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

Abstract

Disclosed are: a genetically modified microorganism capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid at a high yield; and said genetically modified microorganism. The genetically modified microorganism is obtained by enhancing the reaction from producing acetyl-CoA from pyruvic acid, and reducing the function of a catalyst of a pyruvic acid kinase and / or phosphotransferase-based catalyst, in a microorganism having an ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid.

Description

Technical Field

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

Background Art

[0002] 3-Hydroxyadipic acid (IUPAC name: 3-hydroxyhexanedioic acid) and α-hydroxymuconic acid (IUPAC name: (E)-hex-2-enedioic acid) are dicarboxylic acids having 6 carbon atoms. These can be used as raw materials for polyesters by polymerization with polyhydric alcohols and as raw materials for polyamides by polymerization with polyvalent amines. Also, compounds obtained by adding ammonia to the ends thereof and lactamizing them can be used as raw materials for polyamides.

[0003] The following documents are known in relation to the production of 3-hydroxyadipic acid or α-hydroxymuconic acid using microorganisms.

[0004] As a document related to the production of dicarboxylic acids having 6 carbon atoms 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 for catalyzing the reduction reaction from 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. Regarding the biosynthesis pathway of these substances, it is described that it passes through an enzymatic reaction for reducing 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. On the other hand, all the genes modified in Patent Document 1 are limited to the reactions on the above biosynthesis pathway in the cell, and there is no description about the decrease or enhancement of the enzymatic activity in the metabolic pathway upstream of them.

[0005] Patent Document 2 describes a method for producing 1,3-butadiene using microorganisms with modified metabolic pathways. In this document, 3-hydroxyadipic acid (3-hydroxyadipate) is described as a metabolic intermediate in the metabolic pathway in which 1,3-butadiene is biosynthesized from acetyl-CoA and succinyl-CoA.

[0006] Patent Document 3 describes a method for producing muconic acid using microorganisms with modified metabolic pathways. In this document, α-hydromuconic acid (2,3-dehydroadipate) is described as a metabolic intermediate in the metabolic pathway in which trans,trans-muconic acid is biosynthesized from acetyl-CoA and succinyl-CoA.

[0007] The biosynthetic pathways described in Patent Documents 2 and 3 both involve an enzymatic reaction that reduces 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA.

[0008] Patent documents 4 and 5 describe methods for producing adipic acid and hexamethylenediamine (HMDA) using non-natural microorganisms. These documents describe that these substances share a common biosynthetic pathway in which 3-oxoadipyl-CoA is synthesized from acetyl-CoA and succinyl-CoA. However, different biosynthetic pathways are described for the synthesis of 3-hydroxyadipic acid or α-hydromuconic acid from 3-oxoadipyl-CoA.

[0009] Patent Document 4 describes pyruvate kinase as an additional gene deletion to improve the formation of growth-coupled HMDA in the production of HMDA, and phosphotransferase enzyme as an additional gene deletion to improve the yield. On the other hand, in the production of adipic acid, phosphotransferase enzyme is described as an additional gene deletion to improve the formation of growth-coupled adipic acid, but pyruvate kinase is not mentioned. In both the production of HMDA and adipic acid, there is no description of enhancing the reaction that produces acetyl-CoA from pyruvate.

[0010] Patent Document 5 describes the production of adipic acid using non-natural microorganisms possessing phosphoketolase, including the attenuation or removal of phosphotransferase enzyme and pyruvate kinase activity. It also describes genetic modifications that include enhancing the reaction to produce acetyl-CoA from pyruvate for NADH production in the presence of phosphoketolase.

[0011] Patent Document 6 discloses a method for improving microorganisms based on in silico analysis, which reveals that succinic acid production is increased by deleting the genes pykF, pykA, and ptsG, which encode pyruvate kinase and phosphotransferase enzymes in Escherichia coli, and culturing them under anaerobic conditions.

[0012] Non-patent document 1 discloses that deleting pdhR, a gene encoding a transcriptional repressor of the pyruvate dehydrogenase complex in Escherichia coli, increases the production of 2-oxoglutaric acid. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] International Publication No. 2019 / 107516 [Patent Document 2] Special Publication No. 2013-535203 [Patent Document 3] U.S. Patent Application Publication No. 2011 / 0124911A1 [Patent Document 4] Japanese Patent Publication No. 2015-146810 [Patent Document 5] U.S. Patent Application Publication No. 2017 / 0298363A1 [Patent Document 6] Special Publication No. 2008-527991 [Non-patent literature]

[0014] [Non-Patent Document 1] J Biosci Bioeng. 2017 Apr;123(4):437-443. [Overview of the project] [Problems that the invention aims to solve]

[0015] Patent Document 1 discloses the enhancement of enzyme gene expression involved in improving the productivity of 3-hydroxyadipic acid or α-hydromuconic acid. However, the enzyme genes whose expression is enhanced are limited to reactions downstream of acetyl-CoA and succinyl-CoA in the biosynthetic pathway, and there is no description of enhancing enzyme activity in metabolic pathways upstream of these. Patent Documents 2 or 3 describe metabolic pathways in which 3-hydroxyadipic acid or α-hydromuconic acid can be produced in microorganisms, but there is no description of stopping the metabolism at 3-hydroxyadipic acid or α-hydromuconic acid and secreting it into the culture medium. Furthermore, there has been no verification of whether 3-hydroxyadipic acid or α-hydromuconic acid can actually be produced when using microorganisms into which enzyme genes that catalyze the reaction of reducing 3-oxoadipyl-CoA to 3-hydroxyadipic-CoA, as described in Patent Documents 2 to 5, have been introduced. Patent Document 6 and Non-Patent Document 1 do not contain any description of 3-hydroxyadipic acid or α-hydromuconic acid.

[0016] Therefore, the present invention aims to provide a genetically modified microorganism that enhances the enzyme activity by introducing a gene encoding 3-oxoadipyl-CoA reductase or by increasing the expression of said gene, and further modifying the upstream metabolic pathway to produce 3-hydroxyadipic acid and / or α-hydromuconic acid in high yield, as well as a method for producing such a substance using the modified microorganism. [Means for solving the problem]

[0017] As a result of intensive studies to achieve the above object, the present inventors have found that a genetically modified microorganism having the ability to produce 3-hydroxyadipic acid and / or α-hydroxymuconic acid, enhancing the reaction for generating acetyl-CoA from pyruvic acid, and reducing the functions of pyruvate kinase and / or phosphotransferase enzyme has excellent productivity of 3-hydroxyadipic acid and / or α-hydroxymuconic acid, and thus have completed the present invention.

[0018] That is, the present invention provides the following. (1) A genetically modified microorganism having the ability to produce 3-hydroxyadipic acid and / or α-hydroxymuconic acid, wherein the reaction for generating acetyl-CoA from pyruvic acid is enhanced and the functions of pyruvate kinase and / or phosphotransferase enzyme are reduced. (2) The genetically modified microorganism according to (1), wherein the enhancement of the reaction for generating acetyl-CoA from pyruvic acid is the enhancement of the reaction catalyzed by pyruvate dehydrogenase complex and / or the enhancement of the reaction catalyzed by pyruvate formate lyase. (3) The genetically modified microorganism according to (2), wherein the enhancement of the reaction catalyzed by the pyruvate dehydrogenase complex is the enhancement by increasing the expression level of the pyruvate dehydrogenase complex and / or enhancing the activity of the pyruvate dehydrogenase complex. (4) The genetically modified microorganism according to (3), wherein the increase in the expression level of the pyruvate dehydrogenase complex is achieved by reducing the function of a pyruvate dehydrogenase complex transcriptional repressor. (5) The genetically modified microorganism according to (3), wherein the enhancement of the activity of the pyruvate dehydrogenase complex is achieved by reducing the sensitivity of the pyruvate dehydrogenase complex to NADH. (6) The genetically modified microorganism according to (2), wherein the enhancement of the reaction catalyzed by pyruvate formate lyase is the enhancement by increasing the expression level of pyruvate formate lyase. (7) The genetically modified microorganism according to any one of (1) to (6), in which the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA is enhanced. (8) The genetically modified microorganism according to any one of (1) to (7), in which the reaction of phosphoenolpyruvate carboxykinase is enhanced. (9) The genetically modified microorganism according to any one of (1) to (8), which is a microorganism that does not perform sugar metabolism through the phosphoketolase pathway. (10) A method for producing 3-hydroxyadipic acid and / or α-hydroxymuconic acid, comprising the step of culturing the genetically modified microorganism according to any one of (1) to (9). (11) A method for producing a genetically modified microorganism having the ability to produce 3-hydroxyadipic acid and / or α-hydroxymuconic acid, comprising the step of enhancing or reducing an inherent function of a microorganism by genetic modification, wherein the step includes: a step (a) of enhancing the reaction of producing acetyl-CoA from pyruvate, and a step (b) of reducing the functions of pyruvate kinase and / or phosphotransferase enzyme. (12) The method according to (11), further comprising a step (c) of enhancing the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. (13) The method according to (11) or (12), further comprising a step (d) of enhancing the reaction of phosphoenolpyruvate carboxykinase. [Effect of the Invention]

[0019] In a microorganism having the ability to produce 3-hydroxyadipic acid and / or α-hydroxymuconic acid, by genetic modification that enhances the reaction of producing acetyl-CoA from pyruvate and reduces the functions of pyruvate kinase and / or phosphotransferase enzyme, 3-hydroxyadipic acid and / or α-hydroxymuconic acid can be produced in a higher yield as compared with the microorganism of the parental strain that has not been modified with the gene. [Modes for Carrying Out the Invention]

[0020] In this invention, we have found that in microorganisms capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid, enhancing the reaction that generates acetyl-CoA from pyruvate in the microorganism, and reducing the function of pyruvate kinase and / or phosphotransferase enzymes, allows for the production of 3-hydroxyadipic acid and / or α-hydromuconic acid in high yield.

[0021] Hereinafter, in this specification, 3-hydroxyadipic acid may be abbreviated as 3HA, and α-hydromuconic acid as HMA. Also, 3-oxoadipyl-CoA may be abbreviated as 3OA-CoA, 3-hydroxyadipyl-CoA as 3HA-CoA, and 2,3-dehydroadipyl-CoA as HMA-CoA. Furthermore, phosphoenolpyruvic acid may be abbreviated as PEP. 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, the complex of proteins encoded by the aceE, aceF, and lpd genes in the presence of a single promoter may be described as the pyruvate dehydrogenase complex and abbreviated as PDHc. Furthermore, the aceE, aceF, and lpd genes may be collectively referred to as the PDHc gene group. Furthermore, pyruvate formate lyase and pyruvate formate lyase activating enzyme are collectively referred to as pyruvate formate lyase and are sometimes abbreviated as PFL or Pfl. Pyruvate kinase is sometimes abbreviated as Pyk, and phosphotransferase enzyme as PTS. Nucleic acids that encode functional polypeptides are sometimes referred to as genes.

[0022] The genetically modified microorganisms of the present invention can biosynthesize 3-hydroxyadipic acid and / or α-hydromuconic acid using acetyl-CoA and succinyl-CoA as intermediates, as shown in the following metabolic pathways. The metabolic pathway from glucose to acetyl-CoA is well known as glycolysis, and the metabolic pathway to succinyl-CoA is well known as the TCA cycle.

[0023] [ka]

[0024] The metabolic pathways for producing 3-hydroxyadipic acid and / or α-hydromuconic acid from acetyl-CoA obtained in glycolysis and succinyl-CoA obtained in the TCA cycle are shown below, with the metabolites represented by their chemical formulas. Here, reaction A represents the reaction that produces 3-oxoadipyl-CoA from acetyl-CoA and succinyl-CoA. Reaction B represents the reaction that reduces 3-oxoadipyl-CoA to produce 3-hydroxyadipic-CoA. Reaction C represents the reaction that produces 2,3-dehydroadipyl-CoA from 3-hydroxyadipic-CoA. Reaction D represents the reaction that produces 3-hydroxyadipic acid from 3-hydroxyadipic-CoA. Reaction E represents the reaction that produces α-hydromuconic acid from 2,3-dehydroadipyl-CoA. The enzymes that catalyze each reaction in the following metabolic pathways, as well as methods for creating microorganisms capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid using these metabolic pathways, are described in detail in International Publication No. 2019 / 107516.

[0025] [ka]

[0026] One method for enhancing the reaction that produces acetyl-CoA from pyruvate in this invention is to enhance the reaction catalyzed by the pyruvate dehydrogenase complex (PDHc). The PDHc to be enhanced is not particularly limited as long as it has catalytic activity to produce acetyl-CoA from pyruvate. For example, in bacteria of the genera Escherichia and Serratia, PDHc consists of pyruvate dehydrogenase (E1, EC1.2.4.1), dihydrolipoyl transacetylase (E2, EC2.3.1.12), and dihydrolipoyl dehydrogenase (E3, EC1.8.1.4), which are encoded by the aceE, aceF, and lpd genes, respectively. PDHc as a whole catalyzes the reaction that produces acetyl-CoA, CO2, and NADH from pyruvate, coenzyme A, and NAD+. These PDHc genes form an operon, and the expression of these PDHc genes is regulated by the PDHc transcription repressor (PdhR) encoded by the pdhR gene. In the presence of PdhR, the transcription of the PDHc genes is suppressed, resulting in decreased PDHc expression. Conversely, in the absence of PdhR, the transcription of the PDHc genes is not inhibited, and PDHc expression increases.

[0027] Specific examples of PDHc include AceE (NCBI-ProteinID:NP_414656), AceF (NCBI-ProteinID:NP_414657), and Lpd (NCBI-ProteinID:NP_414658) derived from Escherichia coli str. K-12 substr. MG1655 strain, as well as AceE (SEQ ID NO: 1), AceF (SEQ ID NO: 2), and Lpd (SEQ ID NO: 3) derived from Serratia grimesii NBRC13537 strain, and LpdA (NCBI-ProteinID:ABR75580) derived from Klebsiella pneumoniae subsp. pneumoniae MGH 78578 strain. To determine whether the polypeptide encoded by the gene of the microorganism used in this invention is PDHc, a BLAST search can be performed on public databases such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0028] Specific examples of PDHc transcription repressors include PdhR (NCBI-ProteinID:NP_414655) derived from Escherichia coli str. K-12 substr. MG1655 strain and PdhR (SEQ ID NO: 4) derived from Serratia grimesii NBRC13537 strain. To determine whether a polypeptide encoded by a gene in the microorganism used in this invention is a PDHc transcription repressor, a BLAST search can be performed on public databases such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes).

[0029] One way to enhance the reactions catalyzed by PDHc is to increase the expression level of at least one enzyme that constitutes PDHc. Methods for increasing the expression level of at least one enzyme that constitutes PDHc include, for example, introducing at least one gene that makes up the PDHc gene group from outside the host microorganism into the cell, increasing the copy number of the gene group, or modifying the promoter region or ribosome binding sequence upstream of the coding region of the gene group. These methods may be performed individually or in combination. Furthermore, increasing PDHc expression can also be achieved by reducing the function of PDHc transcription repressors.

[0030] Another way to enhance the reactions catalyzed by PDHc is, for example, to enhance the activity of PDHc. To enhance PDHc activity, the catalytic activity of at least one of the enzymes that make up PDHc should be increased. A specific method for enhancing PDHc activity is, for example, to reduce its sensitivity to NADH. Reduced sensitivity to NADH means, for example, that the Ki value of the enzyme for NADH is more than twice as high as that of the control. PDHc with reduced sensitivity to NADH can be obtained by using the E354K and / or H322Y mutants of Lpd (NCBI-ProteinID:NP_414658) from Escherichia coli str. K-12, as described by Kim et al., J. Bacteriol. 190:3851-3858 (2008), or by using Lpd (NCBI-ProteinID:ABR75580) or a part thereof from Klebsiella pneumoniae, which is known to function under anaerobic conditions. The enzyme gene with enhanced catalytic activity may replace the wild-type gene naturally present in the microorganism used for production, or it may be allowed to coexist with it. Furthermore, the copy number of the enzyme gene may be increased, or the promoter region upstream of the coding region or the ribosome binding sequence may be modified. These methods may be performed individually or in combination.

[0031] Another method for enhancing the reaction that produces acetyl-CoA from pyruvate in this invention is to enhance the reaction catalyzed by pyruvate formate lyase. The pyruvate formate lyase to be enhanced is not particularly limited as long as it has catalytic activity to produce acetyl-CoA from pyruvate. For example, in bacteria of the genera Escherichia and Serratia, pyruvate formate-lyase (EC2.3.1.54) functions in the presence of pyruvate formate-lyase activating enzyme (EC1.97.1.4), which are encoded by the pflB and pflA genes, respectively. Pyruvate formate lyase catalyzes the reaction that produces acetyl-CoA and formate from pyruvate and coenzyme A. pflB and pflA form an operon.

[0032] Specific examples of pyruvate formate lyase include PflB (NCBI-ProteinID:NP_415423) and PflA (NCBI-ProteinID:NP_415422) derived from Escherichia coli str. K-12 substr. MG1655 strain, and PflB (SEQ ID NO: 5) and PflA (SEQ ID NO: 6) derived from Serratia grimesii NBRC13537 strain. To determine whether the polypeptide encoded by the gene of the microorganism used in this invention is pyruvate formate lyase, a BLAST search can be performed on public databases such as NCBI and KEGG.

[0033] Methods to enhance the reaction catalyzed by pyruvate formate lyase include, for example, increasing the catalytic activity of pyruvate formate lyase itself or increasing the expression level of pyruvate formate lyase, but increasing the expression level of pyruvate formate lyase is preferable. Methods to increase the expression level of pyruvate formate lyase include, for example, introducing the pyruvate formate lyase gene from outside the host microorganism into the cell, increasing the copy number of the gene, or modifying the promoter region upstream of the coding region of the gene or the ribosome binding sequence. These methods may be performed individually or in combination.

[0034] Incidentally, U.S. Patent Application Publication No. 2017 / 0298363A1 describes the production of adipic acid using non-natural microorganisms whose metabolism has been modified to improve energy efficiency by having a phosphoketolase pathway. The document states that while glucose metabolism in the glycolysis pathway produces NADH, glucose metabolism in the phosphoketolase pathway does not produce NADH. Therefore, when producing reduced compounds using microorganisms having a phosphoketolase pathway, the expression or activity of PDHc or pyruvate formate lyase and NAD(P)H-producing formate dehydrogenase should be enhanced to improve the NADH deficiency in the metabolism of the microorganisms. Here, since adipic acid is a compound that is in a reduced state than 3-hydroxyadipic acid and α-hydromuconic acid, a person skilled in the art would expect that enhancing the reaction of PDHc and / or pyruvate formate lyase when producing 3-hydroxyadipic acid and / or α-hydromuconic acid would lead to an imbalance in the redox balance and a decrease in the yield of the compound. However, in this invention, when a genetically modified microorganism capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid is cultured in which the reaction for producing acetyl-CoA from pyruvate is enhanced, an unexpected effect is obtained: the production of 3-hydroxyadipic acid and / or α-hydromuconic acid is increased.

[0035] In this invention, reducing the function of pyruvate kinase and / or phosphotransferase enzymes means reducing their enzyme activity. The method for reducing function is not particularly limited, but for example, it can be done by reducing the gene encoding the enzyme through gene mutation treatment using a mutagenetic agent or ultraviolet irradiation, deletion of part or all of the base sequence using site-directed mutagenesis, introduction of frameshift mutations into the base sequence, insertion of stop codons into the base sequence, etc. It can also be reduced by using genetic recombination technology to remove all or part of the base sequence or to replace it with other base sequences. Among these, the method of removing part or all of the base sequence is preferred.

[0036] Pyruvate kinase (Pyk) is classified under EC2.7.1.40 and is an enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate, converting it to pyruvate and ATP. Specifically, this includes PykF (NCBI-ProteinID:NP_416191) and PykA (NCBI-ProteinID:NP_416368) from Escherichia coli str. K-12 substr. MG1655 strain, and PykF (SEQ ID NO: 7) and PykA (SEQ ID NO: 7) from Serratia grimesii NBRC13537 strain.

[0037] 8) are examples. If the microorganism used in the present invention has two or more genes encoding pyruvate kinase, it is preferable to reduce the function of all pyruvate kinases. Whether the polypeptide encoded by the genes of the microorganism used in the present invention is pyruvate kinase can be determined by performing a BLAST search on public databases such as NCBI and KEGG.

[0038] The phosphotransferase system (PTS) is the primary mechanism for taking up carbohydrates such as hexoses, hexitols, and disaccharides into cells. In the PTS, carbohydrates are taken into cells and simultaneously converted to phosphate esters, while the phosphate donor, phosphoenolpyruvate (PEP), is converted to pyruvate.

[0039] PTS enzymes consist of two common enzymes that function regardless of the type of carbohydrate: phosphoenolpyruvate sugar phosphotransferase enzyme I and phosphocarrier protein HPr, and membrane-bound sugar-specific permeases (enzyme II) that are specific to certain carbohydrates. Enzyme II is further composed of sugar-specific IIA, IIB, and IIC components. The enzymes of enzyme II exist as individual or multi-domain proteins linked together, depending on the organism. In microorganisms, phosphoenolpyruvate sugar phosphotransferase enzyme I is encoded by the ptsI gene, phosphocarrier protein HPr by the ptsH gene, glucose-specific IIA by the crr gene, and glucose-specific IIB and IIC by the ptsG gene.

[0040] The enzyme encoded by the ptsG gene is classified under EC2.7.1.199 and is called protein-Npi-phosphohistidine-D-glucose phosphotransferase. Examples include PtsG from Escherichia coli str. K-12 substr. MG1655 strain (NCBI-ProteinID:NP_415619) and PtsG from Serratia grimesii NBRC13537 strain (SEQ ID NO: 9). To determine whether the polypeptide encoded by the gene of the microorganism used in this invention is a PTS enzyme, a BLAST search can be performed on websites such as NCBI or KEGG.

[0041] In this invention, the function of one or more of the above-mentioned PTS enzymes may be reduced. Any of the above-mentioned PTS enzyme functions may be reduced, but it is preferable to reduce the function of the enzyme involved in glucose uptake, and it is particularly preferable to reduce the function of PtsG. Specific examples of ptsG genes include ptsG derived from Escherichia coli str. K-12 substr.MG1655 strain (NCBI-GeneID:945651) and ptsG derived from Serratia grimesii NBRC13537 strain (SEQ ID NO: 10).

[0042] As described below, Escherichia coli is a microorganism capable of producing 3-hydroxyadipic acid and α-hydromuconic acid. Japanese Patent Publication No. 2008-527991 describes the creation of mutant strains of Escherichia coli in which the pykF and pykA genes encoding pyruvate kinase and the ptsG gene encoding phosphotransferase enzyme were deleted. It also states that when these mutant strains were cultured under anaerobic conditions, the yield of succinic acid increased, while the yields of acetic acid and ethanol decreased. Here, acetic acid and ethanol are compounds metabolized from acetyl-CoA, as shown in the metabolic pathway in Scheme 2 above. In other words, it is presumed that in Japanese Patent Publication No. 2008-527991, deleting the ptsG, pkF, and pykA genes of Escherichia coli reduced the supply of acetyl-CoA, resulting in a decrease in the yields of acetic acid and ethanol.

[0043] On the other hand, 3-hydroxyadipic acid and / or α-hydromuconic acid produced by the method of the present invention are compounds metabolized through multiple reactions from 3-oxoadipyl-CoA, which is produced from acetyl-CoA and succinyl-CoA, as described above. Therefore, based on the description in Japanese Patent Publication No. 2008-527991, it is expected that deleting the genes for pyruvate kinase and phosphotransferase enzymes will reduce the supply of acetyl-CoA, thereby reducing the yield of 3-hydroxyadipic acid and / or α-hydromuconic acid. However, contrary to the above expectation, in the present invention, in genetically modified microorganisms in which the reaction for producing acetyl-CoA from pyruvate is enhanced and the reaction for reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipic acid is enhanced, the yield of 3-hydroxyadipic acid and / or α-hydromuconic acid is improved by reducing the function of pyruvate kinase and / or phosphotransferase enzymes.

[0044] Furthermore, in the present invention, it is preferable that the reaction of phosphoenolpyruvate (PEP) carboxykinase (Pck) is enhanced in order to increase the production capacity of 3-hydroxyadipic acid and / or α-hydromuconic acid. Enhancing the reaction of PEP carboxykinase can be achieved, for example, by enhancing the activity of the enzyme that catalyzes this reaction. Methods for enhancing the activity of the enzyme 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 of the gene or the ribosome binding sequence. These methods may be performed individually or in combination, but it is preferable to introduce the enzyme gene into the cell of a host microorganism from outside the cell using the method described in International Publication No. 2019 / 107516 (US Patent Application Publication No. 2020 / 291435A1). International Publication No. 2019 / 107516 (U.S. Patent Application Publication No. 2020 / 291435A1) is incorporated herein by reference.

[0045] PEP carboxykinases are classified under EC4.1.1.49 and are enzymes that catalyze the reaction that produces oxaloacetate and ATP from PEP, carbon dioxide, and ADP. Specifically, examples include Pck (NCBI-ProteinID:NP_417862) from Escherichia coli str. K-12 substr. MG1655 strain, and PckA_1 (SEQ ID NO: 11) and PckA_2 (SEQ ID NO: 12) from Serratia grimesii NBRC13537 strain.

[0046] PEP carboxykinase plays a major role physiologically in the production of glucose from fatty acids during gluconeogenesis. While the reaction catalyzed by phosphoenolpyruvate carboxykinase is reversible, in the production of 3-hydroxyadipic acid and / or α-hydromuconic acid, the reaction proceeds in the direction of converting phosphoenolpyruvate and carbon dioxide into oxaloacetate.

[0047] To determine whether the polypeptide encoded by the enzyme gene used in this invention is phosphoenolpyruvate carboxykinase, a BLAST search can be performed on websites such as NCBI or KEGG.

[0048] Japanese Patent Publication No. 2015-146810 describes that deletion of the PEP carboxykinase gene is effective in creating in silico microbial strains that produce adipic acid in high yield from acetyl-CoA and succinyl-CoA using a metabolic network model. Furthermore, Japanese Patent Publication No. 2015-504688 describes enhancing the activity of PEP carboxykinase to increase the PEP pool in the production of muconic acid biosynthesized via PEP. Additionally, U.S. Patent Application Publication No. 2017 / 0298363A1 describes enhancing the activity of PEP carboxykinase to improve the availability of PEP in the production of adipic acid biosynthesized via PEP. That is, given that the reaction catalyzed by PEP carboxykinase proceeds in the direction of producing PEP from oxaloacetate, a person skilled in the art would expect that enhancing the activity of the PEP carboxykinase gene by increasing its expression would decrease the yield of 3-hydroxyadipic acid and / or α-hydromuconic acid. However, contrary to the above expectations, the present invention has found that when genetically modified microorganisms capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid are cultured in which the reaction for producing acetyl-CoA from pyruvate is enhanced, the reaction for reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipic-CoA is enhanced, and the function of pyruvate kinase and / or phosphotransferase enzymes is reduced, and the reaction of phosphoenolpyruvate carboxykinase is enhanced, the production of 3-hydroxyadipic acid and / or α-hydromuconic acid is improved.

[0049] Furthermore, in the present invention, it is preferable to enhance the reaction that reduces 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA. Enhancing the reaction that reduces 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA means, for example, enhancing the activity of the enzyme that catalyzes this reaction. Methods for enhancing the activity of the enzyme include, for example, introducing the enzyme gene into the cell of a host microorganism from outside the cell, increasing the copy number of the gene, or modifying the promoter region upstream of the coding region or the ribosome binding sequence of the gene. These methods may be performed individually or in combination, but it is preferable to introduce the enzyme gene into the cell of a host microorganism from outside the cell using the method described in International Publication No. 2019 / 107516 (US Patent Application Publication No. 2020 / 291435A1).

[0050] Specific examples of 3-oxoadipyl-CoA reductases that catalyze the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA include enzymes classified as 3-hydroxyacyl-CoA dehydrogenases under EC1.1.1.35, enzymes classified as 3-hydroxybutyryl-CoA dehydrogenases under EC1.1.1.157, and enzymes that have 70% or more sequence identity to any of the amino acid sequences of SEQ ID NOs. 1-6 and 213 disclosed in International Publication No. 2019 / 107516 (US Patent Application Publication No. 2020 / 291435A1) and possess 3-oxoadipyl-CoA reductase activity. Specific examples include PaaH from Pseudomonas putida strain KT2440 (NCBI-ProteinID:NP_745425.1), PaaH from Escherichia coli str. K-12 substr. MG1655 strain (NCBI-ProteinID:NP_415913.1), DcaH from Acinetobacter baylyi strain ADP1 (NCBI-ProteinID:CAG68533.1), PaaH from Serratia plymuthica strain NBRC102599 (NCBI-ProteinID:WP_063197120), and polypeptide from Serratia marcescens strain ATCC13880 (NCBI-ProteinID:KFD11732.1), all of which are enzymes that catalyze the reaction of reducing 3-oxoadipyl-CoA to produce 3-hydroxyadipyl-CoA.

[0051] Furthermore, from the viewpoint of the productivity of 3-hydroxyadipic acid and / or α-hydromuconic acid, the genetically modified microorganism of the present invention is preferably a microorganism that does not metabolize sugars via the phosphoketolase pathway. Such microorganisms exist in nature and can be preferably used as hosts when creating the genetically modified microorganism of the present invention that does not metabolize sugars via the phosphoketolase pathway. Alternatively, after creating the genetically modified microorganism of the present invention, a microorganism that does not metabolize sugars via the phosphoketolase pathway may be created by knocking out the phosphoketolase gene of the microorganism using a method well known to those skilled in the art.

[0052] The following microorganisms are naturally capable of producing 3-hydroxyadipic acid:

[0053] The genus Escherichia includes species such as Escherichia fergusonii and Escherichia coli. Genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia nematodiphila. The genus Pseudomonas includes species such as Pseudomonas chlororaphis, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens. Hafnia species such as Hafnia alvei. Corynebacterium spp. such as Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, Corynebacterium glutamicum. Bacillus genus such as Bacillus badius, Bacillus megaterium, and Bacillus roseus. Streptomyces genus such as Streptomyces vinaceus, Streptomyces karnatakensis, Streptomyces olivaceus. Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus necator, Cupriavidus oxalaticus. The genus Acinetobacter includes species such as Acinetobacter baylyi and Acinetobacter radioresistens. The genus Alcaligenes, such as Alcaligenes faecalis. The genus Nocardioides, including Nocardioides albus. Brevibacterium genus, such as Brevibacterium iodinum. Delftia species such as Delftia acidovorans. The genus Shimwellia, including Shimwellia blattae. The genus Aerobacter, including Aerobacter cloacae. Rhizobium species, such as Rhizobium radiobacter.

[0054] Among microorganisms that inherently possess the ability to produce the aforementioned 3-hydroxyadipic acid, the present invention prefers microorganisms that do not metabolize sugars via the phosphoketolase pathway, such as those belonging to the genera Escherichia, Serratia, Hafnia, Corynebacterium (including Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium ammoniagenes, and Corynebacterium glutamicum), Brevibacterium, Shimwellia, and Aerobacter, and more preferably microorganisms belonging to the genera Escherichia or Serratia.

[0055] The following microorganisms are presumed to inherently possess the ability to produce α-hydromuconate:

[0056] The genus Escherichia includes species such as Escherichia fergusonii and Escherichia coli. Genus Serratia such as Serratia grimesii, Serratia ficaria, Serratia fonticola, Serratia odorifera, Serratia plymuthica, Serratia entomophila or Serratia nematodiphila. The genus Pseudomonas includes species such as Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas azotoformans, and Pseudomonas chlororaphis subsp. aureofaciens. Hafnia species such as Hafnia alvei. The genus Bacillus, including Bacillus badius. Cupriavidus genus such as Cupriavidus metallidurans, Cupriavidus numazuensis, Cupriavidus oxalaticus. The genus Acinetobacter includes species such as Acinetobacter baylyi and Acinetobacter radioresistens. The genus Alcaligenes, such as Alcaligenes faecalis. Delftia species such as Delftia acidovorans. The genus Shimwellia, including Shimwellia blattae.

[0057] Among microorganisms that inherently possess the ability to produce the aforementioned α-hydromuconic acid, the present invention prefers microorganisms of the genera Escherichia, Serratia, Hafnia, and Shimwellia, which do not undergo sugar metabolism via the phosphoketolase pathway, and more preferably microorganisms belonging to the genera Escherichia or Serratia.

[0058] 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 D into the microorganism. Similarly, 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 E into the microorganism.

[0059] 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 and / or α-hydromuconic acid or not, but may belong to the genera Escherichia, Serratia, Hafnia, Psuedomonas, Corynebacterium, Bacillus, Streptomyces, Cupriavidus, Acinetobacter, Alcaligenes, Microorganisms belonging to the genera Brevibacterium, Delftia, Shimwellia, Aerobacter, Rhizobium, Thermobifida, Clostridium, Schizosaccharomyces, Kluyveromyces, Pichia, and Candida are preferred, microorganisms belonging to the genera Escherichia, Serratia, Hafnia, and Pseudomonas are more preferred, and microorganisms belonging to the genera Escherichia or Serratia are particularly preferred.

[0060] 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.

[0061] One or more genes may be introduced. Furthermore, gene introduction and gene expression enhancement may be combined.

[0062] When the gene to be expressed in this invention is incorporated into an expression vector or a 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, and a transcription termination sequence. It may also include a gene that controls promoter activity.

[0063] The promoter used in this invention is not particularly limited as long as it can express a gene within the host microorganism, but examples include the gap promoter, trp promoter, lac promoter, tac promoter, and T7 promoter.

[0064] In the present invention, when using an expression vector to introduce a gene or enhance gene expression, there are no particular limitations as long as it can autonomously replicate within the microorganism, but examples include the pBBR1MCS vector, pBR322 vector, pMW vector, pET vector, pRSF vector, pCDF vector, pACYC vector, and derivatives of the above-mentioned vectors.

[0065] In this invention, when introducing genes or enhancing gene expression using nucleic acids for genome integration, site-directed homologous recombination is used. The method of site-directed homologous recombination is not particularly limited, but examples include a method using λ Red recombinase and FLP recombinase (Proc Natl Acad Sci USA. 2000 Jun 6; 97(12):6640-6645.) and a method using λ Red recombinase and the sacB gene (Biosci Biotechnol Biochem. 2007 Dec; 71(12):2905-11.).

[0066] 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 (Journal of Molecular Biology, 53, 159 (1970)) and the electroporation method (NM Calvin, PC Hanawalt.J. Bacteriol, 170 (1988), pp.2796-2801).

[0067] 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.

[0068] Fermentation raw materials are raw materials that the genetically modified microorganism can metabolize. "Metabolism" refers to the conversion of a chemical compound taken in by a microorganism from outside the cell, or produced from another chemical compound within the cell, into another chemical compound through an enzymatic reaction. Sugars can preferably be used as a carbon source. Specific examples of sugars include monosaccharides such as glucose, sucrose, fructose, galactose, mannose, xylose, and arabinose; disaccharides and polysaccharides formed by the linkage of these monosaccharides; and starch saccharification solutions, molasses, and cellulose-containing biomass saccharification solutions containing these.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] The culture conditions for genetically modified microorganisms to produce 3-hydroxyadipic acid and / or α-hydromuconic 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.

[0073] 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 6.8.

[0074] The range of aeration conditions in culture is not particularly limited as long as 3-hydroxyadipic acid and / or α-hydromuconic acid can be produced, but 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.

[0075] If foaming occurs during liquid culture, defoaming agents such as mineral oil, silicone oil, and surfactants can be appropriately added to the culture medium.

[0076] After 3-hydroxyadipic acid and / or α-hydromuconic acid have been produced in the culture of the aforementioned microorganism to a recoverable amount, the produced product can be recovered. Recovery of the produced product, 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 product 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. [Examples]

[0077] The present invention will be specifically described below with reference to examples.

[0078] Reference example 1 Plasmids expressing enzymes that catalyze the reaction (reaction A) to produce 3OA-CoA and coenzyme A from acetyl-CoA and succinyl-CoA, the reaction (reaction B) to produce 3HA-CoA from 3OA-CoA, and the reaction (reaction D) to produce 3-hydroxyadipic acid from 3HA-CoA and the reaction (reaction E) to produce α-hydromuconic acid from HMA-CoA.

[0079] The autologous replication vector pBBR1MCS-2 (ME Kovach, (1995), Gene 166: 175-176) in Escherichia coli was cleaved with XhoI to obtain pBBR1MCS-2 / XhoI. To incorporate a constitutive expression promoter into this vector, primers were designed (sequence numbers 14, 15) for PCR amplification of the upstream region 200b (sequence number 13) of gapA (NCBI-GeneID: NC_000913.3) using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR reactions were performed according to standard procedures. The obtained fragments and pBBR1MCS-2 / XhoI were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) and introduced into Escherichia coli strain DH5α. The plasmid was extracted from the obtained recombinant E. coli strain, and its sequence was confirmed by a standard method to obtain pBBR1MCS-2::Pgap. Subsequently, pBBR1MCS-2::Pgap was cleaved with ScaI to obtain pBBR1MCS-2::Pgap / ScaI. To amplify the gene encoding the enzyme that catalyzes reaction A, primers were designed (SEQ ID NOs: 16, 17) for PCR amplification of the full-length acyltransferase gene pcaF (NCBI-GeneID: 1041755) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and the PCR reaction was performed according to a standard method. The obtained fragments and pBBR1MCS-2::Pgap / ScaI were ligated using the In-Fusion HD Cloning Kit and introduced into E. coli strain DH5α. The plasmid was extracted from the obtained recombinant strain, and its sequence was confirmed by a standard method to obtain pBBR1MCS-2::AT. Next, pBBR1MCS-2::AT was cut with HpaI to obtain pBBR1MCS-2::AT / HpaI.To amplify the genes encoding the enzymes that catalyze reactions D and E, primers were designed (SEQ ID NOs: 18, 19) for PCR amplification of the continuous sequences containing the full-length CoA transferase genes pcaI and pcaJ (NCBI-GeneID: 1046613, 1046612) using the genomic DNA of Pseudomonas putida strain KT2440 as a template, and PCR reactions were performed according to standard procedures. The resulting fragments and pBBR1MCS-2::AT / HpaI were ligated using the In-Fusion HD Cloning Kit and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pBBR1MCS-2::ATCT.

[0080] pBBR1MCS-2::ATCT was cleaved with ScaI to obtain pBBR1MCS-2::ATCT / ScaI. Using the genomic DNA of Serratia marcescens strain ATCC13880 as a template, primers for amplifying the nucleic acid described in SEQ ID NO: 20 were designed (SEQ ID NOs: 21, 22), and PCR was performed according to standard procedures. The obtained fragments and pBBR1MCS-2::ATCT / ScaI were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pBBR1MCS-2::ATCTOR.

[0081] Example 1 Creation of Serratia microbial mutants with reduced pyruvate kinase function We created Serratia microbial mutants with reduced pyruvate kinase function by deleting the pykF and pykA genes, which encode pyruvate kinase in Serratia microorganisms.

[0082] The deletion methods for pykF and pykA followed the procedure described in Proc Natl Acad Sci USA.2000 Jun 6; 97(12): 6640-6645.

[0083] pykF deficiency PCR was performed using pKD4 as a template and oligoDNA of sequence numbers 23 and 24 as primers to obtain PCR fragments for pykF deficiency. The FRT recombinase expression plasmid pKD46 was introduced into Serratia grimesii strain NBRC13537 to obtain an ampicillin-resistant strain. The obtained strain was inoculated into 5 mL of LB medium containing 500 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 500 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 cells were 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. 25 and 27 were used as primers.

[0084] Next, the kanamycin-resistant strain was inoculated into 5 mL of LB medium and subcultured twice at 37°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 culturing the obtained strains at 40°C, colony-direct PCR was performed, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 26 and 27 were used as primers. The kanamycin-sensitive strain was inoculated into 5 mL of LB medium and subcultured twice at 37°C to remove pCP20. The obtained strain was designated SgΔPf.

[0085] pykA deficiency PCR was performed using pKD4 as a template and oligoDNA sequences 28 and 29 as primers to obtain PCR fragments for pykA deficiency.

[0086] The pykA gene was deleted from the Serratia grimesii NBRC13537ΔpykF strain using the same method as for the creation of pykF-deficient strains. After introducing pKD46 into this strain, a PCR fragment for pykA deficiency was introduced. Colony-direct PCR was performed using the resulting kanamycin-resistant strain, and the deletion of the target gene and insertion of the kanamycin resistance gene were confirmed by band length. OligoDNA of sequence numbers 25 and 31 were used as primers.

[0087] Next, pKD46 was removed to obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony-direct PCR was performed using the obtained strains, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 30 and 31 were used as primers. pCP20 was removed from the kanamycin-sensitive strain. The resulting strain was designated SgΔPP.

[0088] Example 2 Creation of Serratia microbial mutants with reduced phosphotransferase enzyme function. We created Serratia microbial mutants in which the phosphotransferase enzyme function was reduced by deleting the ptsG gene, which encodes phosphotransferase in Serratia microbial microorganisms.

[0089] Using pKD4 as a template, PCR was performed using oligo DNA from sequence numbers 32 and 33 as primers to obtain PCR fragments for ptsG deficiency. After introducing pKD46 into Serratia grimesii NBRC13537 and SgΔPP, the PCR fragments for ptsG deficiency were introduced. Colony-direct PCR was performed using the resulting kanamycin-resistant strains, and deletion of the target gene and insertion of the kanamycin resistance gene were confirmed by band length. Oligo DNA from sequence numbers 25 and 35 were used as primers.

[0090] Next, pKD46 was removed to obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony-direct PCR was performed using the obtained strains, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 34 and 35 were used as primers. pCP20 was removed from the kanamycin-sensitive strain. The resulting strains were designated SgΔG and SgΔPPG, respectively.

[0091] Example 3 Creation of Serratia microbial mutants with reduced function of pyruvate dehydrogenase complex transcription repressors. We created Serratia microbial mutants in which the expression level of PDHc was increased by deleting the pdhR gene (SEQ ID NO: 36), which encodes a transcription repressor of the pyruvate dehydrogenase complex in Serratia microorganisms.

[0092] Using pKD4 as a template, PCR was performed using oligo DNA sequences 37 and 38 as primers to obtain PCR fragments for pdhR deficiency. After introducing pKD46 into SgΔPP, SgΔG, and SgΔGPP, the PCR fragments for pdhR deficiency were introduced. Colony-direct PCR was performed using the resulting kanamycin-resistant strains, and deletion of the target gene and insertion of the kanamycin resistance gene were confirmed by band length. Oligo DNA sequences 25 and 40 were used as primers.

[0093] Next, pKD46 was removed to obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony-direct PCR was performed using the obtained strains, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 39 and 40 were used as primers. pCP20 was removed from the kanamycin-sensitive strain. The resulting strains were designated SgΔPPR, SgΔGR, and SgΔGPPR, respectively.

[0094] Example 4 Creation of Serratia microbial mutants in which the function of the pyruvate dehydrogenase complex transcription repressor is reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. The strains prepared in Example 3 were each introduced with the plasmids prepared in Reference Example 1 to create Serratia microbial mutants.

[0095] SgΔPPR, SgΔGR, and SgΔGPPR were inoculated into 5 mL of LB medium and incubated with shaking at 30°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium and incubated with shaking at 30°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 afterwards, 1 mL of SOC medium was added and incubated with shaking at 30°C for 1 hour. 50 μL was spread onto LB agar medium containing 25 μg / mL of kanamycin and incubated at 30°C for 1 day. The obtained strains were designated SgΔPPR / 3HA, SgΔGR / 3HA, and SgΔGPPR / 3HA, respectively.

[0096] Example 5 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia microbial mutants into which the function of the pyruvate dehydrogenase complex transcription repressor is reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced. Using the Serratia microbial mutants prepared in Example 4, we conducted production tests for 3-hydroxyadipic acid and α-hydromuconic acid.

[0097] 5 mL of medium I (10 g / L Bacto tryptone (Difco Laboratories), 5 g / L Bacto yeast extract (Difco Laboratories), 5 g / L sodium chloride, 25 μg / mL kanamycin) adjusted to pH 7 (φ18 mm glass test tube, aluminum stopper) was inoculated with one loop of the mutant prepared in Example 2, and cultured with shaking at 30°C for 120 min-1 for 24 hours. 0.25 mL of the culture medium 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) adjusted to pH 6.5 (in an 18 mm diameter glass test tube with an aluminum stopper), and cultured with shaking at 30°C.

[0098] Quantitative analysis of substrates and products 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 the following method to quantify the concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining in the culture medium that were not utilized. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using the following formula (1) based on these results are shown in Table 1.

[0099] Yield (%) = Amount of product produced (mol) / Amount of sugar consumed (mol) × 100 ... Equation (1). (Quantitative analysis of 3-hydroxyadipic acid and α-hydromuconic acid by LC-MS / MS) • HPLC: 1290 Infinity (manufactured by Agilent Technologies) Column: Synergi hydro-RP (Phenomenex), 100 mm length, 3 mm inner diameter, 2.5 μm particle size Mobile phase: 0.1% formic acid aqueous solution / methanol = 70 / 30 Flow rate: 0.3mL / min Column temperature: 40℃ LC detector: 1260DAD VL+ (210nm) • MS / MS: Triple-Quad LC / MS (manufactured by Agilent Technologies) Ionization method: ESI negative mode.

[0100] Quantitative analysis of organic acids by HPLC • HPLC: LC-10A (manufactured by Shimadzu Corporation) Column: Shim-pack SPR-H (manufactured by Shimadzu GLC Co., Ltd.), length 250 mm, inner diameter 7.8 mm, particle size 8 μm Shim-pack SCR-101H (manufactured by Shimadzu GLC Co., Ltd.): Length 250mm, inner diameter 7.8mm, particle size 10μm Mobile phase: 5 mM p-toluenesulfonic acid Reaction solution: 5 mM p-toluenesulfonic acid, 0.1 mM EDTA, 20 mM Bis-Tris Flow rate: 0.8mL / min Column temperature: 45℃ Detector: CDD-10Avp (manufactured by Shimadzu Corporation).

[0101] Quantitative analysis of sugars by HPLC • HPLC: Shimazu Prominence (manufactured by Shimadzu Corporation) Column: Shodex Sugar SH1011 (manufactured by Showa Denko Corporation), length 300 mm, inner diameter 8 mm, particle size 6 μm Mobile phase: 0.05M sulfuric acid aqueous solution Flow rate: 0.6mL / min Column temperature: 65℃ Detector: RID-10A (manufactured by Shimadzu Corporation).

[0102] Reference example 2 Creation of Serratia microbial mutants in which the function of the pyruvate dehydrogenase complex transcription repressor is not impaired, and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. The strains prepared in Examples 1 and 2 were introduced with the plasmids prepared in Reference Example 1 using the same method as in Example 4, to create Serratia microbial mutants. The resulting strains were designated SgΔPP / 3HA, SgΔG / 3HA, and SgΔGPP / 3HA, respectively.

[0103] Comparative Example 1 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced, while the function of the pyruvate dehydrogenase complex transcription repressor is not impaired. The Serratia microbial mutants prepared in Reference Example 2 were cultured using the same method as in Example 5. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the culture medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 1.

[0104] Table 1 shows that in Serratia microbial mutants in which the function of the pyruvate dehydrogenase complex transcription repressor was reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, the yields of 3-hydroxyadipic acid and α-hydromuconic acid were improved.

[0105] [Table 1]

[0106] Example 6 Creation of Echerichia microbial mutants with reduced pyruvate kinase function We created Echerichia microbial mutants with reduced pyruvate kinase function by deleting the pykF (NCBI-GeneID:946179) and pykA (NCBI-GeneID:946527) genes, which encode pyruvate kinase in Echerichia microorganisms.

[0107] The deletion methods for pykF and pykA followed the procedure described in Proc Natl Acad Sci USA.2000 Jun 6; 97(12): 6640-6645.

[0108] pykF deficiency PCR was performed using pKD4 as a template and oligoDNA of sequence numbers 41 and 42 as primers to obtain PCR fragments for pykF deficiency. The FRT recombinase expression plasmid pKD46 was introduced into Escherichia coli str. K-12 substr. MG1655 strain 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 cells were 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. 25 and 44 were used as primers.

[0109] Next, the kanamycin-resistant strain was inoculated into 5 mL of LB medium and subcultured twice at 37°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 culturing the obtained strains at 40°C, colony-direct PCR was performed, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 43 and 44 were used as primers. The kanamycin-sensitive strain was inoculated into 5 mL of LB medium and subcultured twice at 37°C to remove pCP20. The obtained strain was designated as EcΔPf.

[0110] pykA deficiency PCR was performed using pKD4 as a template and oligoDNA sequences 45 and 46 as primers to obtain a PCR fragment for pykA deficiency.

[0111] Using the same method as for creating pykF-deficient strains, pykA was deleted from the Escherichia coli str. K-12 substr. MG1655ΔpykF strain. After introducing pKD46 into this strain, a PCR fragment for pykA deletion was introduced. Colony-direct PCR was performed using the resulting kanamycin-resistant strain, and deletion of the target gene and insertion of the kanamycin resistance gene were confirmed based on band length. OligoDNA of sequence numbers 25 and 48 were used as primers.

[0112] Next, pKD46 was removed to obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony-direct PCR was performed using the obtained strains, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 47 and 48 were used as primers. pCP20 was removed from the kanamycin-sensitive strain. The resulting strain was designated EcΔPP.

[0113] Example 7 Creation of Escherichia microbial mutants with reduced phosphotransferase enzyme function. We created Escherichia microbial mutants in which the phosphotransferase enzyme function was reduced by deleting the ptsG gene, which encodes phosphotransferase in Escherichia microorganisms.

[0114] PCR was performed using pKD4 as a template and oligo DNA of sequence numbers 49 and 50 as primers to obtain PCR fragments for ptsG deficiency. After introducing pKD46 into Escherichia coli str. K-12 substr. MG1655 and EcΔPP, the PCR fragments for ptsG deficiency were introduced. Colony-direct PCR was performed using the resulting kanamycin-resistant strains, and deletion of the target gene and insertion of the kanamycin resistance gene were confirmed by band length. Oligo DNA of sequence numbers 25 and 52 were used as primers.

[0115] Next, pKD46 was removed to obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony-direct PCR was performed using the obtained strains, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 51 and 52 were used as primers. pCP20 was removed from the kanamycin-sensitive strain. The resulting strains were designated EcΔG and EcΔPPG, respectively.

[0116] Example 8 Creation of Escherichia microbial mutants with reduced function of pyruvate dehydrogenase complex transcription repressors. We created Escherichia microbial mutants in which the expression level of PDHc was increased by deleting the pdhR gene (NCBI-GeneID:944827), which encodes a transcriptional repressor of the pyruvate dehydrogenase complex in Escherichia microorganisms.

[0117] Using pKD4 as a template, PCR was performed using oligo DNA sequences 53 and 54 as primers to obtain PCR fragments for pdhR deficiency. After introducing pKD46 into EcΔPP, EcΔG, and EcΔGPP, the PCR fragments for pdhR deficiency were introduced. Colony-direct PCR was performed using the resulting kanamycin-resistant strains, and deletion of the target gene and insertion of the kanamycin resistance gene were confirmed by band length. Oligo DNA sequences 25 and 56 were used as primers.

[0118] Next, pKD46 was removed to obtain an ampicillin-sensitive strain. pCP20 was introduced into the ampicillin-sensitive strain to obtain an ampicillin-resistant strain again. Colony-direct PCR was performed using the obtained strains, and the removal of the kanamycin resistance gene was confirmed by band length. OligoDNA of SEQ ID NOs. 55 and 56 were used as primers. pCP20 was removed from the kanamycin-sensitive strain. The resulting strains were designated EcΔPPR, EcΔGR, and EcΔGPPR, respectively.

[0119] Example 9 Creation of Escherichia microbial mutants in which the function of the pyruvate dehydrogenase complex transcription repressor is reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. Escherichia microbial mutants were created by introducing the plasmids prepared in Reference Example 1 into the strains prepared in Example 8.

[0120] EcΔPPR, EcΔGR, and EcΔGPPR were inoculated into 5 mL of LB medium and incubated with shaking at 30°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium and incubated with shaking at 30°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 afterwards, 1 mL of SOC medium was added and incubated with shaking at 30°C for 1 hour. 50 μL was spread onto LB agar medium containing 25 μg / mL of kanamycin and incubated at 30°C for 1 day. The obtained strains were designated EcΔPPR / 3HA, EcΔGR / 3HA, and EcΔGPPR / 3HA, respectively.

[0121] Example 10 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which the function of the pyruvate dehydrogenase complex transcription repressor is reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced. The Escherichia microbial mutants prepared in Example 9 were cultured using the same method as in Example 5. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the culture medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 2.

[0122] Reference example 3 Creation of Escherichia microbial mutants in which the function of the pyruvate dehydrogenase complex transcription repressor is not impaired, and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. The strains prepared in Examples 6 and 7 were introduced with the plasmids prepared in Reference Example 1 using the same method as in Example 9, to create Escherichia microbial mutants. The resulting strains were designated EcΔPP / 3HA, EcΔG / 3HA, and EcΔGPP / 3HA, respectively.

[0123] Comparative Example 2 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced, while the function of the pyruvate dehydrogenase complex transcription repressor is not impaired. The Escherichia microbial mutants prepared in Reference Example 3 were cultured using the same method as in Example 5. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the culture medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 2.

[0124] Table 2 shows that in Escherichia microbial mutants in which the function of the pyruvate dehydrogenase complex transcription repressor was reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, the yields of 3-hydroxyadipic acid and α-hydromuconic acid were improved.

[0125] [Table 2]

[0126] Reference example 4 Creation of Escherichia microbial mutants in which only the function of the pyruvate dehydrogenase complex transcription repressor is reduced. The pdhR gene was deleted from Escherichia coli str. K-12 substr. MG1655 using the same method as in Example 8. The resulting strain was designated EcΔR.

[0127] Reference example 5 The creation of Escherichia microbial mutants in which only the function of the pyruvate dehydrogenase complex transcription repressor is reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced, and Escherichia microbial mutants in which plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. EcΔR strain and Escherichia coli str. K-12 substr. MG1655 were introduced with the plasmids prepared in Reference Example 1 using the same method as in Example 9, to create Escherichia microbial mutants. The resulting strains were designated EcWT / 3HA and EcΔR / 3HA, respectively.

[0128] Reference example 6 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which only the function of the pyruvate dehydrogenase complex transcription repressor is reduced and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced, and Escherichia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. The Escherichia microbial mutants prepared in Reference Example 5 were cultured using the same method as in Example 5. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the culture medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 3.

[0129] Table 3 shows that in Escherichia microbial mutants in which only the function of the pyruvate dehydrogenase complex transcription repressor was reduced, and plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, the yields of 3-hydroxyadipic acid and α-hydromuconic acid decreased compared to the control strain.

[0130] [Table 3]

[0131] Reference example 7 Plasmid creation for PDHc expression The expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.), which can autonomously replicate in E. coli, was cleaved with SacI to obtain pMW119 / SacI. To incorporate a constitutive expression promoter into this vector, primers were designed (sequence numbers 57, 58) for PCR amplification of the upstream region 200b (sequence number 13) of gapA (NCBI-GeneID: NC_000913.3) using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR reactions were performed according to standard procedures. The obtained fragments and pMW119 / SacI were ligated using the In-Fusion HD Cloning Kit (manufactured by Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant E. coli strain, and the plasmid whose nucleotide sequence was confirmed by standard procedures was designated as pMW119::Pgap. Next, pMW119::Pgap was cleaved with SphI to obtain pMW119::Pgap / SphI. To amplify the gene encoding the pyruvate dehydrogenase complex, primers were designed (SEQ ID NOs. 59, 60) for PCR amplification of the region containing the full-length aceE (NCBI-GeneID: 944834), aceF (NCBI-GeneID: 944794), and lpd (NCBI-GeneID: 944854) regions using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR reactions were performed according to standard procedures. The obtained fragments and 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 ampicillin-resistant strain, and its nucleotide sequence was confirmed by standard procedures. The obtained plasmid was named "pMW119::PDHc".

[0132] Reference example 8 Plasmid creation for PFL expression To amplify the gene encoding PFL, primers were designed (SEQ ID NOs. 61, 62) for PCR amplification of the region containing the full-length pflB (NCBI-GeneID: 945514) and pflA (NCBI-GeneID: 945517) using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template, and PCR reactions were performed according to standard procedures. The resulting fragments and pMW119::Pgap / SphI were ligated using the In-Fusion HD Cloning Kit (Takara Bio Inc.) and introduced into E. coli strain DH5α. The plasmid was extracted from the resulting recombinant strain, and its nucleotide sequence was confirmed by standard procedures. The obtained plasmid was named "pMW119::PFL".

[0133] Example 11 Creation of Serratia microbial mutants by introducing plasmids that increase the expression levels of PDHc or PFL and express enzymes that catalyze reactions A, B, D, and E. The strains prepared in Reference Example 2 were introduced with the plasmids prepared in Reference Examples 7 and 8, respectively, to create Serratia microbial mutants.

[0134] SgΔPP / 3HA, SgΔG / 3HA, and SgΔGPP / 3HA were inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and incubated with shaking at 30°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and incubated with shaking at 30°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 pMW119::PDHc or pMW119::PFL, 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 30°C for 1 hour. 50 μL of the cells were spread onto LB agar containing kanamycin 25 μg / mL and ampicillin 500 μg / mL, and incubated at 30°C for 1 day. The resulting pMW119::PDHc-introduced strains were designated SgΔPP / 3HAPDHc, SgΔG / 3HAPDHc, and SgΔGPP / 3HAPDHc, respectively. The resulting pMW119::PFL-introduced strains were designated SgΔPP / 3HAPFL, SgΔG / 3HAPFL, and SgΔGPP / 3HAPFL, respectively.

[0135] Example 12 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced, resulting in increased expression levels of PDHc or PFL. The Serratia microbial mutants prepared in Example 11 were cultured in the same manner as in Example 5, except that 500 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 4.

[0136] Reference example 9 Creation of Serratia microbial mutants that do not show increased PDHc or PFL expression levels, and into which plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced. A Serratia microbial mutant was created by introducing pMW119 as a negative control to the strain prepared in Reference Example 3.

[0137] pMW119 was introduced into SgΔPP / 3HA, SgΔG / 3HA, and SgΔGPP / 3HA using the same method as in Example 11. The resulting strains were designated as SgΔPP / 3HApMW, SgΔG / 3HApMW, and SgΔGPP / 3HApMW, respectively.

[0138] Comparative Example 3 Production trials of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia microbial mutants that do not show increased PDHc or PFL expression and have been introduced with plasmids expressing enzymes that catalyze reactions A, B, D, and E. The Serratia microbial mutants prepared in Reference Example 9 were cultured in the same manner as in Example 5, except that 500 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 4.

[0139] Table 4 shows that in Serratia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, the yields of 3-hydroxyadipic acid and α-hydromuconic acid were improved.

[0140] [Table 4]

[0141] Example 13 Creation of Escherichia microbial mutants by introducing plasmids that increase PFL expression and express enzymes that catalyze reactions A, B, D, and E. Escherichia microbial mutants were created by introducing the plasmids prepared in Reference Examples 7 and 8, respectively, into the strain prepared in Reference Example 3.

[0142] EcΔPP / 3HA, EcΔG / 3HA, and EcΔGPP / 3HA were inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and incubated with shaking at 30°C for 1 day. 0.5 mL of the culture solution was inoculated into 5 mL of LB medium containing 25 μg / mL kanamycin and incubated with shaking at 30°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 pMW119::PFL, 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 incubated with shaking at 30°C for 1 hour. 50 μL was spread onto LB agar medium containing kanamycin 25 μg / mL and ampicillin 100 μg / mL, and incubated at 30°C for 1 day. The resulting pMW119::PFL-introduced strains were designated EcΔPP / 3HAPFL, EcΔG / 3HAPFL, and EcΔGPP / 3HAPFL, respectively.

[0143] Example 14 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, resulting in increased PFL expression levels. The Escherichia microbial mutants prepared in Example 13 were cultured in the same manner as in Example 5, except that 100 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 5.

[0144] Reference example 10 Creation of Escherichia microbial mutants that do not show increased PDHc or PFL expression levels, and into which plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced. A Serratia microbial mutant was created by introducing pMW119 as a negative control to the strain prepared in Reference Example 3.

[0145] pMW119 was introduced into EcΔPP / 3HA, EcΔG / 3HA, and EcΔGPP / 3HA using the same method as in Example 13. The resulting strains were designated EcΔPP / 3HApMW, EcΔG / 3HApMW, and EcΔGPP / 3HApMW, respectively.

[0146] Comparative Example 4 Production trials of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants that do not show increased PDHc or PFL expression and have been introduced with plasmids expressing enzymes that catalyze reactions A, B, D, and E. The Escherichia microbial mutants prepared in Reference Example 10 were cultured in the same manner as in Example 5, except that 100 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 5.

[0147] Table 5 shows that Escherichia microbial mutants, into which plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, exhibited increased PFL expression and improved yields of 3-hydroxyadipic acid and α-hydromuconic acid.

[0148] [Table 5]

[0149] Reference example 11 Pck and plasmids expressing enzymes that catalyze reactions A, B, D, and E. To incorporate a promoter that constitutively expresses Pck, primers were designed (sequence numbers 64 and 65) for PCR amplification of the upstream region 200b (sequence number 63) of gapA (NCBI Gene ID: NC_000913.3) using the genomic DNA of Escherichia coli K-12 MG1655 as a template, and PCR reactions were performed according to standard procedures. The obtained fragments and the fragments obtained by cleaving pBBR1MCS-2::ATCTOR prepared in Reference Example 1 with SacI 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::ATCTORPgap. Next, to amplify the gene encoding Pck, primers were designed (sequence numbers 67, 68) for PCR amplification of a continuous sequence containing the full length of the pck gene (sequence number 66) using the genomic DNA of Serratia grimesii strain NBRC13537 as a template, and PCR was performed according to standard procedures. The resulting fragments and the fragments obtained by cleaving pBBR1MCS-2::ATCTORPgap with SacI 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::ATCTORPCK.

[0150] Example 15 Creation of Escherichia microbial mutants with increased Pck expression levels and plasmids expressing enzymes that catalyze reactions A, B, D, and E. In the same manner as in Example 13, pMW119::ATCTORPCK or pMW119 as a control was introduced into EcΔPPR / 3HA and EcΔGR / 3HA prepared in Example 9. The resulting strains were designated EcΔPPR / 3HAPCK, EcΔGR / 3HAPCK, EcΔPPR / 3HApMW, and EcΔGR / 3HApMW, respectively.

[0151] Example 16 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced, resulting in increased Pck expression. The EcΔPPR / 3HAPCK, EcΔGR / 3HAPCK, EcΔPPR / 3HApMW, and EcΔGR / 3HApMW prepared in Example 15 were cultured in the same manner as in Example 14. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the culture medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 6.

[0152] Table 6 shows that Escherichia microbial mutants, in which Pck expression levels were increased and plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, showed further improvements in the yields of 3-hydroxyadipic acid and α-hydromuconic acid.

[0153] [Table 6]

[0154] Reference example 12 Plasmids for expressing Lpd from Escherichia coli or LpdA from Klebsiella pneumoniae. The expression vector pMW119 (manufactured by Nippon Gene Co., Ltd.), which can autonomously replicate in E. coli, was cleaved with SacI and KpnI to obtain pMW119 / SacI and KpnI.

[0155] To amplify the gene encoding Lpd from Escherichia coli, primers were designed (SEQ ID NOs: 69, 70) for PCR amplification of the entire lpd gene (NCBI-GeneID: 944854) and its 5' 0.5kb and 3' 0.1kb regions, using the genomic DNA of Escherichia coli str. K-12 substr. MG1655 as a template. PCR reactions were performed according to standard procedures. The resulting fragments, along with pMW119 / SacI and 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 its nucleotide sequence was confirmed by standard procedures. The obtained plasmid was named "pMW119::EcLPD".

[0156] To amplify the gene encoding LpdA from Klebsiella pneumoniae, the full-length lpdA gene (NCBI-GeneID:CP000647,REGION:142460..143884) from Klebsiella pneumoniae subsp. pneumoniae strain MGH78578, as well as the region containing the 5' 0.5kb and 3' 0.5kb of lpd from Escherichia coli str. K-12 substr. MG1655, were synthesized. Primers for PCR amplification of these regions were designed (SEQ ID NOs. 69, 71), and PCR reactions were performed according to standard procedures. The resulting fragments, along with pMW119 / SacI and 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 its nucleotide sequence was confirmed by standard procedures. The obtained plasmid was named "pMW119::KpLPD".

[0157] Example 17 Creation of Escherichia microbial mutants with increased Lpd expression and plasmids expressing enzymes that catalyze reactions A, B, D, and E. Escherichia microbial mutants were created by introducing the plasmids prepared in Reference Example 12 into the strains prepared in Reference Example 3.

[0158] Using the same method as in Example 13, pMW119::EcLPD was introduced into EcΔG / 3HA prepared in Reference Example 3, and pMW119::KpLPD was introduced into EcΔPP / 3HA. The resulting strains were designated EcΔG / 3HAEcLPD and EcΔPP / 3HAKpLPD, respectively.

[0159] Example 18 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E have been introduced, resulting in increased Lpd expression. The Escherichia microbial mutants prepared in Example 17 were cultured in the same manner as in Example 5, except that 100 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 7.

[0160] Table 7 shows that Escherichia microbial mutants into which plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, showed improved yields of 3-hydroxyadipic acid and α-hydromuconic acid.

[0161] [Table 7]

[0162] Reference example 13 Creation of a plasmid to express Lpd with reduced sensitivity to NADH. Amino acid variants of pMW119::EcLPD obtained in Reference Example 12 were constructed. Using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs), pMW119::EcLPD was used as a template, and oligonucleotides of SEQ ID NOs. 72 and 73 were used as primers to substituted glutamic acid (E) at position 354 with lysine (K). The resulting plasmid was named pMW119::EcLPDE354K.

[0163] Similarly, using pMW119::EcLPD as a template and oligonucleotides of sequence numbers 74 and 75 as primers, the histidine (H) at position 322 was substituted with tyrosine (Y). The resulting plasmid was named pMW119::EcLPDH322Y.

[0164] Example 19 Creation of Escherichia microbial mutants by introducing plasmids that express Lpd with reduced sensitivity to NADH and enzymes that catalyze reactions A, B, D, and E. Escherichia microbial mutants were created by introducing the plasmids prepared in Reference Example 13 into the strains prepared in Reference Example 3.

[0165] Using the same method as in Example 13, pMW119::EcLPDE354K or pMW119::EcLPDH322Y was introduced into the EcΔG / 3HA prepared in Reference Example 3, respectively. The resulting strains were named EcΔG / 3HAEcLPDE354K and EcΔG / 3HAEcLPDH322Y, respectively.

[0166] Example 20 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which plasmids expressing Lpd with reduced sensitivity to NADH and enzymes that catalyze reactions A, B, D, and E have been introduced. The Escherichia microbial mutants prepared in Example 19 were cultured in the same manner as in Example 5, except that 100 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 8.

[0167] Table 8 shows that Escherichia microbial mutants into which plasmids expressing Lpd with reduced sensitivity to NADH and enzymes that catalyze reactions A, B, D, and E were introduced showed further improved yields of 3-hydroxyadipic acid and α-hydromuconic acid.

[0168] [Table 8]

[0169] Example 21 Construction of Serratia microbial mutants lacking one type of pyruvate kinase, with increased Lpd expression, and incorporating plasmids expressing enzymes that catalyze reactions A, B, D, and E. In Example 1, pBBR1MCS-2::ATCTOR was introduced into SgΔPf using the same method as in Example 4, and pMW119::EcLPD was introduced using the same method as in Example 11. The resulting strain was named SgΔPf / 3HAEcLPD.

[0170] Example 22 Construction of Escherichia microbial mutants lacking one type of pyruvate kinase, with increased Lpd expression, and incorporating plasmids expressing enzymes that catalyze reactions A, B, D, and E. In Example 6, pBBR1MCS-2::ATCTOR was introduced into EcΔPf using the same method as in Example 9, and pMW119::EcLPD was introduced using the same method as in Example 17. The resulting strain was named EcΔPf / 3HAEcLPD.

[0171] Example 23 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Serratia or Escherichia microbial mutants that lack one type of pyruvate kinase, have increased Lpd expression, and have plasmids expressing enzymes that catalyze reactions A, B, D, and E. The Serratia or Escherichia microbial mutants prepared in Examples 21 and 22 were cultured in the same manner as in Example 5, except that 500 μg / mL or 100 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 9.

[0172] Table 9 shows that Serratia and Escherichia microbial mutants lacking one type of pyruvate kinase, with increased Lpd expression, and introduced plasmids expressing enzymes that catalyze reactions A, B, D, and E, also showed improved yields of 3-hydroxyadipic acid and α-hydromuconic acid.

[0173] [Table 9]

[0174] Reference example 14 Creation of Escherichia microbial mutants in which only PFL expression levels are increased and plasmids expressing enzymes that catalyze reactions A, B, D, and E are introduced. In the EcWT / 3HA prepared in Reference Example 5, pMW119::PFL prepared in Reference Example 8, or pMW119 as a control, was introduced using the same method as in Example 13. The resulting strains were designated EcWT / 3HAPFL and EcWT / 3HApMW.

[0175] Reference example 15 Production experiments of 3-hydroxyadipic acid and α-hydromuconic acid using Escherichia microbial mutants into which only PFL expression levels were increased and plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced. EcWT / 3HAPFL and EcWT / 3HApMW prepared in Reference Example 14 were cultured in the same manner as in Example 5, except that 100 μg / mL of ampicillin was added to the culture medium. The concentrations of 3-hydroxyadipic acid and α-hydromuconic acid accumulated in the culture supernatant, other products, and sugars remaining unused in the medium were quantified. Furthermore, the yields of 3-hydroxyadipic acid and α-hydromuconic acid calculated using formula (1) based on these results are shown in Table 10.

[0176] Table 10 shows that in Escherichia microbial mutants into which only PFL expression levels were increased and plasmids expressing enzymes that catalyze reactions A, B, D, and E were introduced, the yields of 3-hydroxyadipic acid and α-hydromuconic acid decreased compared to the control strain.

[0177] [Table 10]

Claims

1. A genetically modified microorganism belonging to the genus Escherichia or Serratia, which has the ability to produce 3-hydroxyadipic acid and / or α-hydromuconic acid, wherein the reaction catalyzed by the pyruvate dehydrogenase complex and / or the reaction catalyzed by pyruvate formate lyase is enhanced, and the function of pyruvate kinase is reduced and / or the ptsG gene is deleted, wherein the productivity of the genetically modified microorganism in producing 3-hydroxyadipic acid and / or α-hydromuconic acid is increased compared to the unmodified parent strain.

2. The genetically modified microorganism according to claim 1, wherein the enhancement of the reaction catalyzed by the pyruvate dehydrogenase complex is achieved by increasing the expression level of the pyruvate dehydrogenase complex and / or enhancing the activity of the pyruvate dehydrogenase complex.

3. The genetically modified microorganism according to claim 2, wherein the increase in the expression level of the pyruvate dehydrogenase complex is achieved by reducing the function of the pyruvate dehydrogenase complex transcription repressor.

4. The genetically modified microorganism according to claim 2, wherein the enhancement of the activity of the pyruvate dehydrogenase complex is achieved by reducing the sensitivity of the pyruvate dehydrogenase complex to NADH.

5. The genetically modified microorganism according to claim 1, wherein the enhancement of the reaction catalyzed by pyruvate formate lyase is achieved by increasing the expression level of pyruvate formate lyase.

6. The genetically modified microorganism according to claim 1, wherein the functional impairment of the pyruvate kinase is due to the removal of part or all of the base sequence encoding the pyruvate kinase.

7. Furthermore, the genetically modified microorganism according to any one of claims 1 to 6, wherein the expression of the gene encoding 3-hydroxyacyl-CoA dehydrogenase is enhanced.

8. Furthermore, the genetically modified microorganism according to any one of claims 1 to 7, wherein the expression of the gene encoding phosphoenolpyruvate carboxykinase is enhanced.

9. A genetically modified microorganism according to any one of claims 1 to 8, which is a microorganism that does not metabolize sugars via the phosphoketolase pathway.

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

11. A method for producing a genetically modified microorganism capable of producing 3-hydroxyadipic acid and / or α-hydromuconic acid, comprising the steps of enhancing or reducing the intrinsic function of a microorganism belonging to the genus Escherichia or Serratia by genetic modification, wherein the steps include (a) enhancing a reaction catalyzed by a pyruvate dehydrogenase complex and / or a reaction catalyzed by pyruvate formate lyase, and (b) reducing the function of pyruvate kinase and / or deleting the ptsG gene.

12. The method according to claim 11, further comprising step (c) enhancing the expression of a gene encoding 3-hydroxyacyl-CoA dehydrogenase.

13. The method according to claim 11 or 12, further comprising the step (d) of enhancing the expression of a gene encoding phosphoenolpyruvate carboxykinase.

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