Modified microorganisms and methods for producing compounds

Abstract

To provide a novel modified microorganism having a C6 compound production ability and a novel method for producing a C6 compound.SOLUTION: A modified microorganism includes a genetic modification that suppresses a transcription factor to control expression of pyruvate dehydrogenase, having a production pathway of a C6 compound, in which the C6 compound is at least one compound selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, 6-hydroxyhexanoic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to modified microorganisms and methods for producing compounds. [Background technology]

[0002] C6 compounds such as adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, and 6-hydroxyhexanoic acid are used as raw materials for, for example, polyamides and polyurethanes, and are considered important compounds in the chemical industry. In recent years, there have been concerns about the depletion of fossil fuels and their role in global warming. Therefore, in chemical production processes, there is a desire to shift from fossil fuel-derived raw materials to renewable raw materials, such as biomass-derived raw materials. A production method using fermentation production by microorganisms with metabolically modified genetic engineering has been proposed.

[0003] On the other hand, production processes using microorganisms can suffer from a lack of productivity. Another problem with production processes using microorganisms is the potential for the generation of by-products, such as compounds originally produced by the microorganism but different from the target compound, or compounds produced by side reactions from metabolic intermediates. The generation of by-products can reduce the yield of the target compound and further increase the burden and cost of the separation and purification processes. Therefore, various studies have been conducted to improve the productivity of the target compound and reduce the generation of by-products. One such method proposed is the genetic modification of host microorganisms, as shown below.

[0004] For example, an example of adipic acid producing bacteria and adipic acid production has been proposed in which an adipic acid biosynthesis gene is introduced into Escherichia coli as a host in which the ldhA, poxB, pta, adhE, and sucD genes have been disrupted (Non-Patent Document 1).

[0005] Furthermore, for example, a gene deletion set that can increase the production of compounds such as 6-aminocaproic acid, hexamethylenediamine, and adipic acid, predicted by an in silico method, has been disclosed (Patent Document 1).

[0006] Furthermore, genetic modifications have been proposed that can suppress by-products in genetically modified microorganisms having pathways for producing hexamethylenediamine, 6-aminohexanoic acid, adipic acid, caprolactam, caprolactone, levulinic acid, and 1,6-hexanediol (Patent Document 2).

[0007] However, the modification of host microorganisms using the above-mentioned conventional techniques has been limited to genetic modification of enzymes that drive chemical reactions directly involved in the production of target compounds or by-products. Microorganisms with C6 compound production pathways using the above-mentioned conventional techniques do not necessarily have sufficient productivity for industrial use. Therefore, there is a need to improve the productivity of microorganisms. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5773990 specification [Patent Document 2] International Publication No. 2016 / 209883 [Non-patent literature]

[0009] [Non-Patent Document 1] Cheong, Seokjung, James M. Cromburg, and Ramon Gonzalez. “Energy-and carbon-efficient synthesis of functionalized small molecules in bacteria using non-decarboxylative Claisen condensation reactions.”Nature biotechnology” 34.5 (2016): 556-561. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a novel modified microorganism capable of producing a C6 compound and a novel method for producing a C6 compound. [Means for solving the problem]

[0011] As a result of further investigations, the present inventors have focused on transcription factors that control gene expression, in addition to methods for directly modifying enzyme genes involved in metabolic pathway reactions, and have found that suppressing a specific transcription factor in a microorganism that has a C6 compound production pathway can improve the C6 compound production ability. Furthermore, they have found that suppressing this transcription factor can reduce the production of valine, which accumulates as a by-product.

[0012] That is, the present invention provides: [1] A genetic modification that suppresses the transcription factor that controls the expression of pyruvate dehydrogenase. It has a production pathway for C6 compounds, the modified microorganism, wherein the C6 compound is at least one compound selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, 6-hydroxyhexanoic acid, 3-oxoadipate, 3-hydroxyadipate, and 2,3-dehydroadipate; [2] The gene modification that suppresses the transcription factor that controls the expression of pyruvate dehydrogenase is a modification that suppresses the expression of a gene encoding said transcription factor, and A modification that reduces the activity of the transcription factor compared to a non-reduced strain; The modified microorganism according to [1], which is one or more selected from the following: [3] The modified microorganism is selected from the group consisting of Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, The modified microorganism according to [1] or [2], which belongs to a genus selected from the group consisting of the genera Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, and Aspergillus; [4] The modified microorganism according to any one of [1] to [3], wherein the modified microorganism is Escherichia coli; [5] The modified microorganism according to any one of [1] to [4], wherein the transcription factor controlling the expression of pyruvate dehydrogenase is PdhR; [6] The PdhR (A-1) DNA consisting of SEQ ID NO: 128, (A-2) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 128 and encodes a protein having PdhR activity; (A-3) DNA consisting of a nucleotide sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 128, and encoding a protein having PdhR activity; (A-4) DNA encoding a protein having PdhR activity, which is a nucleotide sequence encoding a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added relative to the amino acid sequence of the protein encoded by the nucleotide sequence of SEQ ID NO: 128, or (A-5) DNA consisting of a degenerate isomer of the base sequence of SEQ ID NO: 128 or a protein encoded by (B-1) a protein consisting of the amino acid sequence of SEQ ID NO: 129; (B-2) a protein having an amino acid sequence that has 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence of SEQ ID NO: 129 and having PdhR activity; or (B-3) A protein having PdhR activity, which consists of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 129. [5] modified microorganisms; [7] A method for producing a C6 compound, comprising a culture step of culturing the modified microorganism described in any one of [1] to [6]. [Effects of the Invention]

[0013] The present invention provides a novel modified microorganism capable of producing a C6 compound and a novel method for producing a C6 compound. [Brief explanation of the drawings]

[0014] [Figure 1]FIG. 1 shows an example of a biosynthetic pathway from acetyl-CoA and succinyl-CoA to C6 compounds in the modified microorganism of the present invention. [Figure 2] FIG. 2 shows the amino acid sequence of the enzyme. [Figure 3] FIG. 3 shows the base sequences of the primers. [Figure 4] FIG. 4 shows the amino acid sequence of the enzyme. [Figure 5] FIG. 5 shows the amino acid sequence of the enzyme. [Figure 6] FIG. 6 shows the amino acid sequence of the enzyme. [Figure 7] FIG. 7 shows the amino acid sequence of the enzyme. [Figure 8] FIG. 8 shows the base sequences of the primers. [Figure 9] FIG. 9 shows the base sequences of the primers. [Figure 10] FIG. 10 shows the base sequences of the primers. [Figure 11] FIG. 11 shows the base sequence encoding the enzyme. [Figure 12] FIG. 12 shows the base sequences of the promoter, linker, and terminator. [Figure 13] FIG. 13 shows the base sequences of the primers. [Figure 14] FIG. 14 shows the base sequences of the primers. [Figure 15] FIG. 15 shows the base sequences of the primers. [Figure 16] FIG. 16 shows the base sequence encoding the enzyme. [Figure 17] FIG. 17 shows the base sequences of the primers. [Figure 18] FIG. 18 shows the nucleotide sequence encoding the enzyme (PdhR) and the amino acid sequence of the enzyme. DETAILED DESCRIPTION OF THE INVENTION

[0015] Embodiments of the present invention are described in detail below. The present invention is not limited to the following embodiments, and various conditions can be modified and varied within the scope of the present invention. Furthermore, unless otherwise specified, the DNA acquisition, vector preparation, transformation, and other genetic manipulations described herein can be performed using methods described in known publications such as Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience), and Genetic Engineering Experiment Notes (Yodosha, Takaaki Tamura). Unless otherwise specified, nucleotide sequences are written in the 5' to 3' direction herein. The terms "polypeptide" and "protein" are used interchangeably herein. The term "modified microorganism" is synonymous with "genetically modified microorganism," and the term "genetically modified microorganism" is also simply referred to as "recombinant microorganism."

[0016] In this specification, a numerical range indicated using "to" indicates a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range in one stage can be arbitrarily combined with the upper limit or lower limit of a numerical range in another stage.

[0017] The modified microorganism of the present invention is a microorganism that contains a genetic modification that suppresses a transcription factor that controls the expression of pyruvate dehydrogenase and has a pathway for producing C6 compounds.

[0018] As used herein, the term "C6 compound" includes: Adipic acid (CAS No. 124-04-9), Hexamethylenediamine (CAS No. 124-09-4), 1,6-Hexanediol (CAS No. 629-11-8), 6-aminohexanoic acid (CAS No. 60-32-2), 6-amino-1-hexanol (CAS No. 4048-33-3), 6-hydroxyhexanoic acid (CAS No. 1191-25-9), 3-oxoadipic acid (CAS No. 689-31-6), 3-Hydroxyadipic acid (CAS No. 14292-29-6) 2,3-Dehydroadipic acid (CAS No. 4440-68-0) The term "C6 compound" refers to one or more compounds selected from the group consisting of these compounds.

[0019] It will be understood by those skilled in the art that the "C6 compound" herein can be in any neutral or ionized form, including any salt form, and that this form is pH dependent.

[0020] With respect to the term "C6 compound production pathway," the "C6 compound" is at least one compound selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, 6-hydroxyhexanoic acid, 3-oxoadipic acid, 3-hydroxyadipic acid, and 2,3-dehydroadipic acid.

[0021] As used herein, with respect to a C6 compound, "having a production pathway" means that the genetically modified microorganism of the present invention expresses sufficient amounts of enzymes for each reaction step in the production pathway of the C6 compound to proceed and is capable of biosynthesizing the compound. That is, the recombinant microorganism of the present invention expresses sufficient amounts of enzymes for each reaction step in the production pathway of the C6 compound to proceed and is capable of biosynthesizing the C6 compound. The recombinant microorganism of the present invention may be one that uses a host microorganism that inherently has the ability to produce a C6 compound, or it may be one that has been modified from a host microorganism that does not inherently have the ability to produce the compound so that it has the ability to produce a C6 compound.

[0022] As used herein, the terms "endogenous" or "endogenous" are used to mean that a host microorganism that has not been genetically modified possesses the referenced gene or the protein encoded thereby (typically an enzyme or transcription factor) functionally sufficient to drive a dominant biochemical reaction within the host microorganism.

[0023] As used herein, the terms "foreign" or "exogenous" refer to the introduction of a gene or nucleic acid sequence according to the present invention into a host when the host microorganism does not have the gene to be introduced according to the present invention before genetic recombination, does not substantially express the protein (typically an enzyme or transcription factor) encoded by the gene, or has a different gene encoding the amino acid sequence of the protein but does not express comparable amounts of the endogenous protein after genetic recombination. The terms "foreign" and "exogenous" are used interchangeably herein.

[0024] As used herein, with respect to a compound, "having a production pathway" means that the modified microorganism of the present invention expresses sufficient amounts of enzymes for each reaction step in the production pathway of the compound to proceed, and is therefore capable of biosynthesizing the compound.

[0025] As used herein, the term "host microorganism" refers to a microorganism capable of having a pathway for producing a target compound. The terms "host microorganism" and "host" are used interchangeably herein. The host microorganism of the present invention is not particularly limited and may be either a prokaryote or a eukaryote. Any microorganism that has already been isolated and preserved, a microorganism newly isolated from nature, or a microorganism that has been genetically modified may be selected.

[0026] For example, the host microorganism may be selected from the group consisting of Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, and the like. The host microorganism preferably belongs to a genus selected from the group consisting of Saccharomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, and Aspergillus. Escherichia coli is preferably used as the host microorganism in the present invention.

[0027] Therefore, the modified microorganism according to the present invention can be, for example, a host microorganism of the genus Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, or the like. The bacterium belongs to a genus selected from the group consisting of the genera Saccharomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, and Aspergillus, and is preferably Escherichia coli.

[0028] As described above, the modified microorganism of the present invention comprises a genetic modification that suppresses a transcription factor that controls the expression of pyruvate dehydrogenase. As used herein, the term "transcription factor that controls the expression of pyruvate dehydrogenase" is also referred to simply as "transcription factor."

[0029] An example of a transcription factor that controls the expression of pyruvate dehydrogenase is PdhR of Escherichia coli. PdhR is a protein encoded by the pdhR gene of Escherichia coli, and acts as a transcription factor that controls the expression of enzyme genes including the pyruvate dehydrogenase complex. pdhR is also synonymously referred to as aceC, genA, and yacB. The amino acid sequence of PdhR is, for example, · PdhR from Escherichia coli strain BL21(DE3), registered under the GenBank accession number ACT42013.1; PdhR from Escherichia coli K-12 strain MG1655, registered under GenBank accession number AAC73224.1, etc. Examples of the nucleotide sequence of the coding region of the pdhR gene include, but are not limited to, The pdhR gene of Escherichia coli K-12 MG1655 strain, registered with NCBI GeneID 944827, etc. These include, but are not limited to:

[0030] In one embodiment, PdhR is (A-1) DNA consisting of SEQ ID NO: 128, (A-2) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 128 and encodes a protein having PdhR activity; (A-3) DNA consisting of a nucleotide sequence having 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 128, and encoding a protein having PdhR activity; (A-4) DNA encoding a protein having PdhR activity, which is a nucleotide sequence encoding a protein consisting of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added relative to the amino acid sequence of the protein encoded by the nucleotide sequence of SEQ ID NO: 128, or (A-5) DNA consisting of a degenerate isomer of the base sequence of SEQ ID NO: 128 The protein is encoded by (Figure 18).

[0031] In a preferred embodiment, PdhR is (A-1) DNA consisting of SEQ ID NO: 128, (A-2) DNA that hybridizes under stringent conditions with DNA consisting of a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 128 and encodes a protein having PdhR activity; (A-3) DNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 128 and encoding a protein having PdhR activity; (A-4) DNA encoding a protein having PdhR activity, which has a nucleotide sequence encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of the protein encoded by the nucleotide sequence of SEQ ID NO: 128, or (A-5) DNA consisting of a degenerate isomer of the base sequence of SEQ ID NO: 128 It is a protein encoded by

[0032] In a more preferred embodiment, PdhR is a protein encoded by the DNA consisting of (A-1) SEQ ID NO: 128.

[0033] As used herein, "stringent conditions" refers to conditions such as "1xSSC, 0.1% SDS, 60°C," more stringent conditions such as "0.1xSSC, 0.1% SDS, 60°C," and even more stringent conditions such as "0.1xSSC, 0.1% SDS, 68°C."

[0034] In another embodiment, PdhR is (B-1) a protein consisting of the amino acid sequence of SEQ ID NO: 129; (B-2) a protein having an amino acid sequence that has 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 129 and having PdhR activity; or (B-3) A protein having PdhR activity, which consists of an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids are deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 129 (Figure 18).

[0035] In a preferred embodiment, PdhR is (B-1) a protein consisting of the amino acid sequence of SEQ ID NO: 129; (B-2) a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 129 and having PdhR activity, or (B-3) A protein having PdhR activity, which consists of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence of SEQ ID NO: 129.

[0036] In a more preferred embodiment, PdhR is a protein consisting of the amino acid sequence of SEQ ID NO: 129 (B-1).

[0037] As used herein, with respect to a transcription factor, "genetic modification that suppresses" the transcription factor includes not only modifications that suppress the expression of a gene encoding the transcription factor, but also modifications that reduce the activity of the transcription factor. That is, the modified microorganism of the present invention has been modified so that the expression of a gene encoding the transcription factor is suppressed or the activity of the transcription factor is reduced.

[0038] As used herein, with respect to genes encoding transcription factors, "suppression of expression" includes "reduction of expression." Furthermore, with respect to transcription factors, "suppression of activity" is synonymous with "suppression of function," "reduction of function," and "reduction of activity," and these terms are used interchangeably. Furthermore, with respect to microorganisms, the terms "non-mutant strain" and "non-reduced strain" are used interchangeably.

[0039] Without intending to be limited by any theory, in the present invention, the transcription factor that controls the expression of pyruvate dehydrogenase negatively regulates the expression of pyruvate dehydrogenase, which catalyzes the reaction from pyruvate to acetyl-CoA. For example, by suppressing the expression of the transcription factor, the suppression of pyruvate dehydrogenase expression is relieved, and the expression of pyruvate dehydrogenase is promoted. As a result, it is thought that the production of C6 compounds biosynthesized from acetyl-CoA as a precursor is promoted and / or the production of valine biosynthesized from pyruvate as a precursor is reduced (Figure 1).

[0040] In a preferred embodiment, the genetic modification that suppresses the transcription factor that controls the expression of pyruvate dehydrogenase is a modification that suppresses the expression of the gene encoding the transcription factor; and Modification that reduces the activity of the transcription factor compared to a non-decreased strain One or more selected from the following:

[0041] In a more preferred embodiment, the genetic modification that suppresses the transcription factor that controls the expression of pyruvate dehydrogenase is a modification that suppresses the expression of the gene encoding the transcription factor, or Modification that reduces the activity of the transcription factor compared to a non-decreased strain is.

[0042] In a further preferred embodiment, the genetic modification that suppresses a transcription factor that controls the expression of pyruvate dehydrogenase is a modification that reduces the activity of the transcription factor.

[0043] Modifications that suppress transcription factors can be achieved, for example, by reducing the expression of the gene encoding the transcription factor. More specifically, reduced gene expression may refer to a reduction in the amount of gene transcription (mRNA amount) and / or a reduction in the amount of gene translation (transcription factor amount). Reduced gene expression also includes cases where the gene is not expressed at all.

[0044] The reduction in gene expression may be, for example, a reduction in the transcription level of the gene, a reduction in the translation level of the gene, or a combination thereof. The reduction in transcription level can be achieved, for example, by modifying transcriptional regulatory regions such as the promoter and operator regions of the gene. The reduction in gene transcription level can be evaluated by methods well known to those skilled in the art, such as quantitative RT-PCR and Northern blotting. The transcription level of the gene may be reduced to, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% compared to a non-reduced strain.

[0045] Methods for reducing translation include, for example, modifying translation regulatory regions such as ribosome binding sites (RBSs) and inserting riboswitch regions upstream of genes to suppress translation. A riboswitch is an RNA that selectively binds to a specific small molecule compound, called a ligand. In the absence of a ligand, the riboswitch forms a secondary structure through RNA base pairing, affecting the nucleic acid around the riboswitch. In particular, when a riboswitch contains a ribosome binding site downstream, it prevents ribosomes from accessing the ribosome binding site, thereby inhibiting the translation of mRNA for genes located further downstream. On the other hand, in the presence of a ligand, the ribosome can access the ribosome binding site through the dissolution of the secondary structure associated with ligand binding. Therefore, in the absence of a ligand, the gene's mRNA is not translated, resulting in suppression of target gene expression. The reduction in gene translation can be assessed by methods well known to those skilled in the art, such as Western blotting and ELISA. The amount of translation of the gene may be reduced to, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% compared to a non-reduced strain.

[0046] Modifications that suppress a transcription factor can also be achieved, for example, by modifications that reduce the activity of the transcription factor.

[0047] Modifications that reduce the activity of a transcription factor include, for example, modifications that reduce or eliminate the activity of the transcription factor produced. The reduction in transcription factor activity can be evaluated by methods well known to those skilled in the art, such as gel shift assay. For example, the reduction in transcription factor activity may be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 0% compared to the transcription factor activity of a non-reduced strain.

[0048] Modifications that reduce the activity of a transcription factor also include modifications that result in complete abolishment of transcription factor production. The amount of transcription factor produced can be evaluated by methods well known to those skilled in the art, such as Western blotting and ELISA.

[0049] Modification to reduce the activity of a transcription factor can be achieved, for example, by disrupting the gene encoding the transcription factor. Disruption of a gene means modifying the gene so that an active transcription factor is not produced, and includes modifications that reduce or eliminate the activity of the transcription factor that is produced, and modifications of the gene so that the transcription factor is not produced at all.

[0050] Modifications that reduce the activity of a transcription factor can be achieved, for example, by deleting part or all of the coding region of the gene on the chromosome (i.e., disrupting the gene). Furthermore, the entire gene may be deleted, including the sequences before and after the gene on the chromosome. The region to be deleted may be any of the N-terminal region, internal region, and C-terminal region, as long as the reduction in transcription factor activity can be achieved.

[0051] Gene disruption can also be achieved by, for example, introducing an amino acid substitution (missense mutation) into the coding region of a gene on a chromosome, introducing a stop codon (nonsense mutation), or introducing a frameshift mutation that adds or deletes 1 to 2 bases.

[0052] Furthermore, gene disruption can also be achieved by inserting other sequences into the coding region of the gene on the chromosome, including antibiotic resistance genes and transposons, but is not limited thereto as long as they reduce the activity.

[0053] Gene disruption can be achieved by a method utilizing homologous recombination, such as a method using λ-phage Red recombinase (Datsenko, Kirill A., and Barry L. Wanner. “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.” Proceedings of the National Academy of Sciences 97.12 (2000): 6640-6645.), a method using a suicide vector containing a temperature-sensitive replication origin (Blomfield et al., Molecular microbiology 5.6 (1991): 1447-1457.), or a method using the CRISPR-Cas9 system (Jiang, Yu, et al. “Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system.” Appl. Environ. Microbiol. 81.7 (2015): 2506-2514.), but is not limited to these methods.

[0054] Alternatively, gene disruption may be achieved by mutagenesis, which may include physical treatments such as X-ray treatment, ultraviolet treatment, and gamma-ray treatment, as well as chemical treatments using mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, and methyl methanesulfonate, but is not particularly limited as long as it reduces activity.

[0055] Gene disruption can be confirmed by methods well known to those skilled in the art, for example, by determining the nucleotide sequence or sequence length of the coding region of the gene.

[0056] As used herein, the term "non-reduced strain" refers to a strain in which the transcription factor controlling the expression of pyruvate dehydrogenase is not repressed, and is also referred to as a "non-mutant strain." Non-reduced strains include, but are not limited to, wild-type and standard strains of each microbial strain, and derivatives including strains obtained by breeding. For example, E. coli strains include, but are not limited to, K-12, B, C, and W strains, as well as derivatives thereof, such as BL21(DE3), W3110, MG1655, JM109, DH5α, and HB101.

[0057] In a preferred embodiment, the modified microorganism of the present invention has a C6 compound production pathway that further expresses enzymes that catalyze reaction steps in the C6 compound production pathway. An example of a C6 compound production pathway that the modified microorganism of the present invention may have is shown in Figure 1.

[0058] In one embodiment, the modified microorganism of the present invention includes the following gene encoding an "enzyme that catalyzes a reaction step in a C6 compound production pathway": · The gene encoding 3-oxoadipyl CoA thiolase, the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, · The gene encoding 3-hydroxyadipyl-CoA dehydratase, · The gene encoding 2,3-dehydroadipyl-CoA reductase, a gene encoding a thioester hydrolase, · CoA-transferase-encoding genes, -genes encoding phosphate acyltransferases, -genes encoding phosphotransferases, - a gene encoding an acid thiol ligase, genes encoding dehydrogenases, · a gene encoding carboxylic acid reductase, Genes encoding transaminases (aminotransferases), and the gene encoding alcohol dehydrogenase, By including such a gene, the modified microorganism can efficiently produce C6 compounds.

[0059] In a preferred embodiment, the modified microorganism of the present invention contains at least one of the following genes encoding "enzymes that catalyze reaction steps in the C6 compound production pathway": · The gene encoding 3-oxoadipyl CoA thiolase, the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, the gene encoding 3-hydroxyadipyl-CoA dehydratase, and The gene encoding 2,3-dehydroadipyl-CoA reductase By including such a gene, the modified microorganism can efficiently produce adipic acid.

[0060] In another preferred embodiment, the modified microorganism of the present invention contains at least one of the following genes encoding "enzymes catalyzing reaction steps in a C6 compound production pathway": · The gene encoding 3-oxoadipyl CoA thiolase, the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, the gene encoding 3-hydroxyadipyl-CoA dehydratase, and · The gene encoding 2,3-dehydroadipyl-CoA reductase, · a gene encoding carboxylic acid reductase, The gene encoding alcohol dehydrogenase By including such genes, the modified microorganism can efficiently produce 6-hydroxyhexanoic acid and / or 1,6-hexanediol.

[0061] In another preferred embodiment, the modified microorganism of the present invention contains at least one of the following genes encoding "enzymes catalyzing reaction steps in a C6 compound production pathway": · The gene encoding 3-oxoadipyl CoA thiolase, the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, · The gene encoding 3-hydroxyadipyl-CoA dehydratase, · The gene encoding 2,3-dehydroadipyl-CoA reductase, a gene encoding carboxylic acid reductase, and Genes encoding transaminases (aminotransferases) By including such genes, the modified microorganism can efficiently produce 6-aminohexanoic acid and / or hexamethylenediamine (1,6-diaminohexane).

[0062] 3-Oxoadipyl-CoA thiolase performs the reaction in step A of Figure 1. 3-Hydroxyadipyl-CoA dehydrogenase performs the reaction shown in step B of Figure 1. 3-Hydroxyadipyl-CoA dehydratase performs the reaction shown in step C of Figure 1. 2,3-Dehydroadipyl-CoA reductase performs the reaction shown in step D of Figure 1. The thioester hydrolase can perform at least one of steps E, W, X, and Y in Figure 1. CoA transferase can perform at least one of the reactions in steps E, W, X, Y, S, and T in Figure 1. The combination of a phosphate acyltransferase and a phosphotransferase may be used to perform at least one of the reactions in steps E, W, X, Y, S, and T of Figure 1; The acid thiol ligase may be used to carry out at least one of the reactions in steps E, W, X, Y, S, and T of Figure 1. The dehydrogenase may perform at least one of steps F, U, and V in Figure 1. Carboxylic acid reductase can perform at least one of steps G, I, K, and N in Figure 1. Transaminases (aminotransferases) perform at least one of the reactions in steps J, M, P, and R in Figure 1. Alcohol dehydrogenase performs at least one of steps H, L, O, and Q in Figure 1. Catalyze.

[0063] If the modified microorganism of the present invention expresses sufficient amounts of an enzyme that catalyzes a reaction step in the C6 compound production pathway without the introduction of an exogenous gene, the reaction may proceed using an enzyme encoded by an endogenous gene.

[0064] The enzymes that catalyze each reaction step in the C6 compound production pathway possessed by the modified microorganism of the present invention may be encoded by a single gene or multiple genes.

[0065] The enzymes that catalyze each reaction in the C6 compound production pathway are described below with reference to FIG.

[0066] In step A of Figure 1, succinyl-CoA and acetyl-CoA are condensed to form 3-oxoadipyl-CoA. Examples of enzymes that can catalyze this conversion include β-ketothiolases. Furthermore, enzymes classified into groups such as EC 2.3.1.9 (acetoacetyl-CoA thiolase), EC 2.3.1.16 (3-ketoacyl-CoA thiolase), and EC 2.3.1.174 (3-oxoadipyl-CoA thiolase) can be exemplified as enzymes that can be active in this conversion. The enzymes used in the present invention are not limited as long as they are active in this conversion, and examples include 3-oxoadipyl-CoA thiolases. In one embodiment, PaaJ derived from Escherichia coli, consisting of the amino acid sequence set forth in SEQ ID NO: 1, is used (Figure 2).

[0067] In step B of Figure 1, 3-oxoadipyl-CoA is converted to 3-hydroxyadipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in the EC 1.1.1 group. For example, enzymes classified in groups such as EC 1.1.1.35 (3-hydroxyacyl-CoA dehydrogenase), EC 1.1.1.36 (acetoacetyl-CoA dehydrogenase), EC 1.1.1.157 (3-hydroxybutanoyl-CoA dehydrogenase), EC 1.1.1.211 (long-chain 3-hydroxyacyl-CoA dehydrogenase), and EC 1.1.1.259 (3-hydroxypimeloyl-CoA dehydrogenase) can be exemplified as enzymes that can be active in this conversion. The enzyme used in the present invention is not limited as long as it has the activity for this conversion, and an example is 3-hydroxyadipyl-CoA dehydrogenase. In one embodiment, PaaH derived from Escherichia coli and consisting of the amino acid sequence set forth in SEQ ID NO: 2 is used.

[0068] In step C of Figure 1, 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA. Examples of enzymes that can catalyze this conversion include hydro-lyases classified in the EC 4.2.1 group. For example, enzymes classified in the groups EC 4.2.1.17 (enoyl-CoA hydratase), EC 4.2.1.55 (3-hydroxybutanoyl-CoA dehydratase), and EC 4.2.1.74 (long-chain enoyl-CoA hydratase) can be exemplified as enzymes that can be active in this conversion. The enzyme used in the present invention is not limited as long as it is active in this conversion, but an example is 3-hydroxyadipyl-CoA dehydratase. In one embodiment, PaaF derived from Escherichia coli, consisting of the amino acid sequence set forth in SEQ ID NO: 3, is used (Figure 2). In another embodiment, the enzyme used is PaaF (L3), a 3-hydroxyadipyl-CoA dehydratase enzyme encoded by the nucleotide sequence (783 bp) of a PCR amplification product using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template and the nucleotides set forth in SEQ ID NO: 4 and SEQ ID NO: 5 as primers (Figure 3). The Burkholderia sp. LEBP-3 strain (hereinafter also referred to as "L3 strain") was submitted for deposit with the National Institute of Technology and Evaluation's Patent Microorganisms Depositary (NPMD) on December 4, 2020 (accession number: NITE ABP-03334) and is internationally deposited under "accession number: NITE BP-03334."

[0069] The DNA polymerase used to prepare the PCR amplification product may be any known in the art, including, but not limited to, Taq DNA polymerase, hot-start-adjusted DNA polymerase, and proofreading DNA polymerases that have 3'-5' exonuclease activity in addition to polymerase activity. In a preferred embodiment of the PCR amplification product preparation conditions, a specific nucleotide is used as a forward primer and a reverse primer at 1 μM each, and PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio) is used as the enzyme. A 25 μL volume of PCR amplification product is prepared by performing 30 cycles of heat treatment at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 72°C for 5 seconds / kb.

[0070] In step D of Figure 1, 2,3-dehydroadipyl-CoA is converted to adipyl-CoA. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in the EC 1.3.1 group. For example, EC 1.3.1.8 (acyl-CoA dehydrogenase (NADP) + )), EC 1.3.1.9 (enoyl-ACP reductase (NADH)), EC 1.3.1.38 (trans-2-enoyl-CoA reductase (NADP + )) (Ter), EC 1.3.1.44 (trans-2-enoyl-CoA reductase (NAD + Examples of enzymes that may be active in this conversion include enzymes classified into the groups EC 1.3.1.86 (crotonyl-CoA reductase), EC 1.3.1.93 (long-chain acyl-CoA reductase), and EC 1.3.1.104 (enoyl-ACP reductase (NADPH)). The enzyme used in the present invention is not limited as long as it has activity in this conversion, and is, for example, 2,3-dehydroadipyl-CoA reductase.

[0071] The 2,3-dehydroadipyl-CoA reductase used in the present invention is not particularly limited. Typical enzymes that have been reported to have 2,3-dehydroadipyl-CoA reductase activity include, for example, the enzyme DcaA derived from Acinetobacter baylyi (Kallscheuer, Nicolai, et al. "Improved production of adipate with Escherichia coli by reversal of β-oxidation," Applied microbiology and biotechnology 101.6 (2017): 2371-2382.), and enoyl-CoA reductases derived from biological species such as Candida tropicalis, Euglena gracilis, Clostridium beijerinckii, and Yarrowia lipolytica (JP Patent Publication No. 2011-512868). These enzymes can also be used in the present invention. In another embodiment, the enzyme that can be used is MmgC (L3), a 2,3-dehydroadipyl-CoA reductase encoded by the base sequence (1155 bp) of a PCR amplification product using the chromosomal DNA of Burkholderia sp. L3 strain as a template, and using the nucleotides shown in SEQ ID NO: 6 and SEQ ID NO: 7 as primers ( FIG. 3 ).

[0072] In addition, enzymes derived from biological species such as Candida auris, Kluyveromyces marxianus, Pichia kudriavzevii, Thermothelomyces thermophilus, Thermothielavioides terrestris, Chaetomium thermophilum, Podospora anserina, Purpureocillium lilacinum, and Pyrenophora teres can be used, and preferably at least one enzyme having an amino acid sequence set forth in SEQ ID NOs: 8 to 16 is used (Figure 4).

[0073] In the reactions of steps E, W, X, and Y in Figure 1, coenzyme A (CoA) is eliminated. Examples of enzymes that can catalyze this conversion include thioester hydrolases classified in the EC 3.1.2 group. For example, enzymes classified in groups such as EC 3.1.2.1 (acetyl-CoA hydrolase) and EC 3.1.2.20 (acyl-CoA hydrolase) can be exemplified as enzymes that can have activity in this conversion. There is no limitation on the enzymes that can be used in the present invention, as long as they have activity in this conversion.

[0074] Further, examples of enzymes that can catalyze the reactions of steps E, W, X, and Y in Figure 1 include CoA transferases classified into the EC 2.8.3 group. For example, enzymes classified into groups such as EC 2.8.3.5 (3-oxoacid CoA-transferases), EC 2.8.3.6 (3-oxoadipate CoA-transferases), and EC 2.8.3.18 (succinyl-CoA:acetyl-CoA-transferases) can be exemplified as enzymes that may have activity for this conversion. There is no limitation on the enzymes that can be used in the present invention, as long as they have activity for this conversion.

[0075] Another example of an enzymatic conversion that can catalyze the reactions of steps E, W, X, and Y in Figure 1 is a pathway in which acyl acyltransferases classified in the EC 2.3.1 group transfer the acyl group of acyl-CoA to phosphate to generate acylphosphate, followed by dephosphorylation by phosphotransferases classified in the EC 2.7.2 group. For example, enzymes classified in the EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase) groups are examples of acyltransferases that may be active in this conversion. For example, enzymes classified in the EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butanoate kinase) groups are examples of phosphotransferases that may be active in this conversion. There is no particular limitation on the enzymes that can be used in the present invention, as long as they have the activity for this conversion.

[0076] Additionally, examples of other enzymes capable of catalyzing the reactions of steps E, W, X, and Y in Figure 1 include acid thiol ligases classified in the EC 6.2.1 group. For example, enzymes classified into groups such as EC 6.2.1.1 (acetyl-CoA synthetase), EC 6.2.1.13 (acetyl-CoA synthetase), EC 6.2.1.4 (succinyl-CoA synthetase), EC 6.2.1.5 (succinyl-CoA synthetase), and EC 6.2.1.14 (pimeloyl-CoA synthetase) can be exemplified as enzymes that may have activity for this conversion. The enzymes used in the present invention are not limited as long as they have activity for this conversion.

[0077] In steps F and M of Figure 1, adipyl-CoA is converted to adipate semialdehyde. Examples of enzymes that can catalyze this conversion include those classified in the EC 1.2.1 group. For example, enzymes classified into groups such as EC 1.2.1.10 (acetaldehyde dehydrogenase (acetylation)), EC 1.2.1.17 (glyoxylate dehydrogenase (acylation)), EC 1.2.1.42 (hexadecanal dehydrogenase (acylation)), EC 1.2.1.44 (cinnamoyl-CoA reductase (acylation)), EC 1.2.1.75 (malonyl-CoA reductase (malonic acid semialdehyde formation)), and EC 1.2.1.76 (succinic acid semialdehyde dehydrogenase (acylation)) catalyze a conversion reaction in which CoA is eliminated to produce an aldehyde, similar to this conversion, and therefore can be used as examples of enzymes that may be active in this conversion. The enzyme used in the present invention is not limited as long as it has the activity for this conversion. For example, sucD derived from Clostridium kluyveri and consisting of the amino acid sequence set forth in SEQ ID NO: 17 is used (FIG. 5).

[0078] In the reactions of steps G, I, K, and N in Figure 1, a carboxyl group is converted to an aldehyde. Enzymes that can catalyze this conversion include, for example, carboxylic acid reductases (CARs). For example, EC 1.2.1.30 (carboxylic acid reductase (NADP) +)), EC 1.2.1.31 (L-aminoadipate semialdehyde dehydrogenase), EC 1.2.1.95 (L-2-aminoadipate reductase), and EC 1.2.99.6 (carboxylic acid reductase) catalyze a conversion reaction that produces an aldehyde from a carboxylic acid, similar to this conversion, and therefore can be used as examples of enzymes that may be active in this conversion. Typical examples of biological species from which the enzymes can be derived include, but are not limited to, Nocardia iowensis, Nocardia asteroides, Nocardia brasiliensis, Nocardia farcinica, Segniliparus rugosus, Segniliparus rotundus, Tsukamurella paurometabola, Mycobacterium marinum, Mycobacterium neoaurum, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium chelonae, Mycobacterium immunogenum, Mycobacterium smegmatis, Serpula lacrymans, Heterobasidion annosum, Coprinopsis cinerea, Aspergillus flavus, Aspergillus terreus, Neurospora crassa, and Saccharomyces cerevisiae. The enzymes used in the present invention are not limited as long as they have activity for this conversion. For example, at least one enzyme consisting of the amino acid sequence set forth in any of SEQ ID NOs: 18 to 22 (Figure 5) may be used. Preferably, at least one of the enzyme MaCar derived from Mycobacterium abscessus consisting of the amino acid sequence set forth in SEQ ID NO: 20 and MaCar(m), a variant of MaCar consisting of the amino acid sequence set forth in SEQ ID NO: 22, is used. More preferably, at least MaCar(m) consisting of the amino acid sequence set forth in SEQ ID NO: 22 is used.

[0079] Furthermore, carboxylic acid reductase can be converted into an active holoenzyme by phosphopantetheinylation (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)). Phosphopantetheinylation is catalyzed by phosphopantetheinyl transferase (PT). Enzymes that can catalyze this reaction include, for example, enzymes classified in EC 2.7.8.7. Therefore, the microorganism of the present invention may be further modified to increase the activity of phosphopantetheinyl transferase. Methods for increasing the activity of phosphopantetheinyl transferase include, but are not limited to, introducing an exogenous phosphopantetheinyl transferase gene and enhancing the expression of an endogenous phosphopantetheinyl transferase gene. The enzymes used in the present invention are not limited as long as they have phosphopantetheinyl group transfer activity. Typical examples of such enzymes include EntD from Escherichia coli, Sfp from Bacillus subtilis, Npt from Nocardia iowensis (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)), and Lys5 from Saccharomyces cerevisiae (Ehmann et al., Biochemistry 38.19 (1999): 6171-6177). The enzymes used in the present invention are not limited as long as they have the activity for this conversion. For example, at least one of the enzymes consisting of the amino acid sequences set forth in SEQ ID NOs: 23 to 26 may be used ( FIG. 6 ). Preferably, Npt from Nocardia iowensis, derived from Nocardia iowensis and consisting of the amino acid sequence set forth in SEQ ID NO: 24, is used.

[0080] The reactions in steps J, M, P, and R in Figure 1 are transamination reactions. Examples of enzymes that can catalyze this conversion include transaminases (aminotransferases) classified in the EC 2.6.1 group. For example, enzymes classified in groups such as EC 2.6.1.19 (4-aminobutanoate-2-oxoglutarate transaminase), EC 2.6.1.29 (diamine transaminase), and EC 2.6.1.48 (5-aminovalerate transaminase) can be exemplified as enzymes that can also have activity in this conversion. The enzymes used in the present invention are not particularly limited as long as they have the conversion activity for each step. For example, YgjG, a putrescine aminotransferase from Escherichia coli that has been reported to transaminate cadaverine and spermidine (Samsonova, et al., BMC microbiology 3.1 (2003): 2), SpuC, a putrescine aminotransferase from the genus Pseudomonas (Lu et al., Journal of bacteriology 184.14 (2002): 3765-3773; Galman et al., Green Chemistry 19.2 (2017): 361-366), GabT, a GABA aminotransferase from Escherichia coli, and PuuE may be used. Furthermore, ω-transaminases derived from species such as Ruegeria pomeroyi, Chromobacterium violaceum, Arthrobacter citreus, Sphaerobacter thermophilus, Aspergillus fischeri, Vibrio fluvialis, Agrobacterium tumefaciens, and Mesorhizobium loti have also been reported to have transamination activity toward diamine compounds such as 1,8-diaminooctane and 1,10-diaminodecane, and may be used in the present invention (Sung et al., Green Chemistry 20.20 (2018): 4591-4595., Sattler et al., Angewandte Chemie 124.36 (2012): 9290-9293.).The enzyme used in the present invention is not limited as long as it has activity for this conversion, and for example, at least one of the enzymes consisting of the amino acid sequences set forth in SEQ ID NOS: 27 to 32 may be used (FIG. 7). Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, and glycine.

[0081] In steps H, L, O, and Q of Figure 1, an aldehyde is converted to an alcohol. Examples of enzymes that can catalyze this conversion include oxidoreductases classified in the EC 1.1.1 group. For example, EC 1.1.1.1 (alcohol dehydrogenase) and EC 1.1.1.2 (alcohol dehydrogenase (NADP)) are oxidoreductases. + )), EC 1.1.1.71 (alcohol dehydrogenase [NAD(P) + ]) catalyze the conversion of aldehydes to alcohols, similar to this conversion, and are therefore examples of enzymes that may also have activity for this conversion. Enzymes that can be used in the present invention are not limited as long as they have activity for this conversion, and an example is Ahr derived from Escherichia coli, as set forth in SEQ ID NO: 32 (Figure 7).

[0082] In the reactions of steps S and T in Figure 1, CoA is added to a carboxyl group. Examples of enzymes that can catalyze this conversion include CoA transferases classified in the EC 2.8.3 group, a combination of a phosphate acyltransferase classified in the EC 2.3.1 group and a phosphotransferase classified in the EC 2.7.2 group, and acid thiol ligases classified in the EC 6.2.1 group. The enzymes used in the present invention are not limited as long as they have the activity for this conversion.

[0083] The genes encoding the above-mentioned enzymes that can be used in the present invention may be derived from organisms other than those exemplified, or may be artificially synthesized, as long as they are capable of expressing substantial enzymatic activity in the microbial body.

[0084] Furthermore, the amino acid sequences of the above-mentioned enzymes that can be used in the present invention, or the nucleotide sequences of the genes encoding the enzymes, may contain all mutations that can occur in nature and / or artificially introduced mutations and modifications, such as deletions, substitutions, insertions, and additions, so long as they are capable of expressing substantial enzymatic activity in the microorganism. For example, the amino acid sequences may contain one or more, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 7, particularly preferably 1 to 5, and even more particularly preferably 1 to 3 amino acids deleted, substituted, inserted, and / or added to the amino acid sequence of the above-mentioned enzymes.

[0085] In addition, it is known that there are redundant codons for various codons that encode specific amino acids, and therefore, alternative codons that ultimately translate into the same amino acid may be used in the present invention. In other words, because the genetic code is degenerate, multiple codons can be used to encode a specific amino acid, and therefore, an amino acid sequence can be encoded by any set of similar DNA oligonucleotides. Furthermore, since it is known that most organisms preferentially use a subset of specific codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), "codon optimization" may also be useful in the present invention.

[0086] Therefore, the microorganism of the present invention may contain a nucleotide sequence having, for example, 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the nucleotide sequence of the enzyme gene, provided that it can express substantial enzymatic activity. Alternatively, the microorganism may contain a gene encoding an amino acid sequence having, for example, 80% or more, 85% or more, 88% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence of the enzyme.

[0087] As used herein, the percentage (%) of "sequence identity" of a comparison amino acid sequence to a reference amino acid sequence is defined as the percentage of amino acid residues in the comparison sequence that are identical to those in the reference sequence, after aligning the sequences to maximize identity between the two sequences and, if necessary, introducing gaps into one or both of the two sequences. Conservative substitutions are not considered part of the sequence identity. Sequence identity can be determined using publicly available computer software, for example, using an alignment search tool such as BLAST (Basic Local Alignment Search Tool). Those skilled in the art can determine appropriate parameters for aligning the comparison sequences to maximize alignment. The "sequence identity" of nucleotide sequences can also be determined using a similar method.

[0088] In the present invention, the enzyme genes involved in the biosynthesis of the above-mentioned C6 compounds are introduced into a host microorganism as an "expression cassette," thereby achieving more stable and higher levels of enzyme activity. As used herein, "expression cassette" refers to a nucleotide sequence containing a nucleic acid sequence that regulates transcription and translation and is operably linked to the nucleic acid or gene to be expressed. Typically, the expression cassette of the present invention contains a promoter sequence 5' upstream of the coding sequence, a terminator sequence 3' downstream, and, optionally, additional conventional regulatory elements operably linked thereto. In this case, the nucleic acid or gene to be expressed is introduced into the microorganism.

[0089] A promoter is defined as a DNA sequence that allows RNA polymerase to bind to DNA and initiate RNA synthesis, regardless of whether it is a constitutive or inducible promoter. A strong promoter is one that initiates mRNA synthesis at a high frequency and is suitable for use in the present invention. In E. coli, promoters that can be used include the lac, trp, tac, or trc systems, the major operator and promoter region of λ phage, the regulatory region of fd coat protein, promoters for glycolytic enzymes (e.g., 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamic acid decarboxylase A, and serine hydroxymethyltransferase, and the promoter region of RNA polymerase derived from T7 phage. Terminators that can be used include the T7 terminator, rrnBT1T2 terminator, and lac terminator. In addition to promoter and terminator sequences, examples of other regulatory elements include selectable markers, amplification signals, and replication origins. Suitable regulatory sequences are described, for example, in "Gene Expression Technology: Methods in Enzymology 185", Academic Press (1990).

[0090] The expression cassettes described above are incorporated into vectors, such as plasmids, phages, transposons, IS elements, fosmids, cosmids, or linear or circular DNA, and then inserted into a host microorganism. Plasmids and phages are preferred. These vectors may be autonomously replicated in the host microorganism or may replicate chromosomally. Suitable plasmids include, for example, E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11, or pBdCI. Other usable plasmids are described in "Gene Cloning and DNA Analysis, 7th Edition," Wiley-Blackwell (2016). Introduction of an expression cassette into a vector can be carried out by a conventional method including excision with an appropriate restriction enzyme, cloning, and ligation. Each expression cassette may be located on one vector, or may be located on two or more vectors.

[0091] After the vector carrying the expression cassette of the present invention is constructed as described above, the vector can be introduced into a host microorganism by a conventional method, such as, but not limited to, the calcium chloride method, electroporation, or conjugal transfer.

[0092] The modified microorganism of the present invention may include any genetic manipulation, as long as it includes a genetic modification that suppresses a transcription factor that controls the expression of pyruvate dehydrogenase and has a C6 compound production pathway, including, but not limited to, enhanced gene expression, suppressed gene expression, enhanced enzyme activity, suppressed enzyme activity, enhanced transcription factor activity, and suppressed transcription factor activity.

[0093] The modified microorganism obtained as described above is cultured and maintained under conditions suitable for its growth and / or maintenance to produce the desired C6 compound. Suitable medium compositions, culture conditions, and culture times for transformants derived from various host microorganisms can be easily determined and selected by those skilled in the art.

[0094] Therefore, a second aspect of the present invention relates to a method for producing a target compound, comprising a culturing step of culturing the modified microorganism described above. Specifically, the production method comprises a culturing step of culturing the modified microorganism described above to obtain a culture and / or an extract of the culture of the modified microorganism.

[0095] In the culturing step, the modified microorganism is cultured in a medium containing a carbon source and a nitrogen source to obtain a culture containing the bacterial cells. The modified microorganism of the present invention is cultured under conditions suitable for compound production and for the growth and maintenance of the microorganism. Suitable medium composition, culture time, and culture conditions can be easily determined by those skilled in the art.

[0096] Carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, molasses, fats and oils (e.g., soybean oil, sunflower oil, peanut oil, coconut oil, etc.), fatty acids (e.g., palmitic acid, linoleic acid, oleic acid, linolenic acid, etc.), alcohols (e.g., glycerol, ethanol, etc.), organic acids (e.g., acetic acid, lactic acid, succinic acid, etc.), corn hydrolyzed liquid, and cellulose hydrolyzed liquid. D-glucose, sucrose, or glycerol is preferred. These carbon sources can be used individually or in combination.

[0097] Compounds produced using biomass-derived raw materials can be clearly distinguished from synthetic raw materials derived from, for example, petroleum, natural gas, and coal by measuring their biobased carbon content using Carbon-14 (radiocarbon) analysis as specified in ISO 16620-2 or ASTM D6866.

[0098] Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, casamino acids, tryptone, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, amino acids, and urea), or inorganic compounds (e.g., aqueous ammonia, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.) These nitrogen sources can be used individually or in combination.

[0099] Furthermore, if the modified microorganism expresses a useful additional trait, for example, if it has an antibiotic resistance marker, the medium may contain the corresponding antibiotic. This reduces the risk of contamination by other bacteria during culture. Examples of antibiotics include, but are not limited to, β-lactam antibiotics such as ampicillin, aminoglycoside antibiotics such as kanamycin, macrolide antibiotics such as erythromycin, tetracycline antibiotics, and chloramphenicol.

[0100] If the modified microorganism cannot utilize the above carbon sources, such as cellulose and polysaccharides, it can be adapted to produce a target compound using these carbon sources by using known genetic engineering techniques, such as introducing foreign genes, such as cellulase genes and amylase genes.

[0101] Culturing may be performed in either a batch or continuous manner. In either case, additional carbon sources or the like may be replenished at appropriate times during the culture. Furthermore, culturing may be performed while controlling suitable conditions, such as temperature, oxygen concentration, and pH. For example, the suitable culture temperature for a transformant derived from a typical Escherichia coli strain is usually 15°C to 55°C, preferably 25°C to 40°C. When the host microorganism is aerobic, shaking (e.g., flask culture) or stirring / aeration (e.g., jar fermenter culture) may be performed to ensure an appropriate oxygen concentration during fermentation. Those skilled in the art can easily determine these culture conditions.

[0102] The production method of the present invention preferably further comprises a mixing step of mixing the culture and / or an extract of the culture with a substrate compound to obtain a mixed solution.

[0103] As a result of the reaction, a target compound is produced in the culture and / or the mixed solution. Therefore, in a more preferred embodiment, the production method of the present invention further comprises a recovery step of recovering the target compound from the culture and / or the mixed solution.

[0104] The step of recovering the target compound from the culture is carried out by any method for the purpose of separation and / or purification. Examples include, but are not limited to, centrifugation, membrane filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation, ion exchange, and various types of chromatography. Separation and / or purification may be carried out by a single method or by a combination of multiple methods.

[0105] As described above, the present invention provides novel modified microorganisms capable of producing C6 compounds and novel methods for producing C6 compounds. Furthermore, by incorporating a genetic modification that suppresses the expression of a transcription factor controlling pyruvate dehydrogenase in a host microorganism, the production of the byproduct valine can be suppressed and / or adipic acid production in the C6 compound production pathway can be promoted, resulting in modified microorganisms with excellent C6 compound production capabilities. The modified microorganisms and production methods of the present invention enable efficient production of target C6 compounds. Furthermore, various target compounds can be obtained by performing conversion using additional enzymes depending on the desired target compound. Furthermore, because the modified microorganisms and / or production methods of the present invention have excellent target compound production capabilities, they are expected to be capable of producing target compounds on an industrial scale. The C6 compounds produced by the modified microorganisms and production methods of the present invention can be used, in particular, as monomer raw materials for polymer production.

[0106] While the embodiments for carrying out the present invention have been described above, the above embodiments are merely examples and are not intended to limit the scope of the invention. The above embodiments can be carried out in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. [Example]

[0107] The present invention will be described below based on examples, but the present invention is not limited to these examples.

[0108] The Burkholderia genus strain LEBP-3 (hereinafter also referred to as "strain L3") was applied for deposit with the National Patent Microorganisms Depositary (NPMD) of the National Institute of Technology and Evaluation on December 4, 2020, and has been internationally deposited under accession number NITE BP-03334.

[0109] <Construction of plasmid for gene disruption> Gene disruption, mutagenesis, and gene insertion in E. coli were performed using homologous recombination with pHAK1 (deposited on March 18, 2019, at the National Institute of Technology and Evaluation, National Patent Microorganism Depositary (NPMD) under accession number NITE P-02919; international deposit number: NITE BP-02919). pHAK1 contains a temperature-sensitive mutant repA gene, a kanamycin resistance gene, and the Bacillus subtilis-derived levansucrase gene sacB. The levansucrase gene is lethal to the host microorganism in the presence of sucrose. PCR fragment amplification was performed using PrimeSTAR Max DNA Polymerase (Takara Bio) or KOD FX Neo (Toyobo), and plasmid preparation was performed using the E. coli HST08 strain.

[0110] Using genomic DNA from E. coli BL21(DE3) as a template, a PCR product containing the 5' homologous region, coding region, and 3' homologous region of the disruption target gene was obtained. The combinations of target genes and primer sequences are shown in Table 1 below.

[0111]

Table 1

[0112] Next, this PCR product was inserted into the pHAK1 plasmid fragment amplified using the primers of SEQ ID NOs: 55 and 56 (Figure 9) with the In-Fusion HD cloning kit (product name, manufactured by Clontech) and circularized. It was transformed into Escherichia coli strain HST08, and the plasmid was extracted from the obtained transformant.

[0113] Using the pHAK1 plasmid inserted with the DNA fragments of the 5' homologous region, coding region, and 3' homologous region of the disrupted target gene extracted above as a template, PCR was performed using the primers described in Table 2 below and Figure 10 to obtain a pHAK1 plasmid fragment in which a partial or entire region of the coding region of the disrupted target gene was removed and the 5' homologous region and 3' homologous region were included.

[0114]

Table 2

[0115] <0OO0531>The obtained plasmid fragment was circularized by terminal phosphorylation and self-ligation. It was transformed into Escherichia coli strain HST08, and the plasmid was extracted from the obtained transformant to obtain a plasmid for gene disruption.

[0116] <Construction of Plasmid for Insertion of Enzyme Gene in C6 Compound Production Pathway> A polynucleotide (SEQ ID NO: 79) encoding Eshcherichia coli PaaJ (Ec) (SEQ ID NO: 1) was obtained by cloning from the genomic DNA of the Eshcherichia coli W3110 strain (NBRC12713). A polynucleotide (SEQ ID NO: 80) encoding Eshcherichia coli PaaH (Ec) (SEQ ID NO: 2) was obtained by cloning from the genomic DNA of the Eshcherichia coli W3110 strain (NBRC12713) (FIGS. 2 and 11).

[0117] A plasmid for gene insertion was designed so that the paaJ(Ec) and paaH(Ec) genes could be inserted as an expression cassette into the pflB gene region. The expression cassette, which contained a promoter region (SEQ ID NO: 81), paaJ(Ec) gene coding region, linker sequence (SEQ ID NO: 82), paaH gene coding region, and terminator region (SEQ ID NO: 83) arranged in this order, was inserted between the 5' and 3' homologous regions of the pflB gene coding region contained in the previously constructed pflB gene disruption plasmid (Fig. 12).

[0118] The polynucleotide encoding PaaF (L3) of the L3 strain was obtained by optimizing the base sequence for expression in E. coli and using the artificial gene synthesis service of Eurofins Genomics. The polynucleotide encoding MmgC (L3) of the L3 strain was obtained by optimizing the base sequence for expression in E. coli and using the artificial gene synthesis service of Eurofins Genomics.

[0119] A gene insertion plasmid was designed so that the optimized sequences of the paaF (L3) and mmgC (L3) gene coding regions could be inserted into the yahK gene region as an expression cassette. The expression cassette, which contained the promoter region (SEQ ID NO: 84), paaF (L3) gene coding region, linker sequence (SEQ ID NO: 85), mmgC (L3) gene coding region, and terminator region (SEQ ID NO: 86) arranged in this order, was inserted between the 5' and 3' homologous regions of the yahK gene coding region contained in the previously constructed yahK gene disruption plasmid (Figure 12).

[0120] <Construction of a plasmid for inserting the mutated gltA gene> Using the genomic DNA of E. coli BL21(DE3) strain as a template and primers of SEQ ID NOs: 53 and 54 (FIG. 8), a PCR product containing the 5′ homologous region, coding region, and 3′ homologous region of the gltA gene was obtained.

[0121] Next, this PCR product was inserted into a pHAK1 plasmid fragment amplified using the primers of SEQ ID NOs: 55 and 56 using an In-Fusion HD cloning kit (product name, manufactured by Clontech), and circularized. The resulting fragment was transformed into Escherichia coli HST08, and the plasmid was extracted from the resulting transformant (Figure 9).

[0122] Using the pHAK1 plasmid extracted above, into which DNA fragments of the 5' homologous region, coding region, and 3' homologous region of the gltA gene had been inserted, as a template, PCR was performed using the primers shown in SEQ ID NOs: 87 and 88 (Figure 13), to obtain a pHAK1 plasmid fragment containing the 5' homologous region, the mutated gltA gene coding region, and the 3' homologous region.

[0123] <Construction of modified E. coli strains> E. coli BL21(DE3) was transformed with a plasmid for disrupting the desired gene by electroporation (see Genetic Engineering Experiment Notes, Yodosha, by Takaaki Tamura). The resulting transformant was then plated onto LB agar medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 15 g / L agar powder) containing 100 mg / L kanamycin sulfate and incubated overnight at 30°C to obtain single colonies. One loopful of the transformant was inoculated into 1 mL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride) containing 100 mg / L kanamycin sulfate and cultured with shaking at 30°C. The resulting culture was plated onto LB agar medium containing 100 mg / L kanamycin sulfate and incubated overnight at 42°C. The resulting colonies showed that the plasmid had been integrated into the genome by a single crossover. A loopful of colony was inoculated into 1 mL of LB liquid medium and cultured with shaking at 30°C. The resulting culture was spread onto LB agar medium containing 20% ​​sucrose and incubated at 30°C for 2 days. The resulting colonies were confirmed by colony direct PCR using the primer sets shown in Table 3 and Figure 14 to determine whether the desired gene had been disrupted or inserted.

[0124] The above procedures were repeated to construct E. coli strains containing multiple gene disruptions and gene insertions. The constructed E. coli strains are shown in Table 4. In the table, genes marked with a Δ indicate that the enzyme gene is deleted. "Gene name A::gene name B" indicates that the gene A region has been replaced with a region containing gene B. "gltA R164L" indicates that the 164th arginine in the GltA protein encoded by the gltA gene has been replaced with leucine.

[0125] [Table 3]

[0126] [Table 4]

[0127] <Construction of Plasmid for Expression of Enzyme Genes in C6 Compound Production Pathway> For amplification of PCR fragments, PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio) or KOD FX Neo (product name, manufactured by Toyobo) was used. For plasmid preparation, Escherichia coli JM109 strain was used. For optimization of nucleotide sequences, GeneArt GeneOptimizer (software name, manufactured by Thermo Fisher Scientific) or the artificial gene synthesis service of Eurofins Genomics was used.

[0128] The polynucleotide encoding PaaJ(Ec) of Escherichia coli (SEQ ID NO: 1) was obtained by cloning from the genomic DNA of Escherichia coli W3110 strain (NBRC12713). PCR was performed using the oligonucleotides of SEQ ID NOs: 111 and 112 as primers (Figure 15), and a PCR product containing the coding region (SEQ ID NO: 79) of the paaJ(Ec) gene (Figure 11) was obtained. Next, PCR was performed using pRSFDuet-1 (product name, manufactured by Merck) as a template and the oligonucleotides of SEQ ID NOs: 113 and 114 as primers (Figure 15) to obtain a pRSFDuet-1 fragment. The DNA fragment containing the paaJ(Ec) gene coding region and the pRSFDuet-1 fragment were ligated using the In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and plasmids were extracted from the obtained transformants. "paaJ(Ec)-pRSFDuet" was obtained as the PaaJ(Ec) expression plasmid.

[0129] A polynucleotide encoding Eshcherichia coli PaaH(Ec) (SEQ ID NO: 2) (Figure 2) was obtained by cloning from the genomic DNA of the Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using the oligonucleotides SEQ ID NOs: 115 and 116 as primers (Figure 15), yielding a PCR product containing the coding region of the paaH(Ec) gene (SEQ ID NO: 80) (Figure 11). Next, PCR was performed using "paaJ(Ec)-pRSFDuet" as a template and the oligonucleotides SEQ ID NOs: 117 and 118 as primers (Figure 15), yielding the "paaJ(Ec)-pRSFDuet" fragment. The DNA fragment containing the coding region of the paaH(Ec) gene and the "paaJ(Ec)-pRSFDuet" fragment were ligated using the In-Fusion HD cloning kit (product name, Clontech). This was transformed into E. coli JM109, and a plasmid was extracted from the resulting transformant. The PaaJ(Ec) and PaaH(Ec) co-expression plasmid "paaJ(Ec)-paaH(Ec)-pRSFDuet" was obtained.

[0130] The oligonucleotides shown in SEQ ID NOs: 4 and 5 were used as primers (Figure 3), and the chromosomal DNA of the L3 strain was used as a template. The polynucleotide encoding PaaF (L3) of the L3 strain, encoded by the nucleotide sequence of the PCR amplification product, was optimized for expression in E. coli and obtained using Eurofins Genomics' artificial gene synthesis service. PCR was performed using the oligonucleotides shown in SEQ ID NOs: 119 and 120 as primers (Figure 15), yielding a PCR product containing the optimized sequence of the paaF (L3) coding region. PCR was performed using pETDuet-1 (product name, Merck) as a template and the oligonucleotides shown in SEQ ID NOs: 121 and 122 as primers (Figure 15), yielding the pETDuet-1 fragment. The DNA fragment containing the paaF (L3) coding region and the pETDuet-1 fragment were ligated using the In-Fusion HD cloning kit (product name, Clontech). The E. coli JM109 strain was transformed, and the resulting plasmid was extracted from the transformant. The PaaF(L3) expression plasmid "paaF(L3)-pETDuet" was obtained.

[0131] The polynucleotide encoding Thermothelomyces thermophilus Ter (SEQ ID NO: 11) was obtained by optimizing its base sequence for expression in E. coli using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using the oligonucleotides SEQ ID NOs: 123 and 124 as primers (Figure 15), yielding a PCR product containing the ter gene coding region (SEQ ID NO: 125) (Figure 16). PCR was performed using "paaF(L3)-pETDuet" as a template and the oligonucleotides SEQ ID NOs: 126 and 127 as primers (Figure 17), yielding the "paaF(L3)-pETDuet" fragment. The DNA fragment containing the ter gene coding region and the "paaF(L3)-pETDuet" fragment were ligated using the In-Fusion HD cloning kit (product name, Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the resulting transformant. The PaaF(L3)-ter-pETDuet was obtained as a PaaF(L3) and Ter co-expression plasmid.

[0132] <Adipic Acid Production Test (Comparative Example 1 and Example 1)> E. coli strain No. 048 or No. 071 was transformed by electroporation with the adipic acid production pathway enzyme expression plasmids "paaJ(Ec)-paaH(Ec)-pRSFDuet" and "paaF(L3)-ter-pETDuet." Colonies were formed on LB agar medium containing 50 mg / L ampicillin sodium and 30 mg / L kanamycin sulfate at 37°C for one day. A loopful of colonies was inoculated into 2 mL of LB liquid medium (14 mL round-bottom tube) containing 50 mg / L ampicillin sodium and 30 mg / L kanamycin sulfate, and the resulting preculture solution was obtained by shaking at 37°C for 3 to 5 hours. A 250 mL jar culture apparatus (model Bio Jr. 8, Biot) was used to culture the main culture by adding 0.5 mL of the preculture to a synthetic medium (Table 5) containing 50 mg / L carbenicillin sodium, 30 mg / L kanamycin sulfate, and 0.02 mM IPTG. The culture conditions were as follows: culture temperature 37°C; culture pH 7.0; pH adjustment with 10% (w / v) aqueous ammonia; agitation 750 rpm; and aeration 1 vvm. At 23 hours after inoculation of the preculture, 5 mL of a 700 g / L glycerol solution (35 g of glycerol) was added. Strain SC1 was cultured at 45 hours, and strain SC2 was cultured at 88 hours.

[0133] [Table 5]

[0134] The glycerol concentration and adipic acid concentration in the culture medium were analyzed using a high performance liquid chromatograph Prominence system (Shimadzu Corporation) under the following conditions.

[0135] High-performance liquid chromatograph (HPLC) analysis conditions Detector: Differential refractive index detector Column: Shim-Pack Fast-OA (G), two connected Fast-OA columns (Shimadzu Corporation) Oven temperature: 40°C Mobile phase: 8mM methanesulfonic acid aqueous solution Flow rate: 0.6mL / min Injection volume: 10μL

[0136] The valine concentration in the culture medium was analyzed by GC / MS analysis using trimethylsilyl derivatization. 360 μL of extract (water:methanol:chloroform = 5:2:2 (v / v / v)) was added to 40 μL of the culture medium supernatant (disodium sebacate was added as an internal standard to a final concentration of 10 mM) and mixed thoroughly with a vortex mixer. After centrifugation (16,000 × g, 5 min), 40 μL of the supernatant was transferred to a separate microtube and centrifuged to dryness in a centrifugal evaporator for 1 hour. 100 μL of a 20 mg / mL solution of methoxyamine hydrochloride in pyridine was added to the resulting dried product and shaken at 30°C for 90 min. 50 μL of N-methyl-N-trimethylsilyltrifluoroacetamide was added and shaken at 37°C for 30 min. The reaction solution was used as a GC / MS measurement sample and analyzed under the following conditions.

[0137] GC / MS analysis conditions Equipment: GCMS-QP-2020NX (Shimadzu Corporation) Column: Fused silica capillary tube, deactivated tube (length 1 m, outer diameter 0.35 mm, inner diameter 0.25 mm, manufactured by GL Sciences), InertCap 5MS / NP (length 30 m, inner diameter 0.25 mm, film thickness 0.25 μm, manufactured by GL Sciences) Sample injection volume: 1 μL Sample introduction method: Split (split ratio 25:1) Vaporization chamber temperature: 230℃ Carrier gas: Helium Carrier gas linear velocity: 39.0 cm / sec Oven temperature: 80°C, hold for 2 minutes → increase temperature at 15°C / minute → 325°C, hold for 13 minutes Ionization method: Electron Ionization (EI) Ionization energy: 70 eV Ion source temperature: 230℃ Scan range: m / z = 50 to 500

[0138] The results of metabolite concentrations in the culture medium are shown in Table 6. Transformants SC1 and SC2 were introduced with enzyme genes that catalyze the reactions corresponding to steps A, B, C, and D shown in Figure 1, and endogenous enzymes also promoted the reaction corresponding to step E, producing adipic acid. Transformant SC2, which used strain No. 071, in which the pdhR gene was disrupted, showed higher adipic acid accumulation and higher adipic acid yield relative to glycerol than transformant SC1, which used strain No. 048, in which the pdhR gene was not disrupted. Furthermore, while valine accumulated as a by-product in the culture medium of strain SC1, the accumulation of valine in the culture medium of SC2 was reduced.

[0139] [Table 6] [Industrial Applicability]

[0140] The present invention can provide, for example, an efficient process for producing C6 compounds, and is expected to be applicable to industrial-scale production.

Claims

1. a genetic modification that suppresses a transcription factor that controls the expression of pyruvate dehydrogenase; It has a production pathway for C6 compounds, 1. A modified microorganism, wherein the C6 compound is adipic acid; the modified microorganism is Escherichia coli; the transcription factor controlling the expression of pyruvate dehydrogenase is PdhR; The PdhR is (A-1) DNA consisting of SEQ ID NO: 128; (A-3) DNA consisting of a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence of SEQ ID NO: 128 and encoding a protein having PdhR activity; (A-4) DNA encoding a protein having PdhR activity, which is a nucleotide sequence encoding a protein consisting of an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of the protein encoded by the nucleotide sequence of SEQ ID NO: 128, or (A-5) DNA consisting of a degenerate isomer of the base sequence of SEQ ID NO: 128 a protein encoded by (B-1) a protein consisting of the amino acid sequence of SEQ ID NO: 129; (B-2) a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 129 and having PdhR activity, or (B-3) A protein having PdhR activity, which consists of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO:

129. and The modified microorganism contains at least one gene encoding an enzyme that catalyzes a reaction step in the production pathway of the C6 compound, a gene encoding 3-oxoadipyl CoA thiolase, a gene encoding 3-hydroxyadipyl CoA dehydrogenase, a gene encoding 3-hydroxyadipyl CoA dehydratase, and A gene encoding 2,3-dehydroadipyl CoA reductase Including, the genetic modification is a modification in which the pdhR gene encoding the PdhR is disrupted; The modified microorganism.

2. A method for producing a C6 compound, comprising a culturing step of culturing the modified microorganism described in claim 1.

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