gene deletion method was carried out according to the method 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 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.

[0174] 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: 93). Primers for PCR amplification of the nucleic acid fragment obtained by gene synthesis were designed (SEQ ID NOs: 94, 95), 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 entire sacB gene and kanamycin resistance gene (sacB-kan sequence).

[0175] Next, a nucleic acid fragment for removing the sacB-kan sequence was synthesized. This fragment contains the upstream and downstream regions 500b of the ldhA gene on the genome of E. coli MG1655 strain (SEQ ID NO: 96). To PCR amplify the nucleic acid fragment obtained by gene synthesis, a PCR reaction was performed using primers SEQ ID NOs: 94 and 95 according to a standard procedure. The obtained fragment was purified by agarose gel electrophoresis and nucleic acid column purification kit 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. The obtained colonies were cultured at 30°C on LB agar medium and LB agar medium containing 25 μg / mL kanamycin, and strains without kanamycin resistance were selected. The resulting strains were E. coli MG1655 / ldhA gene deletion strains, in which the ldhA gene in the genome was deleted.

[0176] The pBBR1MCS-2::ATCTOR plasmid was introduced into the E. coli MG1655 / ldhA gene deletion 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 pMW119::Pgap plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin.

[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 5) Preparation and culture of ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. In order to prepare a strain with enhanced aceA gene expression, the pMW119::aceA plasmid was introduced by electroporation into ldhA gene-deleted 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.

[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] (Example 6) Preparation and culture of ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::aceBAK plasmid. In order to prepare a strain with enhanced aceBAK operon expression, pMW119::aceBAK plasmid was introduced by electroporation into ldhA gene-deleted Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with 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.

[0181] 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.

[0182] (Comparative Example 8) Preparation and Culture of Escherichia coli str. K-12 substr. W3110 strain with adhE gene deletion by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid First, Escherichia coli str. K-12 substr. W3110 strain was prepared by deleting the entire length of the adhE gene that encodes alcohol dehydrogenase. The gene deletion method was carried out according to the method 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 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.

[0183] 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: 97). Primers for PCR amplification of the nucleic acid fragment obtained by gene synthesis were designed (SEQ ID NOs: 98, 99), 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 entire sacB gene and kanamycin resistance gene (sacB-kan sequence).

[0184] Next, a nucleic acid fragment for removing the sacB-kan sequence was synthesized. This fragment contains the upstream and downstream regions 500b of the adhE gene on the genome of E. coli W3110 strain (SEQ ID NO: 100). To PCR amplify the nucleic acid fragment obtained by gene synthesis, a PCR reaction was performed using primers SEQ ID NOs: 98 and 99 according to a standard procedure. The obtained fragment was purified by agarose gel electrophoresis and nucleic acid column purification kit 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. The obtained colonies were cultured at 30°C on LB agar medium and LB agar medium containing 25 μg / mL kanamycin, and strains without kanamycin resistance were selected. The resulting strains were E. coli W3110 / adhE gene deletion strains, in which the adhE gene was deleted from the genome.

[0185] The pBBR1MCS-2::ATCTOR plasmid was introduced into the E. coli W3110 / adhE gene deletion 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 pMW119::Pgap plasmid by electroporation. After introduction, the strain was cultured at 37°C on LB agar medium containing 25 μg / mL kanamycin and 100 μg / mL ampicillin.

[0186] 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.

[0187] (Example 7) Preparation and culture of adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA plasmid. In order to prepare a strain with enhanced aceA gene expression, pMW119::aceA plasmid was introduced by electroporation into adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain that had been introduced with 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.

[0188] 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.

[0189] (Example 8) Preparation and culture of adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain by introducing pBBR1MCS-2::ATCTOR plasmid and pMW119::aceBAK plasmid. In order to prepare a strain with enhanced aceBAK operon expression, pMW119::aceBAK plasmid was introduced by electroporation into adhE gene-deleted Escherichia coli str. K-12 substr. W3110 strain that had been introduced with 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.

[0190] 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.

[0191]

[0192] The results from Reference Examples 12 and 13 and Comparative Examples 1 and 2 revealed that in strains with suppressed iclR gene expression, the production of 3HA decreased, and the production ratio of 3HA to acetic acid declined.

[0193] From the results of Reference Examples 14 and 15 and Comparative Examples 3 to 6, it became clear that in strains with increased expression levels of the aceB gene or aceK gene, the production of 3HA was the same or decreased, and the production ratio of 3HA to acetic acid decreased.

[0194] From the results of Reference Examples 14 and 15 and Examples 1 to 4, it became clear that in strains with increased expression levels of the aceA gene or aceBAK operon, the production of 3HA increased, and the ratio of 3HA production to acetic acid improved.

[0195] The results from Comparative Example 7 and Examples 5 and 6 revealed that in strains lacking the ldhA gene and with increased expression levels of the aceA gene or aceBAK operon, 3HA production increased, and the 3HA production ratio to acetic acid improved.

[0196] The results from Comparative Example 8 and Examples 7 and 8 revealed that in strains lacking the adhE gene and with increased expression levels of the aceA gene or aceBAK operon, 3HA production increased, and the 3HA production ratio to acetic acid improved.

[0197] (Reference Example 16) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid and pMW119::EH plasmid. Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid was introduced with 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.

[0198] The strain was inoculated into 5 mL of LB medium (containing 10 g / L of Bacto Tryptone (Difco Laboratories), 5 g / L of Bacto Yeast Extract (Difco Laboratories), and 5 g / L of sodium chloride) which was pH 7 and contained 25 μg / mL of kanamycin, 50 μg / mL of streptomycin, and 100 μg / mL of ampicillin. The culture was then incubated with a platinum loop at 30°C and 120 min⁻¹ for 24 hours with shaking. 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 and 120 min⁻¹ for 24 hours.

[0199] The supernatant obtained by centrifugation of bacterial cells from the culture medium was treated with Millex-GV (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.

[0200] [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.

[0201] [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.

[0202] (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. In order to prepare a strain in which the expression of the iclR gene is suppressed, pMW119::EH 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.

[0203] 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 in formula (2) with HMA, is shown in Table 2.

[0204] (Reference Example 17) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::Pgap plasmid and pACYC::EH plasmid. Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid, was introduced with 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.

[0205] 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.

[0206] (Comparative Example 10) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceB plasmid and pACYC::EH plasmid To prepare a strain with enhanced aceB gene expression, pACYC::EH plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceB 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.

[0207] 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.

[0208] (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 prepare a strain with enhanced aceK gene expression, pACYC::EH plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceK 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.

[0209] 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.

[0210] (Example 9) Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceA plasmid and pACYC::EH plasmid. In order to prepare a strain with enhanced aceA gene expression, pACYC::EH plasmid was introduced into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceA 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.

[0211] 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.

[0212] (Example 10) Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceBAK plasmid and pACYC::EH plasmid. In order to prepare a strain with enhanced aceBAK operon expression, pACYC::EH plasmid was introduced by electroporation into Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::aceBAK plasmid. 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.

[0213] 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.

[0214]

[0215] 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.

[0216] From the results of Reference Example 17 and Comparative Examples 10 and 11, it became clear 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.

[0217] From the results of Reference Example 17 and Examples 9 and 10, it became clear 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.

[0218] (Reference Example 18) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pCDF-1b::temp plasmid and pMW119::EHER plasmid. Escherichia coli str. K-12 substr. MG1655 strain, which was introduced with pBBR1MCS-2::ATCTOR plasmid and pCDF-1b::temp plasmid, was introduced with 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.

[0219] The strain was inoculated into 5 mL of LB medium (containing 10 g / L of Bacto Tryptone (Difco Laboratories), 5 g / L of Bacto Yeast Extract (Difco Laboratories), and 5 g / L of sodium chloride) which was pH 7 and contained 25 μg / mL of kanamycin, 50 μg / mL of streptomycin, and 100 μg / mL of ampicillin. The culture was then incubated with a platinum loop at 30°C and 120 min⁻¹ for 24 hours with shaking. 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 and 120 min⁻¹ for 24 hours.

[0220] 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 ADA production ratio to acetic acid, calculated by replacing 3HA with ADA in equation (2), is shown in Table 3.

[0221] [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.

[0222] [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.

[0223] (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. In order to prepare a strain in which the expression of the iclR gene is suppressed, 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.

[0224] After culturing the strain in the same manner as in Reference Example 16, quantitative analysis of acetic acid and ADA accumulated in the culture supernatant was performed in the same manner as in Reference Example 12. The production ratio of ADA to acetic acid, calculated by replacing 3HA in formula (2) with ADA, is shown in Table 3.

[0225] (Reference Example 19) Preparation and Culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::Pgap plasmid and pACYC::EHER plasmid. Escherichia coli str. K-12 substr. MG1655 strain, which had been introduced with pBBR1MCS-2::ATCTOR plasmid and pMW119::Pgap plasmid, was introduced with pACYC::EHER 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.

[0226] 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.

[0227] (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 In order to prepare a strain with enhanced aceB gene expression, pACYC::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 pMW119::aceB plasmid. 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.

[0228] 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.

[0229] (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 In order to prepare a strain with enhanced aceK gene expression, pACYC::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 pMW119::aceK plasmid. 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.

[0230] 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.

[0231] (Example 11) Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceA plasmid and pACYC::EHER plasmid. In order to prepare a strain with enhanced aceA gene expression, pACYC::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 pMW119::aceA plasmid. 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.

[0232] 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.

[0233] (Example 12) Preparation and culture of Escherichia coli str. K-12 substr. MG1655 strain introduced with pBBR1MCS-2::ATCTOR plasmid, pMW119::aceBAK plasmid and pACYC::EHER plasmid. In order to prepare a strain with enhanced aceBAK operon expression, pACYC::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 pMW119::aceBAK plasmid. 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.

[0234] 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.

[0235]

[0236] The results from Reference Example 18 and Comparative Example 12 revealed that in strains with suppressed iclR gene expression, the amount of ADA produced and the ratio of ADA production to acetate were equivalent.

[0237] 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 acetate remained the same or decreased.

[0238] From the results of Reference Example 19 and Examples 11 and 12, it became clear that in strains with increased expression levels of the aceA gene or aceBAK operon, ADA production increased and the ADA production ratio to acetic acid improved.

[0239] (Reference Example 20) Plasmid expression of enzymes that catalyze the reaction (reaction A) to produce 3OA-CoA and CoA from acetyl-CoA and succinyl-CoA, the reaction (reaction B) to produce 3HA-CoA from 3OA-CoA, and the reaction (reaction E) to produce 3HA from 3HA-CoA. The vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166:175-176), which is capable of autonomous replication in E. coli and contains a kanamycin resistance gene, was cleaved with XhoI to obtain pBBR1MCS-2 / XhoI. In order to incorporate a constitutive expression promoter into this vector, Escherichia coli str. K-12 substr. Using the genomic DNA of MG1655 as a template, primers were designed (sequence numbers 4 and 5) to amplify the upstream region 200b (sequence number 3) of gapA (NCBI GeneID: NC_000913.3), and PCR was 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 E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the plasmid whose base sequence was confirmed by standard procedures was designated as pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was cleaved with ScanI to obtain pBBR1MCS-2::Pgap / ScaI. To amplify the gene encoding the enzyme that catalyzes reaction A, primers were designed (sequence numbers 7 and 8) to amplify the full length of the acyltransferase gene pcaF (NCBI GeneID: 1041755, SEQ ID NO: 6) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and PCR was performed according to standard procedures. The resulting fragment and pBBR1MCS-2::Pgap / ScaI were ligated using the "In-Fusion HD Cloning Kit" (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pBBR1MCS-2::AT.

[0240] Next, pBBR1MCS-2::AT was cleaved with HpaI to obtain pBBR1MCS-2::AT / HpaI. To amplify the gene encoding the enzyme that catalyzes reaction E, primers were designed (sequence numbers 11, 12) to amplify the continuous sequences containing the full-length CoA transferase genes pcaI and pcaJ (NCBI GeneID: 1046613, 1046612; SEQ ID NOs: 9, 10) 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 obtained recombinant strain, and its nucleotide sequence was confirmed by a standard method. This plasmid was designated as pBBR1MCS-2::ATCT.

[0241] pBBR1MCS-2::ATCT was cleaved with CaI to obtain pBBR1MCS-2::ATCT / ScaI. To amplify the nucleic acid encoding the polypeptide of SEQ ID NO: 13 that catalyzes reaction B, primers were designed (SEQ ID NOs: 15, 16) to amplify the full length of the enzyme gene (SEQ ID NO: 14) that catalyzes reaction B, using the genomic DNA of Serratia marcescens strain ATCC13880 as a template, and PCR was performed according to a standard procedure. 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 base sequence was confirmed by a standard procedure was designated as pBBR1MCS-2::ATCTOR.

[0242] (Reference Example 21) Preparation of a plasmid expressing YnfM To amplify the gene encoding YnfM and its surrounding region, PCR was performed using the genomic DNA of Escherichia coli str. K-12 substr. BW25113 as a template and the nucleic acids of SEQ ID NOs. 17 and 18 as primers to amplify the nucleic acid containing the full length of the ynfM gene (SEQ ID NO. 2) and its upstream and downstream regions (0.5 kb). This PCR fragment and the fragment obtained by cleaving the expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.), which can autonomously replicate in E. coli, with KpnI were ligated using the In-Fusion HD Cloning Kit and introduced into E. coli DH5α strain. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose base sequence was confirmed by a standard method was designated as pMW::EcYNFM.

[0243] (Reference Example 22) Preparation of a 3HA-producing strain (control strain) without increased YnfM expression A control 3HA-producing strain was prepared by introducing the plasmids pBBR1MCS-2::ATCTOR and pMW119, prepared in Reference Example 1, into E. coli str. K-12 substr. BW25113.

[0244] E. coli str. K-12 substr. BW25113 was inoculated into 5 mL of LB medium and incubated with shaking at 37°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium and incubated with shaking at 37°C for 2 hours. After cooling the culture solution on ice for 20 minutes, the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol, mixed with 1 μL of pBBR1MCS-2::ATCTOR and pMW119, and then cooled on ice in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-rad) (3kV, 200Ω, 25μF), and immediately afterward, 1 mL of SOC medium was added and the cells were incubated with shaking at 37°C for 1 hour. 50 μL of the cells were spread onto LB agar containing kanamycin 25 μg / mL and ampicillin 100 μg / mL and incubated at 37°C for 1 day. The resulting control strain was designated EcWT / 3HA_pMW.

[0245] (Comparative Example 15) 3HA Production Test Using Control Strain The control strain (EcWT / 3HA_pMW) was inoculated into 5 mL of medium I (φ18 mm glass test tube, aluminum stopper) adjusted to pH 7 (10 g / L Bacto tryptone (Difco Laboratories), 5 g / L Bacto yeast extract (Difco Laboratories), 5 g / L sodium chloride, 25 μg / mL kanamycin, 100 μg / mL ampicillin) using a platinum loop, and incubated at 30°C for 120 min. －１ The culture solution was incubated with shaking for 24 hours. 0.25 mL of the culture solution was added to 5 mL of Medium II (50 g / L glucose, 1 g / L ammonium sulfate, 50 mM potassium phosphate, 0.025 g / L magnesium sulfate, 0.0625 mg / L iron sulfate, 2.7 mg / L manganese sulfate, 0.33 mg / L calcium chloride, 1.25 g / L sodium chloride, 2.5 g / L Bacto tryptone, 1.25 g / L Bacto yeast extract, 25 μg / mL kanamycin, 100 μg / mL ampicillin) adjusted to pH 6.5 (φ18 mm glass test tube, aluminum stopper), and incubated with shaking at 30°C.

[0246] The supernatant obtained by centrifugation of bacterial cells from the culture medium was treated with a membrane using "Millex-GV" (0.22 μm, PVDF, Merck), and the permeate was analyzed by LC-MS / MS and HPLC under the following conditions to quantify the concentrations of 3HA accumulated in the culture supernatant and sugars remaining unused in the culture medium. Furthermore, the yield of 3HA calculated using the following formula (3) based on these results is shown in Table 4.

[0247] [Quantitative analysis of 3HA by LC-MS / MS] The quantitative analysis of 3HA by LC-MS / MS was performed under the conditions described in Reference Example 12.

[0248] [Quantitative analysis of sugar by HPLC] HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation) Column: Shodex Sugar SH1011 (manufactured by Showa Denko K.K.), length 300 mm, inner diameter 8 mm, particle size 6 μm Mobile phase: 0.05 M Sulfuric acid aqueous solution flow rate: 0.6 mL / min Column temperature: 65 °C Detector: RID-10A (manufactured by Shimadzu Corporation).

[0249] 3HA yield (%) = Amount of 3HA produced (mol) / Amount of sugar consumed (mol) × 100 ... Equation (3).

[0250] (Example 13) Preparation of a 3HA-producing strain with increased YnfM expression Plasmids pBBR1MCS-2::ATCTOR and pMW::EcYNFM, prepared in Reference Examples 20 and 21, were introduced into E. coli str. K-12 substr. BW25113 using the same method as in Reference Example 22 to prepare a 3HA-producing strain (EcWT / 3HA_EcYNFM) with increased YnfM expression.

[0251] (Example 14) 3HA production test using a 3HA-producing strain with increased YnfM expression To confirm the effect of increased YnfM expression on improving 3HA productivity, a 3HA production test was conducted using the strain (EcWT / 3HA_EcYNFM) prepared in Example 13, in the same manner as in Comparative Example 15. The results are shown in Table 4.

[0252] Comparing the results of Comparative Example 15 and Example 14, the 3HA-producing strain with increased YnfM expression (Example 14) showed improved 3HA yield compared to the control strain (Comparative Example 15). Therefore, it was shown that increasing YnfM expression enhances 3HA efflux function, thereby improving 3HA productivity.

[0253]

[0254] (Reference Example 23) Creation of a mutant lacking YhhS function To eliminate the YhhS function in Escherichia coli str. K-12 substr. BW25113, a mutant was created in which the gene encoding YhhS was deleted.

[0255] The method for deleting the target gene followed the procedure described in Proc. Natl. Acad. Sci. USA. 2000 Jun 6;97(12):6640-6645.

[0256] PCR was performed using pKD4 as a template and oligoDNA of sequence numbers 19 and 20 as primers to obtain a 1.6 kb PCR fragment for yhhS deficiency. pKD46, an FRT recombinase expression plasmid, was introduced into Escherichia coli str. K-12 substr. BW25113 to obtain an ampicillin-resistant strain. The obtained strain was inoculated into 5 mL of LB medium containing 100 μg / mL ampicillin and cultured with shaking at 30°C for 1 day. 0.5 mL of the culture was inoculated into 50 mL of LB medium containing 100 μg / mL ampicillin and 50 mM arabinose and cultured in a circular motion at 30°C for 2 hours. After cooling the culture on ice for 20 minutes, the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol, mixed with 5 μL of PCR fragments, and then cooled on ice in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene Pulser" (Bio-rad) (3 kV, 200 Ω, 25 μF), and immediately afterwards, 1 mL of SOC medium was added, and the mixture was incubated with shaking at 30°C for 2 hours. The entire volume was spread onto LB agar containing 25 μg / mL of kanamycin and incubated at 30°C for 1 day. Colony-direct PCR was performed using the obtained kanamycin-resistant strains, and deletion of the target gene and insertion of the kanamycin resistance gene were confirmed by band length. OligoDNAs of SEQ ID NOs. 21 and 22, and 22 and 23 were used as primers.

[0257] Next, the kanamycin-resistant strain was inoculated into 5 mL of LB medium and subcultured twice at 40°C to remove pKD46 and obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain, and an ampicillin-resistant strain was obtained again. After subculturing the obtained strain twice at 43°C, colony-direct PCR was performed, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 21 and 22 were used as primers. Since the obtained strain was ampicillin-sensitive, it was confirmed that pCP20 had been removed. The obtained mutant lacking the function of YhhS was named EcΔyhhS.

[0258] (Reference Example 24) Creation of mutants lacking the function of YhhS and YnfM In order to eliminate the function of YnfM in EcΔyhhS created in Reference Example 23, a mutant was created in which the gene encoding YnfM was deleted.

[0259] The ynfM gene of EcΔyhhS was deleted using the same method as in Reference Example 23, except that the oligo DNA of SEQ ID NOs. 24 and 25 was used as primers for PCR using pKD4 as a template, and the oligo DNA of SEQ ID NOs. 26 and 27, and 27 and 23 were used as primers for colony-direct PCR to confirm the deletion of the target gene and the insertion of the kanamycin resistance gene. The resulting mutant was named EcΔyhhS_ynfM.

[0260] (Reference Example 25) Preparation of a 3HA-producing strain lacking the function of YhhS or the functions of YhhS and YnfM The plasmid pBBR1MCS-2::ATCTOR prepared in Reference Example 20 was introduced into EcΔyhhS and EcΔyhhS_ynfM prepared in Reference Examples 23 and 24 to prepare a 3HA-producing strain lacking the function of YhhS or the functions of YhhS and YnfM.

[0261] EcΔyhhS and EcΔyhhS_ynfM were each inoculated into 5 mL of LB medium and incubated with shaking at 37°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium and incubated with shaking at 37°C for 2 hours. After cooling the culture solution on ice for 20 minutes, the cells were washed three times with 10% (w / w) glycerol. The washed pellet was suspended in 100 μL of 10% (w / w) glycerol, mixed with 1 μL of pBBR1MCS-2::ATCTOR, and then cooled on ice in an electroporation cuvette for 10 minutes. Electroporation was performed using a "Gene pulser" (Bio-rad) (3 kV, 200 Ω, 25 μF), and immediately afterward, 1 mL of SOC medium was added and incubated with shaking at 37°C for 1 hour. 50 μL was spread onto LB agar medium containing 25 μg / mL kanamycin and incubated at 37°C for 1 day. The resulting strains were designated EcΔyhhS / 3HA and EcΔyhhS_ynfM / 3HA, respectively.

[0262] (Reference Example 26) 3HA production test using 3HA-producing strains lacking the function of YhhS and the functions of YhhS and YnfM. Using the strains prepared in Reference Example 25 (EcΔyhhS / 3HA, EcΔyhhS_ynfM / 3HA), a 3HA production test was conducted in the same manner as in Comparative Example 15, except that kanamycin was used as the antibiotic. The results are shown in Table 5.

[0263] As shown in Table 5, strains lacking both YhhS and YnfM function showed a lower 3HA yield compared to strains lacking only YhhS function. These results indicate that YnfM has a function of 3HA efflux, and that the improvement in 3HA productivity in the examples was due to the enhancement of the 3HA efflux function by increasing the expression level of YnfM.

[0264] (Reference Example 27) Preparation of a 3HA-producing strain with restored YnfM function Plasmids pBBR1MCS-2::ATCTOR and pMW::EcYNFM, prepared in Reference Examples 20 and 21, were introduced into EcΔyhhS_ynfM, which lacks the functions of YhhS and YnfM, using the same method as in Reference Example 22, to prepare a 3HA-producing strain (EcΔyhhS_ynfM / 3HA_EcYNFM) with restored YnfM function.

[0265] (Reference Example 28) 3HA Production Test Using a 3HA-Producing Strain with Restored YnfM Function To further confirm that YnfM has a 3HA excretion function, a 3HA production test was conducted using the strain prepared in Reference Example 27 (EcΔyhhS_ynfM / 3HA_EcYNFM) in the same manner as in Comparative Example 15. The results are shown in Table 5.

[0266] The results in Table 5 show that the strains with restored YnfM function had increased 3HA yields compared to strains lacking YhhS and YnfM function. These results also indicate that YnfM has a function of 3HA efflux, and that the improvement in 3HA productivity in Example 14 was due to the enhancement of the 3HA efflux function by increasing the expression level of YnfM.

[0267]

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 the function of isocitrate lyase 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. The genetically modified microorganism according to claim 1, further comprising enhanced function of malate synthase and / or isocitrate dehydrogenase / phosphatase.

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 been genetically modified to suppress the expression of the isocitrate lyase repressor gene.

8. The genetically modified microorganism according to claim 1, wherein the microorganism is a microorganism belonging to the genus Escherichia or Serratia.

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 described in 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 genetically modified microorganism capable of producing 3-hydroxyadipic acid, in which the expression level of YnfM or its homolog is increased.

16. A genetically modified microorganism according to claim 15, wherein the expression level of YnfM or its homolog is increased by introducing a gene encoding one of the following polypeptides (A) to (C): (A) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (B) A polypeptide having 60% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 1 and having a function to improve the productivity of 3-hydroxyadipic acid (C) A polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having a function to improve the productivity of 3-hydroxyadipic acid 17. The genetically modified microorganism according to claim 15, wherein the microorganism is a microorganism belonging to the genus Escherichia or Serratia.

18. A method for producing 3-hydroxyadipic acid, comprising the step of culturing a genetically modified microorganism according to any one of claims 15 to 17.

19. A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid by the method described in claim 18, and reacting 3-hydroxyadipic acid with hydrogen in the presence of a catalyst.

20. A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid by the method described in claim 18, and reacting 3-hydroxyadipic acid with ammonia and hydrogen in the presence of a catalyst.

21. A method for producing 3-hydroxyadipic acid-3,6-lactone, comprising the steps of producing 3-hydroxyadipic acid by the method described in claim 18, and condensing 3-hydroxyadipic acid to produce 3-hydroxyadipic acid-3,6-lactone.

22. A method for producing adipic acid, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method of claim 21, and reacting 3-hydroxyadipic acid-3,6-lactone with hydrogen in the presence of a catalyst.

23. A method for producing ε-caprolactam, comprising the steps of producing 3-hydroxyadipic acid-3,6-lactone by the method described in claim 21, and reacting 3-hydroxyadipic acid-3,6-lactone with ammonia and hydrogen in the presence of a catalyst.

24. A method for producing ε-caprolactam, comprising the steps of producing adipic acid by the method of claim 9, 10, 13, 19, or 22, and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst.

25. A method for producing hexamethylenediamine, comprising the steps of producing adipic acid by the method of claim 9, 10, 13, 19, or 22, and reacting adipic acid with ammonia and hydrogen in the presence of a catalyst.

26. A method for producing a polyamide, comprising the steps of producing adipic acid by the method of claim 9, 10, 13, 19, or 22, and polycondensing adipic acid and a diamine.

27. The method for producing a polyamide according to claim 26, wherein the diamine is a diamine containing 1,4-butanediamine, 1,5-pentanediamine, or hexamethylenediamine.

28. A method for producing a polyamide, comprising the steps of producing hexamethylenediamine by the method of claim 25, and polycondensing hexamethylenediamine with a dicarboxylic acid.

29. The method for producing a polyamide according to claim 28, wherein the dicarboxylic acid is adipic acid or sebacic acid.

30. A method for producing polyamide 6,6, comprising the steps of producing adipic acid by the method of claim 9, 10, 13, 19 or 22, producing hexamethylenediamine by the method of claim 25, and polycondensing adipic acid and hexamethylenediamine.

31. A method for producing polyamide 6, comprising the steps of producing ε-caprolactam by the method described in claim 11, 14, 20, 23, or 24, and polycondensing the ε-caprolactam.

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