Recombinant microorganisms and methods for producing C6 compounds

Abstract

[Problem] To provide a recombinant microorganism having 2,3-dehydroadipyl-CoA reductase activity and a method for producing a C6 compound. Also, to provide a recombinant microorganism capable of producing adipic acid or an adipic acid derivative and a method for producing adipic acid or an adipic acid derivative. [Solution] A recombinant microorganism that contains an exogenous gene encoding a protein having an enzymatic activity to reduce 2,3-dehydroadipyl-CoA into adipyl-CoA and / or an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase.

Description

[Technical field]

[0001] The present invention relates to a recombinant microorganism and a method for producing a C6 compound. The present invention also relates to a recombinant microorganism and a method for producing adipic acid or a derivative thereof. [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 polyamides and polyurethanes, and are considered important compounds in the chemical industry. For example, adipic acid (CAS No. 124-04-9) is a monomer compound used as a raw material for polyamides such as nylon-66, urethane, and plasticizers. The current general method for producing adipic acid is chemical synthesis, for example, by oxidizing cyclohexanol alone or a mixture of cyclohexanol and cyclohexanone (K / A oil) with nitric acid.

[0003] In recent years, there have been concerns that fossil fuels will become depleted and are considered to be a factor in global warming. As a result, there is a desire to shift from fossil fuel-derived raw materials to renewable raw materials, such as biomass-derived raw materials, in chemical production processes. For this reason, a production method using fermentation production by microorganisms whose metabolism has been modified by genetic modification has been proposed.

[0004] For example, microorganisms for the biosynthesis of adipic acid, 6-aminocaproic acid, or caprolactam have been reported (Patent Document 1). As an example, the document discloses a method for culturing a microorganism containing "succinyl-CoA:acetyl-CoA acyltransferase" that converts succinyl-CoA and acetyl-CoA into 3-oxoadipyl-CoA; "3-hydroxyacyl-CoA dehydrogenase" that converts 3-oxoadipyl-CoA into 3-hydroxyadipyl-CoA; "3-hydroxyadipyl-CoA dehydratase" that converts 3-hydroxyadipyl-CoA into 5-carboxy-2-pentenoyl-CoA; "5-carboxy-2-pentenoyl-CoA reductase" that converts 5-carboxy-2-pentenoyl-CoA into adipyl-CoA; and "phosphotransadipylase" and "adipate kinase" that convert adipyl-CoA into adipate. However, the document merely shows genes that may promote the reaction as examples of each enzyme, and it has not been demonstrated that the genes for these enzymes can efficiently produce C6 compounds from a biomass-derived carbon source.

[0005] In addition, a method for preparing an adipate thioester, which includes a step of enzymatically converting 2,3-dehydroadipate thioester into an adipate thioester, has also been proposed (Patent Document 2), but only some of the enoyl-CoA reductases derived from biological species such as Candida tropicalis, Euglena gracilis, Clostridium beijerinckii, and Yarrowia lipolytica exhibit enzyme activity, and no examples have been shown that demonstrate the fermentative production of C6 compounds.

[0006] Furthermore, it has been reported that 36 mg / L of adipic acid can be produced using glucose as a carbon source by culturing recombinant Escherichia coli in which the enzymes PaaJ, PaaH, and PaaF derived from Escherichia coli and the enzymes DcaA and TesB derived from Acinetobacter baylyi are co-expressed (Non-Patent Document 1).

[0007] The enzyme activity of trans-enoyl-CoA reductase derived from multiple biological species has been demonstrated, and furthermore, examples have been proposed in which adipic acid and 6-aminocaproic acid were produced by culturing Escherichia coli expressing trans-enoyl-CoA reductase derived from Candida tropicalis or Drosophila melanogaster, or mutants of these enzymes (Patent Document 3).

[0008] However, the above-mentioned examples of fermentation production by the prior art do not necessarily satisfy sufficient productivity for industrial application, and there is a demand for improving the productivity of microorganisms.

[0009] As for adipic acid, a method has been proposed for producing adipic acid using a biomass-derived raw material, in which a biomass-derived carbon source is converted to cis,cis-muconic acid using a genetically modified microorganism, and the resulting cis,cis-muconic acid is then hydrogenated to produce adipic acid (Patent Document 4). Also, a method for producing adipic acid from α-ketoglutaric acid using a genetically modified organism has been proposed (Patent Document 5).

[0010] Furthermore, a method for fermentatively producing adipic acid directly from biomass-derived raw materials has also been proposed (Patent Document 6). In this method, as non-naturally occurring microorganisms capable of producing adipic acid, "succinyl-CoA:acetyl-CoA acyltransferase" that converts succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA, "3-hydroxyacyl-CoA dehydrogenase" that converts 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA, "3-hydroxyadipyl-CoA dehydratase" that converts 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA, and "5-hydroxyadipyl-CoA dehydratase" that converts 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA are used. Although non-naturally occurring microorganisms containing "5-carboxy-2-pentenoyl-CoA reductase" that converts carboxy-2-pentenoyl-CoA to adipyl-CoA and "phosphotransadipylase" / "adipate kinase" that converts adipyl-CoA to adipate are listed, the authors only show genes that code for the enzymes that may promote the reaction for each enzyme, and have not demonstrated that adipic acid can be produced in good yields from biomass-derived carbon materials by using these genes. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Patent No. 5951990 specification [Patent Document 2] Special Publication No. 2011-512868 [Patent Document 3] International Publication No. 2020 / 219859 [Patent Document 4] International Publication No. 1995 / 007996 [Patent Document 5] International Publication No. 2010 / 104391 [Patent Document 6] JP 2011-515111 A [Non-patent literature]

[0012] [Non-Patent Document 1] 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. Summary of the Invention [Problem to be solved by the invention]

[0013] An object of the present invention is to provide a recombinant microorganism having 2,3-dehydroadipyl-CoA reductase activity and a method for efficiently producing a C6 compound.

[0014] It is a further object of the present invention to provide a recombinant microorganism capable of producing adipic acid or an adipic acid derivative, and a method for producing adipic acid or an adipic acid derivative. [Means for solving the problem]

[0015] As a result of intensive research conducted by the present inventors to solve the above-mentioned problems of the conventional technology, they discovered that a recombinant microorganism with good 2,3-dehydroadipyl-CoA reductase activity can be obtained by expressing a specific exogenous enzyme in a host microorganism, and further that C6 compounds can be efficiently produced by culturing this recombinant microorganism, thereby completing the present invention.

[0016] That is, the present invention provides the following. [1] A recombinant microorganism comprising an exogenous gene encoding a protein having an enzymatic activity for reducing 2,3-dehydroadipyl-CoA to adipyl-CoA, The exogenous gene is (i) a DNA encoding a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (ii) DNA 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 shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) DNA consisting of a polynucleotide sequence having 80% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (iv) DNA encoding a protein having 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 a protein encoded by a polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (v) a DNA that hybridizes under stringent conditions to a DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; or (vi) DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 a recombinant microorganism; [2] The exogenous gene is (xi) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16, or (xiii) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 [1] A recombinant microorganism according to [1], [3] The exogenous gene is selected from the group consisting of Candida auris, Kluyveromyces marxianus, Pichia kudriavzevii, Thermothelomyces thermophilus, Thermothielavioides terrestris, Chaetomium thermophilum, Podo The recombinant microorganism according to [1] or [2], which is derived from at least one selected from spora anserina, Purpureocillium lilacinum, and Pyrenophora teres; [4] The recombinant microorganism has a pathway for producing a C6 compound, The recombinant microorganism according to any one of [1] to [3], wherein the C6 compound is at least one selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, and 6-hydroxyhexanoic acid; [5] The recombinant microorganism is selected from the group consisting of Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, and the like. s), Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, or Aspergillus; [6] The recombinant microorganism according to any one of [1] to [5], wherein the recombinant microorganism is Escherichia coli; [7]·The gene encoding 3-oxoadipyl CoA thiolase, · the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, · the gene encoding 3-hydroxyadipyl-CoA dehydratase, A gene encoding carboxylic acid reductase, A gene encoding alcohol dehydrogenase, and Genes that code for transaminases (aminotransferases) The recombinant microorganism according to any one of [1] to [6], further comprising at least one selected from the group consisting of: [8] A method for producing a C6 compound, comprising a culture step of culturing the recombinant microorganism according to any one of [1] to [7], The method for producing the C6 compound is at least one selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, and 6-hydroxyhexanoic acid; [9](a) has the enzyme activity to reduce 2,3-dehydroadipyl-CoA to adipyl-CoA; (b) Below: (i) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (ii) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) encoded by DNA consisting of a polynucleotide sequence having 80% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (iv) an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a protein encoded by a polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (v) encoded by DNA that hybridizes under stringent conditions to DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence set forth in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; and (vi) encoded by DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 a recombinant protein,

[10] (xi) consisting of the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; or (xiii) a recombinant protein encoded by DNA consisting of the polynucleotide sequence set forth in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65;

[11] A method for producing a C6 compound, comprising using a recombinant protein described in [9] or

[10] .

[0017] Furthermore, as a result of extensive research aimed at solving the above problems, the present inventors discovered that adipic acids can be efficiently produced by culturing a genetically modified microbial substance expressing a specific enzyme possessed by Burkholderia sp. strain LEBP-3 (accession number: NITE BP-03334), a microorganism newly isolated from nature, and thus arrived at the present invention.

[0018] That is, the present invention further provides the following:

[12] A recombinant microorganism comprising at least one of an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase;

[13] The recombinant microorganism according to

[12] , wherein at least one of the 3-hydroxyadipyl-CoA dehydratase and the 2,3-dehydroadipyl-CoA reductase is derived from Burkholderia sp. strain LEBP-3 (accession number: NITE BP-03334);

[14] The 3-hydroxyadipyl-CoA dehydratase is produced by using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (1a) a nucleotide sequence represented by SEQ ID NO: 168 and a nucleotide sequence represented by SEQ ID NO: 169, or (1b) a nucleotide containing the base sequence shown in SEQ ID NO: 168 and a nucleotide containing the base sequence shown in SEQ ID NO: 169 and is encoded by DNA having a sequence identity of 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 to a PCR amplification product using the above primers; The recombinant microorganism according to

[12] or

[13] , wherein the DNA has 626 to 940 bp;

[15] The 2,3-dehydroadipyl-CoA reductase is synthesized using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (2a) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (2b) a nucleotide containing the base sequence shown in SEQ ID NO: 170 and a nucleotide containing the base sequence shown in SEQ ID NO: 171 and is encoded by DNA having a sequence identity of 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 to a PCR amplification product using the above primers; The recombinant microorganism according to any one of

[12] to

[14] , wherein the DNA has 924 to 1,386 bp;

[16] The 3-hydroxyadipyl-CoA dehydratase is synthesized using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (1a) a nucleotide sequence represented by SEQ ID NO: 168 and a nucleotide sequence represented by SEQ ID NO: 169, or (1b) a nucleotide containing the base sequence shown in SEQ ID NO: 168 and a nucleotide containing the base sequence shown in SEQ ID NO: 169 The DNA is encoded by PCR amplification using the following primers: The recombinant microorganism according to

[12] or

[13] , wherein the DNA has 783 or 784 bp;

[17] The 2,3-dehydroadipyl-CoA reductase is synthesized using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (2a) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (2b) a nucleotide containing the base sequence shown in SEQ ID NO: 170 and a nucleotide containing the base sequence shown in SEQ ID NO: 171 The DNA is encoded by PCR amplification using the following primers: The recombinant microorganism according to any one of

[12] to

[14] , wherein the DNA has 1155 or 1156 bp;

[18] The recombinant microorganism according to any one of

[12] to

[17] , wherein the recombinant microorganism belongs to a genus selected from the group consisting of Escherichia, Corynebacterium, Bacillus, Acinetobacter, Burkholderia, Pseudomonas, Clostridium, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, and Aspergillus;

[19] The recombinant microorganism according to any one of

[12] to

[18] , having an adipic acid production pathway.

[20] The recombinant microorganism according to any one of

[12] to

[19] , having a hexamethylenediamine production pathway;

[21] The recombinant microorganism according to any one of

[12] to

[19] , having a 1,6-hexanediol production pathway.

[22] The recombinant microorganism according to any one of

[12] to

[19] , having a 6-amino-1-hexanol production pathway.

[23] A method for producing a target compound, comprising a culture step of culturing the recombinant microorganism according to any one of

[12] to

[18] , the target compound is selected from the group consisting of adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol, and 6-amino-1-hexanol; the adipic acid derivative is selected from the group consisting of oxoadipic acid, levulinic acid, 3-hydroxyadipic acid and 2,3-dehydroadipic acid;

[24] A method for producing adipic acid, comprising a culturing step of culturing the recombinant microorganism according to

[19] ;

[25] A method for producing hexamethylenediamine, comprising a culturing step of culturing the recombinant microorganism according to

[20] ;

[26] A method for producing 1,6-hexanediol, comprising a culturing step of culturing the recombinant microorganism according to

[21] ;

[27] A method for producing 6-amino-1-hexanol, comprising a culturing step of culturing the recombinant microorganism according to

[22] ;

[28] (A-1) Using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (a1) a nucleotide having the base sequence shown in SEQ ID NO: 168 and a nucleotide having the base sequence shown in SEQ ID NO: 169, or (a2) a nucleotide containing the base sequence shown in SEQ ID NO: 168 and a nucleotide containing the base sequence shown in SEQ ID NO: 169 A recombinant polypeptide encoded by DNA having a sequence identity of 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 to a PCR amplification product using the following primers: It has 3-hydroxyadipyl CoA dehydratase activity. The DNA has a recombinant polypeptide sequence of 626 to 940 bp; (A-2) Using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (a1) a nucleotide having the base sequence shown in SEQ ID NO: 168 and a nucleotide having the base sequence shown in SEQ ID NO: 169, or (a2) a nucleotide containing the base sequence shown in SEQ ID NO: 168 and a nucleotide containing the base sequence shown in SEQ ID NO: 169 A recombinant polypeptide having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by DNA that is a PCR amplified product using the above primers, It has 3-hydroxyadipyl CoA dehydratase activity. The DNA has a recombinant polypeptide sequence of 626 to 940 bp; or (A-3) Using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (a1) a nucleotide having the base sequence shown in SEQ ID NO: 168 and a nucleotide having the base sequence shown in SEQ ID NO: 169, or (a2) a nucleotide containing the base sequence shown in SEQ ID NO: 168 and a nucleotide containing the base sequence shown in SEQ ID NO: 169 A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using as a primer It has 3-hydroxyadipyl CoA dehydratase activity. The DNA has a recombinant polypeptide sequence of 626 to 940 bp;

[29] (B-1) Using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (b1) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (b2) a nucleotide containing the base sequence shown in SEQ ID NO: 170 and a nucleotide containing the base sequence shown in SEQ ID NO: 171 A recombinant polypeptide encoded by DNA having a sequence identity of 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 to a PCR amplification product using the following primers: It has 2,3-dehydroadipyl CoA reductase activity. The DNA has a recombinant polypeptide sequence of 924 to 1386 bp; (B-2) Using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (b1) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (b2) a nucleotide containing the base sequence shown in SEQ ID NO: 170 and a nucleotide containing the base sequence shown in SEQ ID NO: 171 A recombinant polypeptide having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by DNA that is a PCR amplified product using the above primers, It has 2,3-dehydroadipyl CoA reductase activity. The DNA has a recombinant polypeptide sequence of 924 to 1386 bp; or (B-3) Using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, (b1) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (b2) a nucleotide containing the base sequence shown in SEQ ID NO: 170 and a nucleotide containing the base sequence shown in SEQ ID NO: 171 A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using as a primer It has 2,3-dehydroadipyl CoA reductase activity. The DNA is a recombinant polypeptide having a sequence of 924 to 1386 bp. Effect of the Invention

[0019] The present invention provides a recombinant microorganism having 2,3-dehydroadipyl-CoA reductase activity and a method for producing a C6 compound.

[0020] The present invention also provides a recombinant microorganism capable of producing adipic acid or an adipic acid derivative, and a method for producing adipic acid or an adipic acid derivative. [Brief description of the drawings]

[0021] [Figure 1] FIG. 1 shows examples of biosynthetic pathways from acetyl-CoA and succinyl-CoA to C6 compounds. [Diagram 2] FIG. 2 shows the amino acid sequences of each enzyme and the nucleotide sequences of the primers. [Diagram 3] FIG. 3 shows the amino acid sequences of each enzyme. [Figure 4] FIG. 4 shows the amino acid sequences of each enzyme. [Diagram 5] FIG. 5 shows the amino acid sequences of each enzyme. [Figure 6] FIG. 6 shows the nucleotide sequences encoding each enzyme and the nucleotide sequences of the primers. [Figure 7] FIG. 7 shows the nucleotide sequences encoding each enzyme and the nucleotide sequences of the primers. [Figure 8] FIG. 8 shows the nucleotide sequences encoding each enzyme and the nucleotide sequences of the primers. [Figure 9] FIG. 9 shows the base sequences encoding each enzyme. [Figure 10] FIG. 10 shows the base sequences encoding each enzyme. [Figure 11] FIG. 11 shows the nucleotide sequence of each primer. [Figure 12] FIG. 12 shows the nucleotide sequences encoding each enzyme and the nucleotide sequences of each primer. [Figure 13] FIG. 13 shows the nucleotide sequence of each primer. [Figure 14] FIG. 14 shows the nucleotide sequences encoding each enzyme and the nucleotide sequences of each primer. [Figure 15] FIG. 15 shows the amino acid sequence of each enzyme, the base sequence encoding each enzyme, and the base sequence of each primer. [Figure 16] FIG. 16 shows examples of pathways for producing adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol, and 6-amino-1-hexanol contained in the recombinant microorganism of the present invention. [Figure 17] FIG. 17 shows the genes to be amplified in PCR and the primers used. [Figure 18] FIG. 18 shows the amino acid sequence of each enzyme. [Figure 19A] FIG. 19A shows the nucleotide sequences encoding each enzyme. [Figure 19B] FIG. 19B shows the nucleotide sequences encoding each enzyme. [Figure 20A] FIG. 20A shows the targets to be amplified in PCR and the primers used. [Figure 20B] FIG. 20B shows the targets to be amplified in PCR and the primers used. [Figure 21] FIG. 21 shows the amino acid sequence of the enzyme. [Figure 22A] FIG. 22A shows the amino acid sequence of the enzyme. [Figure 22B] FIG. 22B shows the amino acid sequence of the enzyme. [Figure 22C] FIG. 22C shows the amino acid sequence of the enzyme. [Figure 22D] FIG. 22D shows the amino acid sequence of the enzyme. [Figure 22E] FIG. 22E shows the amino acid sequence of the enzyme. [Figure 23A] FIG. 23A shows the amino acid sequence of the enzyme. [Figure 23B] FIG. 23B shows the amino acid sequence of the enzyme. [Figure 23C] FIG. 23C shows the amino acid sequence of the enzyme. [Figure 23D] FIG. 23D shows the amino acid sequence of the enzyme. [Figure 24A] FIG. 24A shows the amino acid sequence of the enzyme. [Figure 24B] FIG. 24B shows the amino acid sequence of the enzyme. [Figure 24C] FIG. 24C shows the amino acid sequence of the enzyme. [Figure 24D] FIG. 24D shows the amino acid sequence of the enzyme. [Figure 24E] FIG. 24E shows the amino acid sequence of the enzyme. [Diagram 25] FIG. 25 shows the amino acid sequence of the enzyme. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The embodiments of the present invention are described in detail below. The present invention is not limited to the following embodiments, and can be practiced in various modifications within the scope of the gist. Furthermore, unless otherwise specified, the acquisition of DNA described in this specification, the preparation of vectors, and genetic manipulation such as transformation can be performed by methods described in known documents 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 Experimental Notes (Yodosha, Takaaki Tamura). Unless otherwise specified in this specification, nucleotide sequences are described in the 5' to 3' direction. In this specification, the terms "polypeptide" and "protein" are used interchangeably.

[0023] In this specification, a numerical range indicated using "~" indicates a range including the numerical values ​​before and after "~" as the minimum and maximum values, respectively. In the numerical ranges described in stages in this specification, the upper limit or lower limit of a numerical range of a certain stage can be arbitrarily combined with the upper limit or lower limit of a numerical range of another stage.

[0024] As used herein, the terms "endogenous" or "endogenous" are used to mean that the unmodified host microbial organism has the referenced gene or the protein (typically an enzyme) encoded thereby functionally expressed to an extent sufficient to facilitate a dominant biochemical reaction within the host cell.

[0025] As used herein, the terms "foreign" or "exogenous" are used to refer to the introduction of a gene or nucleic acid sequence according to the invention into a host when the host microorganism prior to genetic recombination does not possess the gene to be introduced according to the invention, does not substantially express the enzyme from that gene, and / or encodes the amino acid sequence of the enzyme from that gene or a different gene but does not express the comparable endogenous enzyme activity after genetic recombination.

[0026] <1> Invention A: Recombinant microorganisms and methods for producing C6 compounds The recombinant microorganism of the present invention contains an exogenous gene encoding a protein having an enzymatic activity of reducing 2,3-dehydroadipyl-CoA and converting it to adipyl-CoA. The reaction of reducing 2,3-dehydroadipyl-CoA and converting it to adipyl-CoA is shown in step D of FIG. 1. The "protein having an enzymatic activity of reducing 2,3-dehydroadipyl-CoA and converting it to adipyl-CoA" is also called "2,3-dehydroadipyl-CoA reductase." The enzymatic activity of reducing 2,3-dehydroadipyl-CoA and converting it to adipyl-CoA is also called "2,3-dehydroadipyl-CoA reductase activity" or "2,3-dehydroadipyl-CoA reductase enzyme activity," and these terms are used interchangeably.

[0027] The exogenous gene contained in the recombinant microorganism according to the invention is selected from the following: (i) DNA encoding a protein consisting of an amino acid 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 amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16; (ii) DNA 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 to the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16; (iii) DNA consisting of a polynucleotide 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 polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, or 65; (iv) DNA encoding a protein having an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a protein encoded by a polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, or 65; (v) DNA that hybridizes under stringent conditions with DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (vi) A DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65.

[0028] The exogenous gene contained in a recombinant microorganism according to the invention is preferably selected from the following: (i) a DNA encoding a protein consisting of an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (ii) DNA 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 shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) DNA consisting of a polynucleotide sequence having 80% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (iv) DNA encoding a protein having 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 a protein encoded by a polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (v) DNA that hybridizes under stringent conditions with DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (vi) A DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65.

[0029] More preferably, the exogenous gene contained in the recombinant microorganism according to the present invention is (xi) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16, or (xiii) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 It is.

[0030] The exogenous gene is derived from at least one selected from, for example, Candida auris, Kluyveromyces marxianus, Pichia kudriavzevii, Thermothelomyces thermophilus, Thermothielavioides terrestris, Chaetomium thermophilum, Podospora anserina, Purpureocillium lilacinum, and Pyrenophora teres.

[0031] By containing the above-mentioned exogenous gene, the recombinant microorganism has good 2,3-dehydroadipyl-CoA reductase activity. By using the recombinant microorganism having such enzyme activity, it is possible to produce a target compound. For example, the target compound may be a C6 compound.

[0032] In this specification, 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), and 6-Hydroxyhexanoic acid (CAS No. 1191-25-9) The term "C6 compound" refers to one or more compounds selected from the group consisting of these compounds.

[0033] It will be understood by those of skill in the art that the "C6 compound" herein may be in a neutral or ionized form, including any salt form, and that this form is dependent on pH.

[0034] As described above, the microorganism of the present invention is a genetically modified microorganism into which an exogenous enzyme gene has been introduced. A "genetically modified microorganism" is also simply called a "recombinant microorganism."

[0035] In the present invention, the host microorganism into which the foreign gene of interest is introduced is not particularly limited, and may be either a prokaryote or a eukaryote. It may be arbitrarily selected from those that have already been isolated and preserved, those that have been newly isolated from nature, and those that have been genetically modified. Examples of host microorganisms include those belonging to the genera Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, Streptomyces, The host microorganism belongs to the genus Streptomyces, Rhodococcus, Synechocystis, Alkalihalobacillus, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, or Aspergillus. The host microorganism preferably belongs to the genus Escherichia, and is more preferably Escherichia coli.

[0036] Therefore, the recombinant microorganism according to the present invention, in which an exogenous gene has been introduced into the host microorganism, is, for example, a microorganism belonging to the genus Escherichia, Bacillus, Corynebacterium, Arthrobacter, Brevibacterium, Clostridium, Zymomonas, Pseudomonas, Burkholderia, or the like. The recombinant microorganism of the present invention preferably belongs to the genus Escherichia, and more preferably to Escherichia coli.

[0037] As used herein, "having a production pathway" in relation to a certain compound 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 compound to proceed and is capable of biosynthesizing the compound. The recombinant microorganism of the present invention may be one that uses a host microorganism that inherently has the ability to produce the compound, or may be one that has been modified so that a host microorganism that does not inherently have the ability to produce the compound has the ability to produce the compound.

[0038] As used herein, "derived from" means that a gene or the protein (typically an enzyme) encoded thereby is endogenous to the particular biological species being referred to.

[0039] The genetically modified microorganism of the present invention contains an exogenous gene encoding a protein having an enzyme activity (i.e., 2,3-dehydroadipyl-CoA reductase activity) that reduces 2,3-dehydroadipyl-CoA to adipyl-CoA, and thereby expresses a sufficient amount of the enzyme (i.e., 2,3-dehydroadipyl-CoA reductase) for the reaction to proceed. This enzyme is involved in the reaction of step D in Figure 1.

[0040] Furthermore, by further expressing an enzyme that catalyzes a reaction step in the C6 compound production pathway, the microorganism can become a genetically modified microorganism having a C6 compound production pathway.

[0041] In one embodiment, the recombinant microorganism of the present invention comprises: · the gene encoding 3-oxoadipyl CoA thiolase, · the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, · the gene encoding 3-hydroxyadipyl-CoA dehydratase, a combination of a gene encoding phosphate adipyltransferase and a gene encoding adipate kinase, the gene encoding adipyl-CoA hydrolase, A gene encoding adipic acid CoA transferase, -genes encoding dehydrogenases, A gene encoding carboxylic acid reductase, Genes that code for transaminases (aminotransferases) A gene encoding alcohol dehydrogenase, and · a gene encoding CoA transferase, - a gene encoding an acid thiol ligase, The composition further comprises at least one selected from the group consisting of:

[0042] 3-Oxoadipyl CoA thiolase converts the reaction in step A of Figure 1 into 3-Hydroxyadipyl-CoA dehydrogenase performs the reaction in step B of Figure 1 as follows: 3-Hydroxyadipyl-CoA dehydratase converts the reaction in step C of Figure 1 into The combination of phosphate adipyltransferase and adipate kinase, adipyl-CoA hydrolase, and adipic acid CoA transferase can be used to convert reaction E in Figure 1 to The dehydrogenase can 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. CoA transferase or acid thiol ligase catalyzes at least one of steps S and T Catalyze.

[0043] In a preferred embodiment, the recombinant microorganism according to the invention comprises: · the gene encoding 3-oxoadipyl CoA thiolase, · the gene encoding 3-hydroxyadipyl-CoA dehydrogenase, · the gene encoding 3-hydroxyadipyl-CoA dehydratase, A gene encoding carboxylic acid reductase, A gene encoding alcohol dehydrogenase, and Genes that code for transaminases (aminotransferases) The composition further comprises at least one selected from the group consisting of:

[0044] In addition, when the genetically modified microorganism of the present invention expresses a sufficient amount 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 by an enzyme encoded by an endogenous gene.

[0045] An example of a C6 compound production pathway that the genetically modified microorganism of the present invention may have is shown in Figure 1. Hereinafter, the enzymes that catalyze each reaction step will be described with reference to Figure 1.

[0046] In step A of FIG. 1, succinyl-CoA and acetyl-CoA are condensed to form 3-oxoadipyl-CoA. An example of an enzyme that can catalyze this conversion is β-ketothiolase. Furthermore, for example, 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 also be exemplified as enzymes that can have activity in this conversion. Enzymes that can be used in this conversion are not limited as long as they have activity in this conversion, and examples include succinyl-CoA:acetyl-CoA acyltransferase and 3-oxoadipyl-CoA thiolase. In one embodiment, PaaJ derived from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO: 1 is used.

[0047] In step B of FIG. 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 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 are, for example, 3-hydroxyadipyl-CoA dehydrogenase. In one embodiment, PaaH derived from Escherichia coli consisting of the amino acid sequence set forth in SEQ ID NO:2 is used.

[0048] In step C of FIG. 1, 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA. Examples of enzymes that can catalyze this conversion include hydrolyases classified into the EC 4.2.1 group. For example, enzymes classified into groups such as 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 have activity in this conversion. The enzyme that can be used in the present invention is not limited as long as it has activity in this conversion, and is, for example, 3-hydroxyadipyl-CoA dehydratase. In one embodiment, PaaF derived from Escherichia coli having the amino acid sequence set forth in SEQ ID NO: 3 is used. In another embodiment, the enzyme is 3-hydroxyadipyl CoA dehydratase PaaF (L3) encoded by the base sequence (783 bp) of the PCR amplification product using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template, using the nucleotides shown in SEQ ID NO: 4 and SEQ ID NO: 46 as primers. Burkholderia sp. LEBP-3 strain (hereinafter also abbreviated as "L3 strain") was deposited at the National Institute of Technology and Evaluation (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on December 4, 2020 (Receipt number: NITE ABP-03334) and is internationally deposited under "Deposit number: NITE BP-03334".

[0049] Here, the DNA polymerase used for preparing the PCR amplification product is one known in the art, and examples thereof include, but are not limited to, Taq DNA polymerase, hot start-adjusted DNA polymerase, and proofreading DNA polymerase having 3'-5' exonuclease activity in addition to polymerase activity. In a preferred embodiment of the conditions for preparing the PCR amplification product, 1 μM of each specific nucleotide is used as a forward primer and a reverse primer, and PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio) is used as an enzyme, and the 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 in a volume of 25 μL.

[0050] In step D of FIG. 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 + )), EC 1.3.1.44 (trans-2-enoyl-CoA reductase (NAD + Examples of enzymes that may have activity in this conversion include enzymes classified into groups such as 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)). In the present invention, the aforementioned 2,3-dehydroadipyl-CoA reductase is used for the conversion in step D.

[0051] The 2,3-dehydroadipyl-CoA reductase used in the present invention is, for example, an enzyme derived from the following biological species: Candida auris (SEQ ID NO: 8), Kluyveromyces marxianus (SEQ ID NO: 9), Pichia kudriavzevii (SEQ ID NO: 10), Thermothelomyces thermophilus (SEQ ID NO: 11), Thermothielavioides terrestris (SEQ ID NO: 12), Chaetomium thermophilum (SEQ ID NO: 13), Podospora anserina (SEQ ID NO: 14), Purpureocillium lilacinum (SEQ ID NO: 15), Pyrenophora teres (SEQ ID NO: 16). Preferably, at least one of the enzymes consisting of the amino acid sequences described in SEQ ID NOs: 8 to 16 is used, more preferably at least one of the enzymes consisting of the amino acid sequences described in SEQ ID NOs: 11, 12, 13, 14 and 16 is used (FIG. 3).

[0052] In step E of FIG. 1, adipyl-CoA is converted to adipic acid. Examples of enzymes that can catalyze this conversion include thioester hydrolases classified in the EC 3.1.2 group. For example, enzymes classified in the EC 3.1.2.1 (acetyl-CoA hydrolase) and EC 3.1.2.20 (acyl-CoA hydrolase) groups can be exemplified as enzymes that can have activity in this conversion. Enzymes that can be used in the present invention are not limited as long as they have activity in this conversion, and are, for example, adipyl-CoA hydrolase.

[0053] Another example of an enzyme that can catalyze the reaction of step E in Fig. 1 is CoA-transferase classified into the EC 2.8.3 group. For example, enzymes classified into groups such as EC 2.8.3.5 (3-oxoacid CoA-transferase), EC 2.8.3.6 (3-oxoadipate CoA-transferase), and EC 2.8.3.18 (succinyl-CoA:acetyl-CoA-transferase) can be exemplified as enzymes that can have activity for this conversion. The enzyme that can be used in the present invention is not limited as long as it has activity for this conversion, and is, for example, adipate CoA-transferase.

[0054] Furthermore, as an example of another enzyme conversion that can catalyze the reaction of step E in FIG. 1, an acyltransferase classified in the EC 2.3.1 group transfers the adipyl group of adipyl-CoA to phosphate to generate adipyl phosphate, followed by dephosphorylation by a phosphotransferase classified in the EC 2.7.2 group. For example, as acyltransferase, enzymes classified in the EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase) groups can be exemplified as enzymes that can have activity in this conversion. As phosphotransferase, enzymes classified in the EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butanoate kinase) groups can be exemplified as enzymes that can have activity in this conversion. Enzymes that can be used in the present invention are not limited as long as they have activity in this conversion, and examples include phosphate adipyltransferase and adipate kinase.

[0055] In steps F, U, and V of Figure 1, acyl-CoA is converted to an aldehyde. 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 (formation of malonic semialdehyde)), and EC 1.2.1.76 (succinic semialdehyde dehydrogenase (acylation)) can be exemplified as enzymes that may have activity in this conversion because they catalyze a conversion reaction in which CoA is eliminated to produce an aldehyde, similar to this conversion. The enzyme that can be used in the present invention is not limited as long as it has 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 can be used (FIG. 4).

[0056] In steps G, I, K, and N of FIG. 1, a carboxyl group is converted to an aldehyde. Enzymes that can catalyze this conversion include, for example, carboxylic acid reductase (CAR). 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) can be exemplified as enzymes that may have activity in this conversion because they catalyze a conversion reaction that produces an aldehyde from a carboxylic acid, similar to this conversion. Typical examples of biological species from which the enzymes are 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 that can be used in the present invention are not limited as long as they have activity for this conversion. For example, at least one of the enzymes having the amino acid sequence set forth in any of SEQ ID NOs: 18 to 22 may be used. Preferably, at least one of the enzyme MaCar derived from Mycobacterium abscessus having the amino acid sequence set forth in SEQ ID NO: 20 and MaCar(m), a variant of MaCar having the amino acid sequence set forth in SEQ ID NO: 22, is used. More preferably, MaCar(m) having the amino acid sequence set forth in SEQ ID NO: 22 is used (Figure 4).

[0057] Furthermore, carboxylic acid reductase can be converted to 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 capable of catalyzing this reaction include, for example, enzymes classified in EC 2.7.8.7. Therefore, the microorganism of the present invention may be further modified so that the activity of phosphopantetheinyl transferase is increased. Methods for increasing the activity of phosphopantetheinyl transferase include, but are not limited to, a method of introducing an exogenous phosphopantetheinyl transferase gene and a method of enhancing the expression of an endogenous phosphopantetheinyl transferase gene. Enzymes that can be used in the present invention are not limited as long as they have phosphopantetheinyl group transfer activity, but typical enzymes include, for example, EntD of Escherichia coli, Sfp of Bacillus subtilis, Npt of Nocardia iowensis (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)), and Lys5 of Saccharomyces cerevisiae (Ehmann et al., Biochemistry 38.19 (1999): 6171-6177.). Enzymes that can be used in the present invention are not limited as long as they have activity for this conversion, but 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, and preferably, Npt of Nocardia iowensis derived from Nocardia iowensis consisting of the amino acid sequence set forth in SEQ ID NO: 24 is used (FIG. 5).

[0058] The conversions in steps J, M, P, and R in FIG. 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 have activity in this conversion. The enzymes that can be used in the present invention are not particularly limited as long as they have the conversion activity of each step. For example, putrescine aminotransferase YgjG from Escherichia coli, which has been reported to transaminate cadaverine and spermidine (Samsonova, et al., BMC microbiology 3.1 (2003): 2.), putrescine aminotransferase SpuC 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.), GABA aminotransferase GabT 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 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 that can be 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 31 and 158 may be used (FIGS. 5 and 15). Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, and glycine.

[0059] In steps H, L, O, and Q of FIG. 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 + )), EC 1.1.1.71 (alcohol dehydrogenase [NAD(P) +

[0036] ) can be exemplified as enzymes that may have activity in this conversion because they catalyze the conversion reaction from aldehyde to alcohol, similar to this conversion. Enzymes that can be used in the present invention are not limited as long as they have activity in this conversion, and an example of the enzyme is Ahr derived from Escherichia coli as set forth in SEQ ID NO: 32 (Figure 5).

[0060] In the reactions of steps S and T in Fig. 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 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 activity for this conversion.

[0061] 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 enzyme activity in the host microbial cell.

[0062] The recombinant microorganism of the present invention may be a host microorganism in which any gene has been disrupted as appropriate. Disruption of the target gene is carried out by methods known in the art.

[0063] Furthermore, the amino acid sequence of the above enzyme that can be used in the present invention, or the base sequence of the gene encoding the enzyme, may have all mutations that can occur in nature or artificially introduced mutations and modifications, for example, mutations such as deletion, substitution, insertion, and addition, so long as it can express substantial enzymatic activity in the host microbial cell. For example, the amino acid sequence may include an amino acid sequence in which one or more, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3 amino acids are deleted, substituted, inserted, and / or added to the amino acid sequence of the above enzyme.

[0064] Furthermore, the base sequence of the gene encoding the above enzyme that can be used in the present invention may be a DNA that hybridizes with a DNA having a complementary base sequence under stringent conditions, so long as it can express substantial enzymatic activity in the host microbial cell. "Stringent conditions" are, for example, conditions of about "1xSSC, 0.1% SDS, 60°C," conditions of about "0.1xSSC, 0.1% SDS, 60°C" as more stringent conditions, and conditions of about "0.1xSSC, 0.1% SDS, 68°C" as even more stringent conditions.

[0065] In addition, it is known that there are redundant codons for various codons that code for specific amino acids, and therefore alternative codons that will ultimately be translated into the same amino acid may be used in the present invention. In other words, since the genetic code is degenerate, multiple codons can be used to code for a specific amino acid, and therefore an amino acid sequence can be coded by any set of similar DNA oligonucleotides. It is known that most organisms preferentially use a subset of specific codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), so "codon optimization" depending on the host microorganism may be useful in the present invention.

[0066] Therefore, the genetically modified microorganism of the present invention may contain a base 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 base sequence of the enzyme gene, provided that the genetically modified microorganism can express an enzyme activity. Alternatively, the genetically modified microorganism may contain a gene encoding a protein containing an amino acid sequence having, for example, 80% or more, 85% or more, 88% or more, 90% 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.

[0067] In this specification, 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 when the sequences are aligned to maximize the identity between the two sequences and, if necessary, gaps are introduced into one or both of the two sequences. In this case, conservative substitutions are not considered as part of the sequence identity. Sequence identity can be determined by using publicly available computer software, for example, using an alignment search tool such as BLAST (registered trademark, hereinafter omitted) (Basic Local Alignment Search Tool). Those skilled in the art can determine appropriate parameters for obtaining maximum alignment of the comparison sequences in the alignment. The "sequence identity" of nucleotide sequences can also be determined by a similar method.

[0068] In the present invention, the above-mentioned C6 compound biosynthetic enzyme genes are introduced into the host microbial cell as an "expression cassette", thereby obtaining a more stable and high-level enzyme activity. In this specification, "expression cassette" means a nucleotide sequence containing a nucleic acid sequence that regulates transcription and translation functionally linked to the nucleic acid to be expressed or the gene to be expressed. Typically, the expression cassette of the present invention contains a promoter sequence 5' upstream from the coding sequence, a terminator sequence 3' downstream, and optionally further conventional regulatory elements functionally linked thereto, and in such a case, the nucleic acid to be expressed or the gene to be expressed is introduced into the host microbial cell.

[0069] 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 expression promoter or an inducible expression promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency, and is also preferably used in the present invention. In E. coli, promoters for the lac system, trp system, tac or trc system, the main operator and promoter region of lambda phage, the control region of fd coat protein, promoters for glycolytic enzymes (e.g., 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamic acid decarboxylase A, serine hydroxymethyltransferase, and the promoter region of RNA polymerase derived from T7 phage can be used. In Corynebacterium glutamicum, promoters for the HCE (high-level constitutive expression), cspB promoter, sodA promoter, elongation factor (EF-Tu) promoter, and the like can be used. Terminators that can be used include T7 terminator, rrnBT1T2 terminator, lac terminator, etc. In addition to promoter and terminator sequences, examples of other regulatory elements include selection markers, amplification signals, replication origins, etc. Suitable regulatory sequences are described, for example, in "Gene Expression Technology: Methods in Enzymology 185", Academic Press (1990).

[0070] The expression cassette described above is inserted into a vector, such as a plasmid, a phage, a transposon, an IS element, a fosmid, a cosmid, or a linear or circular DNA, into a host microorganism. Plasmids and phages are preferred. These vectors may be autonomously replicating in the host microorganism or may be replicated chromosomally. Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11, or pBdCI for E. coli; pUB110, pC194, or pBD214 for Bacillus; and pSA77 or pAJ667 for Corynebacterium. Examples of suitable plasmids include pUB110, pC194, or pBD214 for Bacillus species. Other usable plasmids are described in "Gene Cloning and DNA analysis 7th edition", Wiley-Blackwell (2016). The expression cassettes can be introduced into a vector 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.

[0071] After the vector having 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, conjugation, protoplast fusion, etc., and a method suitable for the host microorganism can be selected.

[0072] A second aspect of the present invention relates to a method for producing a target compound, which comprises a culturing step of culturing the aforementioned recombinant microorganism.

[0073] Here, the compound is preferably a C6 compound, which is at least one selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, and 6-hydroxyhexanoic acid.

[0074] The method for producing a C6 compound includes a culture step of culturing a recombinant microorganism according to the present invention. In the culture step, the recombinant microorganism is cultured in a medium containing a carbon source and a nitrogen source to obtain a culture containing bacterial cells. The recombinant microorganism of the present invention is cultured under conditions suitable for C6 compound production and growth and maintenance of the microorganism, and suitable medium composition, culture time, and culture conditions can be easily determined by those skilled in the art.

[0075] Examples of carbon sources include D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, blackstrap 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 decomposition liquid, and cellulose decomposition liquid. Preferred are D-glucose, sucrose, or glycerol. These carbon sources can be used individually or as a mixture.

[0076] C6 compounds produced using biomass-derived feedstocks can be clearly distinguished from synthetic feedstocks derived from, for example, petroleum, natural gas, or coal by measuring the biobased carbon content based on Carbon-14 (radioactive carbon) analysis as specified in ISO 16620-2 or ASTM D6866.

[0077] 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, etc.) 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 mixtures.

[0078] Furthermore, when the recombinant microorganism expresses a useful additional trait, for example, when it has an antibiotic resistance marker, the medium may contain the corresponding antibiotic, thereby reducing the risk of contamination by various bacteria during cultivation. 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.

[0079] The culture may be a batch type or a continuous type. In either case, the carbon source or the like may be additionally replenished at an appropriate time point during the culture. Furthermore, the culture may be performed while controlling suitable conditions such as temperature, oxygen concentration, pH, etc. The suitable culture temperature for a transformant derived from a general microbial host cell is usually in the range of 15°C to 55°C, preferably 25°C to 40°C. When the host microorganism is aerobic, shaking (flask culture, etc.) or stirring / aeration (jar fermenter culture, etc.) may be performed to ensure a suitable oxygen concentration during fermentation. Those culture conditions can be easily set by those skilled in the art.

[0080] The culturing step may include obtaining a culture and / or an extract of the culture of the recombinant microorganism.

[0081] The method for producing a compound according to the present invention may further include a mixing step of mixing the culture and / or an extract of the culture with a substrate compound to obtain a mixed solution. The substrate compound may be appropriately selected depending on the enzyme and the target compound.

[0082] The method for producing a compound according to the present invention may include a step of separating and / or purifying the target compound, C6 compound. The separation and / or purification step may be performed by any method, such as, but not limited to, centrifugation, filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation, ion exchange, and various types of chromatography. In the separation and / or purification step, one method may be selected, or multiple methods may be combined.

[0083] A further aspect of the present invention relates to a protein. In particular, the protein comprises (a) has an enzyme activity that reduces 2,3-dehydroadipyl-CoA to adipyl-CoA; (b) Below: (i) an amino acid 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 amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16; (ii) 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 to the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16; (iii) encoded by DNA consisting of a polynucleotide 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 polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (iv) an amino acid sequence in which 1 to 10, 1 to 7, 1 to 5, or 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a protein encoded by a polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, or 65; (v) encoded by DNA that hybridizes under stringent conditions to DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence set forth in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; and (vi) encoded by DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 The recombinant protein is either

[0084] The protein is preferably (a) has an enzyme activity that reduces 2,3-dehydroadipyl-CoA to adipyl-CoA; (b) Below: (i) an amino acid sequence having 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (ii) an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) encoded by DNA consisting of a polynucleotide sequence having 80% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (iv) an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a protein encoded by a polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (v) encoded by DNA that hybridizes under stringent conditions to DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence set forth in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; and (vi) encoded by DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 The recombinant protein is either

[0085] In a preferred embodiment, the recombinant protein is (xi) consisting of the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; or (xiii) Encoded by DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65.

[0086] Yet another aspect of the present invention relates to a method for producing a C6 compound, comprising using the aforementioned protein.

[0087] As described above, according to the present invention, a recombinant microorganism having good 2,3-dehydroadipyl-CoA reductase activity can be obtained by introducing a gene encoding a protein having 2,3-dehydroadipyl-CoA reductase activity into a host microorganism. In addition, a protein having good 2,3-dehydroadipyl-CoA reductase activity is also provided.

[0088] By using the recombinant microorganism or the protein, the target C6 compound can be efficiently produced. In addition, various C6 compounds can be obtained by performing conversion using additional enzymes depending on the desired C6 compound. Furthermore, since the recombinant microorganism according to the present invention has good C6 compound production ability, it is expected that it can be used for the production of the target compound on an industrial scale.

[0089] <2> Invention B: Recombinant microorganism and method for producing adipic acid or its derivatives The recombinant microorganism of the present invention is a microorganism in which genes have been modified from a host microorganism so as to enable efficient production of adipic acid (ADA) (CAS No. 124-04-9) or an adipic acid derivative.

[0090] In the present specification, the "adipic acid derivative" is an aliphatic carboxylic acid having an aliphatic hydrocarbon group having 3 to 5 carbon atoms and one or more carboxyl groups. The aliphatic hydrocarbon group of the adipic acid derivative is saturated or unsaturated. In the aliphatic hydrocarbon group, a carbon may be substituted with a substituent such as a hydroxyl group, a carboxyl group, a ketone group, or an amino group. The number of carbon atoms of the aliphatic hydrocarbon group of the "adipic acid derivative" is preferably 3 or 4. The number of carboxyl groups of the "adipic acid derivative" is preferably 1 or 2. Specific examples of "adipic acid derivatives" include 3-oxoadipic acid (3-OA), which has an aliphatic hydrocarbon group with four carbon atoms and two carboxylic acids, levulinic acid (LEV), which has an aliphatic hydrocarbon group with three carbon atoms and one carboxylic acid, 3-hydroxyadipic acid (3HA), which has an aliphatic hydrocarbon group with four carbon atoms and two carboxylic acids, and 2,3-dehydroadipic acid (23DA), which has an aliphatic hydrocarbon group with four carbon atoms and two carboxylic acids (see FIG. 16).

[0091] It will be understood by those skilled in the art that adipic acid and adipic acid derivatives herein may be in a neutral or ionized form, including any salt form, and that this form is dependent on pH. Thus, "adipic acid" and "adipic acid derivatives" include free acid forms as well as any salt forms and ionized forms. In this specification, adipic acid and adipic acid derivatives may be collectively referred to as "adipic acids" or "adipic acid analogs."

[0092] It is also possible to synthesize 6-aminocaproic acid, hexamethylenediamine, 1,6-hexanediol, and 6-amino-1-hexanol using adipic acid produced by the recombinant microorganism of the present invention as a raw material. The synthesis method may be a chemical synthesis method or a biological synthesis method using an enzyme or a microorganism containing the enzyme. In addition, as long as the microorganism contains a metabolic pathway for converting adipic acid into the above compounds, metabolic conversion from the raw material using adipic acid as an intermediate within the cells is also possible.

[0093] Specifically, the genetically modified microorganism of the present invention includes at least one of an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase. By including at least one of such exogenous genes, the recombinant microorganism has an adipic acid production pathway and can produce adipic acid or an adipic acid derivative. In this specification, the "genetically modified microorganism" is also simply referred to as "recombinant microorganism."

[0094] At least one of the 3-hydroxyadipyl-CoA dehydratase and the 2,3-dehydroadipyl-CoA reductase is derived from Burkholderia sp. strain LEBP-3 (Accession Number: NITE BP-03334). Preferably, both the 3-hydroxyadipyl-CoA dehydratase and the 2,3-dehydroadipyl-CoA reductase are derived from Burkholderia sp. strain LEBP-3.

[0095] At least one of the exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and the exogenous gene encoding 2,3-dehydroadipyl-CoA reductase is derived from Burkholderia sp. strain LEBP-3 (Accession Number: NITE BP-03334). Preferably, both the exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and the exogenous gene encoding 2,3-dehydroadipyl-CoA reductase are derived from Burkholderia sp. strain LEBP-3 (Accession Number: NITE BP-03334).

[0096] The genetically modified microorganism of the present invention may have at least one of the foreign genes encoding the above enzymes introduced therein. The genetically modified microorganism of the present invention preferably has at least one of the foreign genes encoding the enzymes catalyzing each reaction step of the adipic acid production pathway introduced therein, and expresses the foreign enzyme. Furthermore, when the genetically modified microorganism of the present invention expresses a sufficient amount of the enzyme that catalyzes the reaction step of the adipic acid production pathway without the introduction of a foreign gene, the reaction may proceed by the enzyme encoded by the endogenous gene.

[0097] The recombinant microorganism of the present invention preferably expresses at least one of 3-hydroxyadipyl-CoA dehydratase and 2,3-dehydroadipyl-CoA reductase, and the recombinant microorganism of the present invention expresses at least one of these enzymes, thereby allowing the reaction steps of the adipic acid production pathway to proceed and produce the target compound.

[0098] More preferably, the recombinant microorganism of the present invention expresses both 3-hydroxyadipyl-CoA dehydratase and 2,3-dehydroadipyl-CoA reductase. By expressing these enzymes, the recombinant microorganism of the present invention can proceed through each reaction step of the adipic acid production pathway and produce the target compound.

[0099] Even more preferably, the recombinant microorganism of the present invention expresses an oxidoreductase and / or a β-ketothiolase in addition to 3-hydroxyadipyl-CoA dehydratase and 2,3-dehydroadipyl-CoA reductase. By expressing these enzymes, the recombinant microorganism of the present invention can progress through each reaction step of the adipic acid production pathway, and can efficiently produce the target compound (see FIG. 16).

[0100] Each of the enzymes encoded by the genes contained in the recombinant microorganism of the present invention will be described in detail below.

[0101] <3-Hydroxyadipyl-CoA dehydratase> 3-Hydroxyadipyl-CoA dehydratase is an enzyme that catalyzes the reaction of converting 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA (Step C in Figure 16).

[0102] In one embodiment, 3-hydroxyadipyl-CoA dehydratase is encoded by DNA having a sequence identity of 65% or more, 70% or more, 75% or more, 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 with a PCR amplification product using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and a specific nucleotide as a primer. Here, the DNA has, for example, 626 to 940 bp, preferably 740 to 862 bp, more preferably 743 to 823 bp, even more preferably 767 to 799 bp, particularly more preferably 775 to 791 bp, and most preferably 778 to 788 bp. The primers used for PCR will be described later.

[0103] In a preferred embodiment, 3-hydroxyadipyl-CoA dehydratase is encoded by DNA that is a PCR amplification product using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and specific nucleotides as primers. The DNA has, for example, 626 to 940 bp, preferably 740 to 862 bp, more preferably 743 to 823 bp, even more preferably 767 to 799 bp, particularly preferably 775 to 791 bp, and most preferably 778 to 788 bp.

[0104] In further embodiments, the 3-hydroxyadipyl-CoA dehydratase is selected from: (A-1) A recombinant polypeptide encoded by DNA having a sequence identity of 65% or more, 70% or more, 75% or more, 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 to a PCR amplification product using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and specific nucleotides as primers, It has 3-hydroxyadipyl CoA dehydratase activity. The DNA has the aforementioned number of base pairs (bp), (A-2) A recombinant polypeptide having an amino acid sequence in which, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, and even more preferably 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a polypeptide encoded by DNA that is a PCR amplified product using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and predetermined nucleotides as primers, It has 3-hydroxyadipyl CoA dehydratase activity. The DNA has the aforementioned number of base pairs (bp), and (A-3) A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and specific nucleotides as primers, It has 3-hydroxyadipyl CoA dehydratase activity. The DNA is a recombinant polypeptide having the aforementioned number of base pairs (bp).

[0105] In a further preferred embodiment, the 3-hydroxyadipyl-CoA dehydratase is a recombinant polypeptide encoded by a PCR amplicon using chromosomal DNA of Burkholderia sp. strain LEBP-3 as a template and specific nucleotides as primers, and the base pair number of the amplicon is 783 or 784 bp.

[0106] For 3-hydroxyadipyl-CoA dehydratase, the primers (forward and reverse) used for PCR include the following (see FIG. 17): (1a) a nucleotide having the base sequence shown in SEQ ID NO: 168 and a nucleotide having the base sequence shown in SEQ ID NO: 169, (1b) a nucleotide comprising the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising the base sequence shown in SEQ ID NO: 169; (1c) a nucleotide sequence obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO: 168 and a nucleotide sequence obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO: 169; (1d) A nucleotide comprising a codon-optimized nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 168, and a nucleotide comprising a codon-optimized nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 169.

[0107] The "nucleotide containing the base sequence shown in SEQ ID NO:168 and the nucleotide containing the base sequence shown in SEQ ID NO:169" in (1b) above each have a nucleotide length of, for example, 1 to 20 bp, preferably 1 to 15 bp, more preferably 5 to 15 bp, and even more preferably 10 to 15 bp, in addition to the "base sequence shown in SEQ ID NO:168" and the "base sequence shown in SEQ ID NO:169", respectively.

[0108] The "nucleotides comprising a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 168 and a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 169" in (1d) above respectively comprise a sequence homologous to the vector end in addition to the "base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 168" and the "base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 169", and have a nucleotide length of, for example, an additional 1 to 20 bp, preferably 1 to 15 bp, more preferably 5 to 15 bp, and even more preferably 10 to 15 bp. Examples of the "nucleotides comprising a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 169" in (1d) above include: (1d') A nucleotide having the base sequence shown in SEQ ID NO: 198 and a nucleotide having the base sequence shown in SEQ ID NO: 199 (Figure 20A).

[0109] For 3-hydroxyadipyl-CoA dehydratase, the preferred primers are: (1a) a nucleotide sequence represented by SEQ ID NO: 168 and a nucleotide sequence represented by SEQ ID NO: 169, and (1b) a nucleotide containing the base sequence shown in SEQ ID NO: 168 and a nucleotide containing the base sequence shown in SEQ ID NO: 169 It is.

[0110] The DNA amplified by PCR using the above nucleotides as primers and the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template preferably has a length of 783 or 784 bp. The DNA polymerase used in PCR may be one known in the art, such as Taq DNA polymerase, hot-start adjusted DNA polymerase, or proofreading DNA polymerase having 3'-5' exonuclease activity in addition to polymerase activity.

[0111] <2,3-Dehydroadipyl-CoA reductase> 2,3-Dehydroadipyl-CoA reductase is an enzyme that catalyzes the reaction of converting 2,3-dehydroadipyl-CoA to adipyl-CoA (see step D in Figure 16).

[0112] In one embodiment, the 2,3-dehydroadipyl-CoA reductase is encoded by a DNA having a sequence identity of 65% or more, 70% or more, 75% or more, 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 with a PCR amplification product using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and a specific nucleotide as a primer. The DNA has, for example, 924 to 1386 bp, preferably 1039 to 1249 bp, more preferably 1097 to 1213 bp, even more preferably 1131 to 1179 bp, particularly preferably 1143 to 1167 bp, even more particularly preferably 1145 to 1165 bp, and most preferably 1150 to 1160 bp.

[0113] In a preferred embodiment, the 2,3-dehydroadipyl-CoA reductase is encoded by DNA that is a PCR amplification product using the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and specific nucleotides as primers. The DNA has, for example, 924 to 1386 bp, preferably 1039 to 1249 bp, more preferably 1097 to 1213 bp, even more preferably 1131 to 1179 bp, particularly preferably 1143 to 1167 bp, even more particularly preferably 1145 to 1165 bp, and most preferably 1150 to 1160 bp.

[0114] In further embodiments, the 2,3-dehydroadipyl-CoA reductase is selected from: (B-1) A recombinant polypeptide encoded by DNA having a sequence identity of 65% or more, 70% or more, 75% or more, 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 to a PCR amplification product using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and specific nucleotides as primers, It has 2,3-dehydroadipyl CoA reductase activity. The DNA has the aforementioned number of base pairs (bp), (B-2) A recombinant polypeptide having an amino acid sequence in which, for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, and even more preferably 1 to 3 amino acids have been deleted, substituted, inserted, and / or added to the amino acid sequence of a polypeptide encoded by DNA that is a PCR amplified product using chromosomal DNA of Burkholderia sp. strain LEBP-3 as a template and predetermined nucleotides as primers, It has 2,3-dehydroadipyl CoA reductase activity. The DNA has the aforementioned number of base pairs (bp), and (B-3) A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template and specific nucleotides as primers, It has 2,3-dehydroadipyl CoA reductase activity. The DNA is a recombinant polypeptide having the aforementioned number of base pairs (bp).

[0115] In a further preferred embodiment, the exogenous 2,3-dehydroadipyl-CoA reductase is a PCR amplified product using chromosomal DNA of Burkholderia sp. strain LEBP-3 as a template and predetermined nucleotides as primers, and the base pair number of the amplified product is 1155 or 1156 bp.

[0116] For 2,3-dehydroadipyl-CoA reductase, the primers (forward and reverse) used for PCR include the following (see FIG. 17): (2a) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171; (2b) a nucleotide comprising the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising the base sequence shown in SEQ ID NO: 171; (2c) a nucleotide sequence obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO: 170 and a nucleotide sequence obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO: 171; (2d) A nucleotide sequence comprising a codon-optimized nucleotide sequence shown in SEQ ID NO: 170 and a nucleotide sequence obtained by codon-optimizing the nucleotide sequence shown in SEQ ID NO:171.

[0117] The "nucleotide containing the base sequence shown in SEQ ID NO: 170 and the nucleotide containing the base sequence shown in SEQ ID NO: 171" in (2b) above each have a nucleotide length of, for example, 1 to 20 bp, preferably 1 to 15 bp, more preferably 5 to 15 bp, and even more preferably 10 to 15 bp, in addition to the "base sequence shown in SEQ ID NO: 170" and the "base sequence shown in SEQ ID NO: 171", respectively.

[0118] The "nucleotides comprising a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 170 and a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 171" in (2d) above each contain a sequence homologous to the vector end in addition to the "base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 170" and the "base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 171", and have a nucleotide length of, for example, an additional 1 to 20 bp, preferably 1 to 15 bp, more preferably 5 to 15 bp, and even more preferably 10 to 15 bp. Examples of the "nucleotides comprising a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising a base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 171" in (2d) above include: (2d') A nucleotide having the base sequence shown in SEQ ID NO: 210 and a nucleotide having the base sequence shown in SEQ ID NO: 211 (Figure 20A).

[0119] For 2,3-dehydroadipyl-CoA reductase, the preferred primers are: (2a) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, and (2b) a nucleotide containing the base sequence shown in SEQ ID NO: 170 and a nucleotide containing the base sequence shown in SEQ ID NO: 171 It is.

[0120] The DNA amplified by PCR using the above nucleotides as primers and the chromosomal DNA of Burkholderia sp. LEBP-3 strain as a template preferably has a length of 1155 or 1156 bp. The DNA polymerase used in PCR is one known in the art, such as Taq DNA polymerase, hot-start adjusted DNA polymerase, and proofreading DNA polymerase having 3'-5' exonuclease activity in addition to polymerase activity.

[0121] In this specification, the term "PCR amplification product" refers to a product obtained as a result of performing PCR using a specific nucleotide as a primer. Unless otherwise specified, in this specification, PCR amplification is performed using 1 μM of each specific nucleotide as a forward primer and a reverse primer, and using PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio) as an enzyme, with a volume of 25 μL of solution, and 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.

[0122] In the present invention, the host microorganism into which the foreign gene of interest is introduced is not particularly limited, and may be either a prokaryote or a eukaryote. It can be selected from those that have already been isolated and preserved, those that have been newly isolated from nature, and those that have been genetically modified. The host microorganism belongs to a genus selected from the group consisting of Esserisia, Corynebacterium, Bacillus, Acinetobacter, Burkholderia, Pseudomonas, Clostridium, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, Aspergillus, Klebsiella, Gluconobacter, Zymomonas, Lactobacillus, Lactococcus, Streptococcus, and Streptomyces. The host microorganism of the present invention preferably belongs to a genus selected from the group consisting of Escherichia, Corynebacterium, Bacillus, Acinetobacter, Burkholderia, Pseudomonas, Clostridium, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia and Aspergillus. The host microorganism is more preferably Escherichia coli.

[0123] Therefore, the recombinant microorganism according to the present invention, in which an exogenous gene has been introduced into the host microorganism, is, for example, Esserisia, Corynebacterium, Bacillus, Acinetobacter, Burkholderia, Pseudomonas, Clostridium, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, Aspergillus, Klebsiella, Gluconobacter, Zymomonas, Lactobacillus, Lactobacillus casei ... The recombinant microorganism according to the present invention is preferably a genus selected from the group consisting of Escherichia coli, Corynebacterium, Bacillus, Acinetobacter, Burkholderia, Pseudomonas, Clostridium, Saccharomyces, Schizosaccharomyces, Yarrowia, Candida, Pichia, and Aspergillus.

[0124] The pathways for producing adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol contained in the genetically modified microorganism of the present invention and the enzymes that catalyze each reaction step in the pathways are described below with reference to FIG. 16.

[0125] In the conversion of step A in FIG. 16, for example, succinyl-CoA:acetyl-CoA acyltransferase or 3-oxoadipyl-CoA thiolase is involved, and succinyl-CoA and acetyl-CoA are condensed to convert to 3-oxoadipyl-CoA. Other examples of enzymes that can catalyze this conversion include β-ketothiolases. Furthermore, for example, 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 also be exemplified as enzymes that can have activity in this conversion. The enzyme used in the present invention is not limited as long as it has activity in this conversion, but in one embodiment, PaaJ derived from Escherichia coli consisting of the amino acid sequence shown in SEQ ID NO: 172 is used. In a preferred embodiment, the enzyme is succinyl-CoA:acetyl-CoA acyltransferase encoded by a DNA sequence (1203 bp) that is a PCR amplification product using the chromosomal DNA of Burkholderia sp. strain LEBP-3 as a template and the nucleotides shown in SEQ ID NO:164 and SEQ ID NO:165 as primers.

[0126] In the conversion of step B in FIG. 16, 3-hydroxyadipyl-CoA dehydrogenase is involved, and 3-oxoadipyl-CoA is converted to 3-hydroxyadipyl-CoA. Examples of other enzymes that can catalyze this conversion include oxidoreductases classified into the EC 1.1.1 group. Specifically, for example, enzymes classified into 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 have activity in this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion, and for example, PaaH derived from Escherichia coli having the amino acid sequence shown in SEQ ID NO: 173 is used (FIG. 18). In a preferred embodiment, the enzyme is 3-hydroxyadipyl-CoA dehydrogenase encoded by the DNA sequence (1521 bp) of a PCR amplification product using the chromosomal DNA of Burkholderia sp. strain LEBP-3 as a template and the nucleotides shown in SEQ ID NO: 166 and SEQ ID NO: 167 as primers.

[0127] In the conversion of step C in FIG. 16, for example, 3-hydroxyadipyl-CoA dehydratase is involved, and 3-hydroxyadipyl-CoA is converted to 2,3-dehydroadipyl-CoA. Examples of other enzymes that can catalyze this conversion include hydrolyases classified into the EC 4.2.1 group. Specifically, for example, enzymes classified into groups such as 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 have activity in this conversion. In a preferred embodiment, the enzyme is the aforementioned 3-hydroxyadipyl-CoA dehydratase.

[0128] In the conversion of step D in FIG. 16, for example, 2,3-dehydroadipyl-CoA reductase is involved, and 2,3-dehydroadipyl-CoA is converted to adipyl-CoA. Other examples of enzymes that can catalyze this conversion include oxidoreductases classified in the EC 1.3.1 group. Specifically, 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 + )), EC 1.3.1.44 (trans-2-enoyl-CoA reductase (NAD + Examples of enzymes that may have activity in this conversion include enzymes classified into groups such as 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)). In a preferred embodiment, the enzyme is the aforementioned 2,3-dehydroadipyl-CoA reductase.

[0129] In the conversion of step E in FIG. 16, adipyl-CoA is converted to adipic acid. An example of an enzyme that can catalyze this conversion is a thioester hydratase classified in the EC 3.1.2 group. For example, enzymes classified in groups such as EC 3.1.2.1 (acetyl-CoA hydratase) and EC 3.1.2.20 (acyl-CoA hydratase) can be exemplified as enzymes that can have activity for this conversion. In a preferred embodiment, the enzyme is not limited as long as it has activity for this conversion, but tesB (Ab) from Acinetobacter ADP1 strain shown in SEQ ID NO: 179 is used. Depending on the substrate specificity of the enzyme used, CoA is released from 3-oxoadipyl-CoA, 3-hydroxyadipyl CoA, and 2,3-dehydroadipyl CoA to produce 3-oxoadipate, 3-hydroxyadipate, and 2,3-dehydroadipate, respectively.

[0130] Another example of an enzyme that can catalyze the conversion in step E of Figure 16 is CoA-transferase classified into the EC 2.8.3 group. For example, enzymes classified into groups such as EC 2.8.3.5 (3-oxoacid CoA-transferase), EC 2.8.3.6 (3-oxoadipate CoA-transferase), and EC 2.8.3.18 (succinyl-CoA:acetate CoA-transferase) can be exemplified as enzymes that can have activity in this conversion.

[0131] Furthermore, as an example of another enzyme conversion that can catalyze the conversion in step E of Figure 16, a pathway in which the adipyl group of adipyl-CoA is transferred to phosphate by an acyltransferase classified in the EC 2.3.1 group to generate adipyl phosphate, followed by dephosphorylation by a phosphotransferase classified in the EC 2.7.2 group, can be exemplified. For example, enzymes classified in the EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase) groups as acyltransferases, and EC 2.7.2.1 (acetate kinase) and EC 2.7.2.7 (butanoate kinase) groups as phosphotransferases can be exemplified as enzymes that can have activity in this conversion.

[0132] In step T of Figure 16, 3-oxoadipate is converted to levulinic acid. This conversion is either catalyzed by an enzyme (3-oxoadipate decarboxylase) or occurs spontaneously.

[0133] The conversion in step F of Figure 16 converts adipyl-CoA to adipic 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 (formation of malonic semialdehyde)), and EC 1.2.1.76 (succinic semialdehyde dehydrogenase (acylation)) can be exemplified as enzymes that may have activity in this conversion because they catalyze a conversion reaction in which CoA is eliminated to produce an aldehyde, similar to this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion. For example, sucD derived from Clostridium kluyveri and consisting of the amino acid sequence set forth in SEQ ID NO: 236 is used (FIG. 21).

[0134] In the conversions of steps G, J, and Q in FIG. 16, a carboxyl group is converted to an aldehyde. An example of an enzyme that can catalyze this conversion is carboxylate reductase (CAR). For example, enzymes classified into groups such as EC 1.2.1.30 (carboxylate 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 (carboxylate reductase) can be exemplified as enzymes that can have activity in this conversion because they catalyze a conversion reaction that produces an aldehyde from a carboxylic acid, similar to this conversion. Typical examples of biological species from which the enzymes are 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 enzyme used in the present invention is not limited as long as it has activity for this conversion, but for example, at least one type of enzyme consisting of the amino acid sequence set forth in any of SEQ ID NOs: 237 to 241 may be used (Figures 22A to 22E), and preferably, at least one of the enzyme MaCar derived from Mycobacterium abscessus consisting of the amino acid sequence set forth in SEQ ID NO: 239 and MaCar(m), a mutant of MaCar consisting of the amino acid sequence set forth in SEQ ID NO: 241, is used, and more preferably, MaCar(m) consisting of the amino acid sequence set forth in SEQ ID NO: 241 is used.

[0135] Furthermore, carboxylic acid reductase can be converted to 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 capable of catalyzing this reaction include, for example, enzymes classified in EC 2.7.8.7. Therefore, the microorganism of the present invention may be further modified so that the activity of phosphopantetheinyl transferase is increased. Methods for increasing the activity of phosphopantetheinyl transferase include, but are not limited to, a method of introducing an exogenous phosphopantetheinyl transferase gene and a method of enhancing the expression of an endogenous phosphopantetheinyl transferase gene. The enzyme used in the present invention is not limited as long as it has phosphopantetheinyl group transfer activity, but typical examples include EntD of Escherichia coli, Sfp of Bacillus subtilis, Npt of Nocardia iowensis (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1, 478-485 (2007)), and Lys5 of Saccharomyces cerevisiae (Ehmann et al., Biochemistry 38.19 (1999): 6171-6177.). The enzyme used in the present invention is not limited as long as it has activity for this conversion, but at least one of the enzymes consisting of the amino acid sequences set forth in SEQ ID NOs: 242 to 245 may be used (FIGS. 23A to 23D), and preferably Npt of Nocardia iowensis derived from Nocardia iowensis consisting of the amino acid sequence set forth in SEQ ID NO: 243 is used.

[0136] The conversions in steps M, N, and S in FIG. 16 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 have activity in this conversion. The enzymes used in the present invention are not particularly limited as long as they have the conversion activity of each step. For example, putrescine aminotransferase YgjG from Escherichia coli, which has been reported to transaminate cadaverine and spermidine (Samsonova, et al., BMC microbiology 3.1 (2003): 2.), putrescine aminotransferase SpuC 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.), GABA aminotransferase GabT from Escherichia coli, and PuuE may be used. Furthermore, ω-transaminases derived from organisms such as Ruegeria pomeroyi, Chromobacterium violaceum, Arthrobacter citreus, Sphaerobacter thermophilus, Aspergillus fischeri, Vibrio fluvialis, Agrobacterium tumefaciens, and Mesorhizobium loti have 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: 246 to 250 may be used (FIGS. 24A to 24E). Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, and glycine.

[0137] In the conversion of steps H, L, and R in FIG. 16, 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 + )), EC 1.1.1.71 (alcohol dehydrogenase [NAD(P) + ]) can be exemplified as enzymes that may have activity in this conversion because they catalyze the conversion reaction from aldehyde to alcohol, similar to this conversion. The enzyme used in the present invention is not limited as long as it has activity in this conversion, and for example, Ahr derived from Escherichia coli as set forth in SEQ ID NO: 251 is used (Figure 25).

[0138] In the conversions of steps I and O in Figure 16, 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 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 activity for this conversion.

[0139] 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 enzyme activity in the host microbial cell.

[0140] The genetically modified microorganism of the present invention preferably has introduced therein, in addition to the exogenous genes encoding the aforementioned 3-hydroxyadipyl-CoA dehydratase and / or 2,3-dehydroadipyl-CoA reductase, an exogenous gene encoding at least one enzyme selected from the group consisting of succinyl-CoA:acetyl-CoA acyltransferase and 3-hydroxyadipyl-CoA dehydrogenase.

[0141] That is, the recombinant microorganism according to the present invention preferably further comprises at least one of an exogenous gene encoding succinyl-CoA:acetyl-CoA acyltransferase and an exogenous gene encoding 3-hydroxyadipyl-CoA dehydrogenase.

[0142] In addition to the above-mentioned exogenous genes, i.e., 3-hydroxyadipyl-CoA dehydratase, 2,3-dehydroadipyl-CoA reductase, succinyl-CoA:acetyl-CoA acyltransferase, and 3-hydroxyadipyl-CoA dehydrogenase, further exogenous genes may be introduced into the recombinant microorganism of the present invention.

[0143] The recombinant microorganism of the present invention may be a host microorganism in which any gene has been disrupted as appropriate. Disruption of the target gene may be carried out according to methods known in the art.

[0144] Furthermore, the enzyme genes that can be used for the purposes of the present invention may have all mutations that can occur in nature, or mutations and modifications that have been artificially introduced, so long as they can express substantial enzyme activity in the host microbial cells. For example, it is known that there are extra codons for various codons that code for specific amino acids. Therefore, alternative codons that will ultimately be translated into the same amino acid may also be used in the present invention. In other words, since the genetic code is degenerate, multiple codons can be used to code for a specific amino acid, and therefore the amino acid sequence can be coded by any set of similar DNA oligonucleotides. Only one member of the set is identical to the gene sequence of the native enzyme, but even mismatched DNA oligonucleotides can hybridize to the native sequence under appropriate stringent conditions (e.g., hybridize at 3xSSC, 68°C, wash at 2xSSC, 0.1% SDS, and 68°C), and DNA encoding the native sequence can be identified and isolated, and such genes can also be used in the present invention. In particular, since most organisms are known to preferentially use a specific subset of codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), performing "codon optimization" depending on the host microorganism may also be useful in the present invention.

[0145] Therefore, the genetically modified microorganism of the present invention may contain a base sequence having, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence of the enzyme gene, provided that the genetically modified microorganism can express substantial enzyme activity. Alternatively, the genetically modified microorganism of the present invention may contain a base sequence having, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence encoding the amino acid sequence of the enzyme.

[0146] In the present invention, the biosynthetic enzyme genes are introduced into the host microbial cell as an "expression cassette", thereby obtaining a more stable and high-level enzyme activity. In this specification, the term "expression cassette" refers to a nucleotide sequence that contains a nucleic acid sequence that regulates transcription and translation and is functionally linked to the nucleic acid to be expressed or the gene to be expressed. Typically, the expression cassette of the present invention contains a promoter sequence 5' upstream from the coding sequence, a terminator sequence 3' downstream, and optionally further conventional regulatory elements in a functionally linked state, and in such a case, the nucleic acid to be expressed or the gene to be expressed is introduced into the host microbial cell.

[0147] In this specification, a promoter is defined as a DNA sequence that binds RNA polymerase to DNA and initiates RNA synthesis, regardless of whether it is a constitutive expression promoter or an inducible expression promoter. A strong promoter is a promoter that initiates mRNA synthesis at a high frequency, and is preferably used in the present invention. In E. coli, the lac system, trp system, tac or trc system, the main operator and promoter region of λ phage, the control region of fd coat protein, promoters for glycolytic enzymes (e.g., 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase), glutamic acid decarboxylase A, serine hydroxymethyltransferase, the promoter region of RNA polymerase derived from T7 phage, etc. can be used. In Corynebacterium glutamicum, the HCE (high-level constitutive expression) promoter, the cspB promoter, the sodA promoter, the elongation factor (EF-Tu) promoter, etc. can be used.

[0148] Terminators that can be used include the T7 terminator, the rrnBT1T2 terminator, and the lac terminator.

[0149] In addition to promoter and terminator sequences, other regulatory elements include, for example, selection markers, amplification signals, and replication origins. Suitable regulatory elements are described, for example, in "Gene Expression Technology: Methods in Enzymology 185", Academic Press (1990).

[0150] The host microorganism used in the present invention may be a microorganism in which a gene encoding an enzyme inherent to the microorganism has been disrupted. Examples of genes encoding the enzyme include the ldh gene encoding lactate dehydrogenase, the atoB gene encoding acetyl-CoA acetyltransferase, and the sucD gene encoding succinyl-CoA synthetase α subunit. In this way, by using a host microorganism in which some of the genes of the enzyme inherent to the host microorganism have been disrupted, it is possible to avoid contamination with impurities and suppress the progress of unintended reactions. Specifically, by disrupting the ldh gene encoding lactate dehydrogenase, it is possible to reduce the production of lactic acid, which is an impurity. By disrupting the atoB gene encoding acetyl-CoA acetyltransferase and the sucD gene encoding succinyl-CoA synthetase α subunit, it is possible to improve the yield of adipic acid.

[0151] The expression cassette described above is inserted into a vector, such as a plasmid, a phage, a transposon, an IS element, a fosmid, a cosmid, or a linear or circular DNA, into a host microorganism. Plasmids and phages are preferred. These vectors may be autonomously replicating in the host microorganism or may be replicated chromosomally. Suitable plasmids include, for example, pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III113-B1, λgt11, or pBdCI for E. coli; pUB110, pC194, or pBD214 for Bacillus; and pSA77 or pAJ667 for Corynebacterium. Examples of suitable plasmids include pUB110, pC194, or pBD214 for Bacillus species. Other usable plasmids are described in "Gene Cloning and DNA analysis 6th edition", Wiley-Blackwell (2016). The expression cassettes can be introduced into a vector 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.

[0152] After the vector having 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, conjugation, protoplast fusion, etc., and a method suitable for the host microorganism can be selected.

[0153] Another aspect of the present invention relates to a method for producing a target compound using the recombinant microorganism, more specifically, a method for producing a target compound, comprising a culture step of culturing the recombinant microorganism.

[0154] Here, the target compound is selected from the group consisting of adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol, and 6-amino-1-hexanol. As the adipic acid derivatives, as described above, for example, 3-oxoadipic acid (3-OA), levulinic acid (LEV), 3-hydroxyadipic acid (3HA), and 2,3-dehydroadipic acid (23DA) can be mentioned.

[0155] In one embodiment, the recombinant microorganism of the present invention has an adipic acid production pathway. In this case, the present invention also relates to a method for producing adipic acid, comprising a culturing step of culturing the recombinant microorganism.

[0156] In one embodiment, the recombinant microorganism of the present invention has a hexamethylenediamine production pathway. In this case, the present invention also relates to a method for producing hexamethylenediamine, comprising a culture step of culturing the recombinant microorganism.

[0157] In one embodiment, the recombinant microorganism of the present invention has a 1,6-hexanediol production pathway. In this case, the present invention also relates to a method for producing 1,6-hexanediol, comprising a culturing step of culturing the recombinant microorganism.

[0158] In one embodiment, the recombinant microorganism of the present invention has a 6-amino-1-hexanol production pathway. In this case, the present invention also relates to a method for producing 6-amino-1-hexanol, comprising a culture step of culturing the recombinant microorganism.

[0159] In the culturing step, the recombinant microorganism is cultured in a medium containing a carbon source and a nitrogen source to obtain a culture containing the bacterial cells. The recombinant microorganism of the present invention is cultured under conditions suitable for adipic acid production and for the growth and maintenance of the microorganism, and suitable medium composition, culture time and culture conditions can be easily determined by those skilled in the art.

[0160] Examples of 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 decomposition liquid, cellulose decomposition liquid, carbon dioxide, carbon monoxide, etc. Preferred are D-glucose, sucrose, or glycerol. These carbon sources can be used individually or as a mixture.

[0161] Adipic acid produced using biomass-derived raw materials can be clearly distinguished from synthetic raw materials derived from, for example, petroleum, natural gas, or coal by measuring the biobased carbon content based on Carbon-14 (radioactive carbon) analysis specified in ISO16620-2 or ASTM D6866.

[0162] Nitrogen sources include nitrogen-containing organic compounds (e.g., peptone, casamino acids, tryptone, yeast extract, meat extract, malt extract, corn steep liquor, soy flour, amino acids, and urea, etc.) 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 mixtures.

[0163] Furthermore, when the recombinant microorganism expresses a useful additional trait, for example, when it has an antibiotic resistance marker, the medium may contain the corresponding antibiotic, thereby reducing the risk of contamination by various bacteria during cultivation. 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.

[0164] The cultivation may be batch-wise or continuous. Also, in any case, it may be in a form where the additional carbon source etc. is replenished at an appropriate point during the cultivation. Further, the cultivation may be carried out while controlling conditions such as suitable temperature, oxygen concentration, pH, etc. The suitable cultivation temperature for transformants derived from common microbial host cells is usually in the range of 15°C to 45°C, preferably 25°C to 37°C. When the host microorganism is aerobic, shaking (such as flask cultivation etc.), stirring / aeration (such as jar fermenter cultivation etc.) may be carried out to ensure an appropriate oxygen concentration during fermentation. Those cultivation conditions can be easily set by those skilled in the art.

[0165] The method for producing the target compound according to the present invention may include a separation and purification step of separating and purifying the produced compound from the culture. In this step, any method can be used, for example, methods such as centrifugation, membrane filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation, and various chromatographies, but it is not limited thereto. The separation and purification step may consist of one type of step or may consist of a combination of multiple steps.

[0166] Those skilled in the art given the above description can fully implement the present invention. Hereinafter, examples are given for the purpose of further explanation. Therefore, the present invention is not limited to the said examples.

Examples

[0167] <1> Invention A Hereinafter, Invention A of the present invention will be described based on examples, but Invention A of the present invention is not limited to these examples.

[0168] The amino acid sequences and nucleotide sequences of the samples used in the examples are shown in FIGS. 2 to 15.

[0169] <Construction of C6 compound production pathway enzyme gene expression plasmid> PrimeSTAR Max DNA Polymerase (product name, Takara Bio) or KOD FX Neo (product name, Toyobo) was used to amplify PCR fragments, and E. coli JM109 strain was used to prepare plasmids. GeneArt GeneOptimizer (software name, Thermo Fisher Scientific) or the artificial gene synthesis service of Eurofins Genomics was used to optimize the base sequence.

[0170] A polynucleotide encoding PaaJ(Ec) (SEQ ID NO: 1) of Eshcherichia coli was obtained by cloning from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using oligonucleotides of SEQ ID NO: 34 and 35 as primers to obtain a PCR product containing the coding region of the paaJ(Ec) gene (SEQ ID NO: 33). Next, PCR was performed using pRSFDuet-1 (product name, manufactured by Merck) as a template and oligonucleotides of SEQ ID NO: 36 and 37 as primers to obtain a pRSFDuet-1 fragment. The DNA fragment containing the coding region of the paaJ(Ec) gene and the pRSFDuet-1 fragment were ligated using In-Fusion (registered trademark, hereafter omitted) HD cloning kit (product name, manufactured by Clontech). The product was transformed into E. coli JM109 strain, and a plasmid was extracted from the obtained transformant. "paaJ(Ec)-pRSFDuet" was obtained as a PaaJ(Ec) expression plasmid.

[0171] A polynucleotide encoding PaaH(Ec) (SEQ ID NO: 2) of Eshcherichia coli was obtained by cloning from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using oligonucleotides of SEQ ID NO: 39 and 40 as primers to obtain a PCR product containing the coding region of the paaH(Ec) gene (SEQ ID NO: 38). Next, PCR was performed using "paaJ(Ec)-pRSFDuet" as a template and oligonucleotides of SEQ ID NO: 41 and 42 as primers to obtain a "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 In-Fusion HD cloning kit (product name, manufactured by Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. The PaaJ(Ec) and PaaH(Ec) co-expression plasmid "paaJ(Ec)-paaH(Ec)-pRSFDuet" was obtained.

[0172] A polynucleotide encoding Eshcherichia coli PaaF (Ec) (SEQ ID NO: 3) was obtained by cloning from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using oligonucleotides of SEQ ID NO: 44 and 45 as primers to obtain a PCR product containing the coding region of the paaF (Ec) gene (SEQ ID NO: 43). The nucleotides encoding the PaaF (L3) of the L3 strain, which are encoded in the nucleotide sequence of the PCR amplified product using the nucleotides of SEQ ID NO: 4 and SEQ ID NO: 46 as primers and the chromosomal DNA of the L3 strain as a template, were obtained by optimizing the nucleotide sequence for expression in E. coli and using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using oligonucleotides of SEQ ID NO: 47 and 48 as primers to obtain a PCR product containing the optimized sequence of the coding region of the paaF (L3) gene. PCR was performed using pETDuet-1 (product name, manufactured by Merck) as a template and oligonucleotides of SEQ ID NO: 49 and 50 as primers to obtain a pETDuet-1 fragment. DNA fragments containing the coding regions of paaF(Ec) and paaF(L3) were ligated to the pETDuet-1 fragment using the In-Fusion HD cloning kit (product name, manufactured by Clontech). The resulting plasmid was transformed into E. coli JM109 strain, and a plasmid was extracted from the resulting transformant. "paaF(Ec)-pETDuet" was obtained as the PaaF(Ec) expression plasmid, and "paaF(L3)-pETDuet" was obtained as the PaaF(L3) expression plasmid.

[0173] The polynucleotide encoding dcaA(Ab) (SEQ ID NO: 5) of Acinetobacter baylyi was obtained by optimizing the base sequence for expression in E. coli using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using oligonucleotides of SEQ ID NO: 52 and 53 as primers to obtain a PCR product containing the coding region of the dcaA(Ab) gene (SEQ ID NO: 51). PCR was performed using "paaF(Ec)-pETDuet" as a template and oligonucleotides of SEQ ID NO: 84 and 85 as primers to obtain a "paaF(Ec)-pETDuet" fragment. The DNA fragment containing the coding region of the dcaA(Ab) gene and the "paaF(Ec)-pETDuet" fragment were ligated using In-Fusion HD cloning kit (product name, manufactured by Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the resulting transformant. The PaaF(Ec)-dcaA(Ab)-pETDuet was obtained as a PaaF(Ec) and DcaA(Ab) co-expression plasmid.

[0174] Ter(Ct) (SEQ ID NO: 6) of Candida tropicalis was cloned as a sequence (SEQ ID NO: 7) with 22 residues removed from the N-terminal region. The encoding polynucleotide was optimized for expression in E. coli and obtained using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using oligonucleotides of SEQ ID NO: 55 and 56 as primers to obtain a PCR product containing the coding region of the ter(Ct) gene (SEQ ID NO: 54). PCR was performed using oligonucleotides of SEQ ID NO: 84 and 85 as primers with "paaF(L3)-pETDuet" as a template to obtain a "paaF(L3)-pETDuet" fragment. The DNA fragment containing the coding region of the ter(Ct) gene and the "paaF(L3)-pETDuet" fragment were ligated using 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) and Ter(Ct) co-expression plasmid "paaF(L3)-ter(Ct)-pETDuet" was obtained.

[0175] Using BLAST Search (https: / / www.genome.jp / tools / blast / ) BLASTP, we selected proteins from the KEGG GENES database that are predicted to have the function of 2,3-dehydroadipyl-CoA reductase. The selected candidate enzyme proteins are shown in Table A-1.

[0176] [Table A-1]

[0177] The polynucleotides encoding the selected 2,3-dehydroadipyl-CoA reductase candidate proteins were optimized for expression in E. coli and obtained using the artificial gene synthesis service of Eurofins Genomics, and DNA fragments containing the coding region of each enzyme gene were obtained by PCR. The base sequence of the coding region of each enzyme gene and the sequence number of the primer set used for PCR are shown in Table 2. PCR was performed using "paaF(L3)-pETDuet" as a template and oligonucleotides of sequence numbers 84 and 85 as primers to obtain the "paaF(L3)-pETDuet" fragment. The DNA fragments containing the coding region of each gene and the "paaF(L3)-pETDuet" fragment were connected using the In-Fusion HD cloning kit (product name, Clontech). The E. coli JM109 strain was transformed, and plasmids were extracted from the obtained transformants. PaaF(L3) and each 2,3-dehydroadipyl-CoA reductase candidate protein A co-expression plasmid was obtained.

[0178] [Table A-2]

[0179] Mycobacterium abscessus carboxylate reductase MaCar (SEQ ID NO: 20) was used as mutant MaCar(m) in which the 283rd tryptophan residue in the amino acid sequence was replaced with an arginine residue and the 303rd alanine residue was replaced with a methionine residue (SEQ ID NO: 22). The polynucleotide encoding MaCar(m) was obtained by optimizing the base sequence for expression in E. coli and using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using oligonucleotides of SEQ ID NO: 87 and 88 as primers to obtain a PCR product containing the coding region of the Macar(m) gene (SEQ ID NO: 86).

[0180] Next, this PCR product was inserted between the restriction enzyme NdeI and AvrII cleavage sites of pACYCDuet-1 (product name, manufactured by Merck) using the In-Fusion HD cloning kit (product name, manufactured by Clontech). The product was transformed into Escherichia coli JM109 strain, and a plasmid was extracted from the resulting transformant. "Macar(m)-pACYCDuet" was obtained as the MaCar(m) expression plasmid.

[0181] The polynucleotide encoding Npt (SEQ ID NO: 24) of Nocardia iowensis was obtained by cloning from the genomic DNA of Nocardia iowensis JCM18299 strain (provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Bioresource Project). PCR was performed using oligonucleotides of SEQ ID NO: 90 and 91 as primers to obtain a PCR product containing the coding region of the npt gene (SEQ ID NO: 89). Next, PCR was performed using "MaCar(m)-pACYCDuet" as a template and oligonucleotides of SEQ ID NO: 92 and 93 as primers to obtain a "Macar(m)-pACYCDuet" fragment. The PCR products were connected to each other using In-Fusion HD cloning kit (product name, manufactured by Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "Macar(m)-npt-pACYCDuet" was obtained as a MaCar(m) and Npt co-expression plasmid.

[0182] The polynucleotide encoding Ahr (SEQ ID NO: 32) of Escherichia coli was obtained by cloning from the genomic DNA of Escherichia coli W3110 strain (NBRC12713). PCR was performed using the oligonucleotides of SEQ ID NOs: 95 and 96 as primers to obtain a PCR product containing the coding region of the ahr gene (SEQ ID NO: 94). Next, using "Macar(m)-npt-pACYCDuet" as a template and the oligonucleotides of SEQ ID NOs: 97 and 98 as primers, PCR was performed to obtain a "macar(m)-npt-pACYCDuet" fragment. The PCR product containing the coding region of each gene and the "macar(m)-npt-pACYCDuet" fragment were ligated using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and plasmids were extracted from the obtained transformants. "ahr-macar(m)-npt-pACYCDuet" was obtained as an Ahr, MaCar, Npt co-expression plasmid, respectively.

[0183] <Construction of C6 compound-producing strains> The constructed plasmid was transformed into Escherichia coli BL21(DE3) strain competent cells (manufactured by Nippon Gene), and spread on an LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 50 mg / L of sodium ampicillin, 30 mg / L of kanamycin sulfate, (for the construction of strain HDO1, an additional 30 mg / L of chloramphenicol), and incubated at 37 °C for 24 hours to obtain single colonies of transformants. The obtained transformants are shown in Table A-3.

[0184]

Table A-3

[0185] <Adipic acid production test by culturing recombinant Escherichia coli> A loopful of transformants ADA1 to ADA11 was inoculated into 2 mL (14 mL round bottom tube) of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L) containing 50 mg / L of sodium ampicillin and 30 mg / L of kanamycin sulfate, and cultured with shaking at 37 ° C for 24 hours to obtain a preculture solution. 10 μL of the obtained preculture solution was inoculated into 1 mL of MM medium (96-well deep well plate) containing 50 mg / L of sodium ampicillin and 30 mg / L of kanamycin sulfate, and cultured with shaking at 37 ° C for 48 hours. The composition of the MM medium is shown in Table A-4.

[0186] [Table A-4]

[0187] After the culture was completed, the concentration of adipic acid in the culture solution was analyzed. The analysis was performed by GC / MS analysis using trimethylsilyl derivatization. 360 μL of an extract mixed with water:methanol:chloroform = 5:2:2 (v / v / v) was added to 40 μL of the culture solution centrifuged supernatant (added with disodium sebacate as an internal standard to a final concentration of 10 mM), and the mixture was thoroughly stirred with a vortex mixer. After centrifugation (16,000 × g, 5 minutes), 40 μL of the supernatant was collected in another microtube and centrifuged and dried for 1 hour with a centrifugal evaporator. 100 μL of a 20 mg / mL pyridine solution of methoxyamine hydrochloride was added to the obtained dried product, and the mixture was shaken at 30 ° C for 90 minutes. 50 μL of N-methyl-N-trimethylsilyl trifluoroacetamide was added, and the mixture was shaken at 37 ° C for 30 minutes. The reaction solution was used as a GC / MS measurement sample, and analysis was performed under the following conditions.

[0188] 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℃, hold for 2 minutes → increase temperature at 15℃ / minute → 325℃, hold for 13 minutes Ionization method: Electron Ionization (EI) Ionization energy: 70 eV Ion source temperature: 230℃ Scan range: m / z=50~500

[0189] The results are shown in Table A-5. The transformants ADA1 to ADA11 are transformants into which expression plasmids for enzyme genes capable of promoting the reactions of steps A, B, C, and D in Fig. 1 have been introduced. The reaction of step E proceeds due to endogenous enzymes of E. coli.

[0190] In the culture of transformant ADA1 expressing PaaJ(Ec), PaaH(Ec), and PaaF(Ec) derived from E. coli, and dcaA(Ab) derived from Acinetobacter baylyi (Comparative Example 1), 0.14 g / L of adipic acid was produced. In the culture of transformant ADA2 expressing PaaJ(Ec), PaaH(Ec) derived from E. coli, PaaF(L3) derived from L3 strain, and Ter(Ct) from Candida tropicalis (Comparative Example 2), 0.33 g / L of adipic acid was produced.

[0191] In the culture of transformants ADA3 to ADA11 (Examples 1 to 9) expressing E. coli-derived PaaJ (Ec), PaaH (Ec), L3 strain-derived PaaF (L3), and Ter(caur), Ter(kmx), Ter(pkz), Ter(mtm), Ter(ttt), Ter(cthr), Ter(pan), Ter(plj), or Ter(pte) as an enzyme that catalyzes the reaction in step D of FIG. 1, adipic acid was produced, and it was shown that Ter(caur), Ter(kmx), Ter(pkz), Ter(mtm), Ter(ttt), Ter(cthr), Ter(pan), Ter(plj), or Ter(pte) has the enzyme activity that promotes the adipic acid production pathway reaction.

[0192] In addition, in the culture of the transformants ADA6 (Example 4), ADA7 (Example 5), ADA8 (Example 6), ADA9 (Example 7) and ADA11 (Example 9), more adipic acid was produced than in the comparative example, indicating that the amount of C6 compound produced was improved by expressing the enzymes Ter(mtm), Ter(ttt), Ter(cthr), Ter(pan) or Ter(pte). In addition, these results show that the microorganisms shown as examples exhibit good 2,3-dehydroadipyl-CoA reductase activity.

[0193] [Table A-5]

[0194] <Production test of 1,6-hexanediol and 6-hydroxyhexanoic acid by recombinant E. coli culture> A loopful of the transformant HDO1 was inoculated into 2 mL of LB liquid medium (14 mL round bottom tube) containing 50 mg / L sodium ampicillin, 30 mg / L kanamycin sulfate, and 30 mg / L chloramphenicol, and cultured with shaking at 37°C for 24 hours to obtain a preculture solution. 10 μL of the obtained preculture solution was inoculated into 1 mL of MM medium (96-well deep well plate) containing 50 mg / L sodium ampicillin, 30 mg / L kanamycin sulfate, and 30 mg / L chloramphenicol, and cultured with shaking at 37°C for 48 hours.

[0195] After the cultivation, the 1,6-hexanediol and 6-hydroxyhexanoic acid concentrations in the culture medium were analyzed. The 6-hydroxyhexanoic acid concentration was analyzed using GC / MS under the same conditions as the adipic acid concentration analysis. The 1,6-hexanediol concentration was analyzed using a high-performance liquid chromatograph Prominence system (Shimadzu Corporation) under the following conditions.

[0196] High performance liquid chromatography (HPLC) analysis conditions Detector: Differential refractive index detector Column: Shim-Pack Fast-OA(G), Fast-OA (Shimadzu Corporation) Oven temperature: 40℃ Mobile phase: 8 mM methanesulfonic acid in water Flow rate: 0.6mL / min Injection volume: 10μL

[0197] The results are shown in Table A-6. The transformant HDO1 is a transformant into which expression plasmids for carboxylic acid reductase and alcohol dehydrogenase were introduced in addition to expression plasmids for enzyme genes capable of promoting the reactions of steps A, B, C, and D in Figure 1. In the culture of the transformant HDO1 (Example 10), 6-hydroxyhexanoic acid and 1,6-hexanediol were produced.

[0198] [Table A-6]

[0199] <Construction of plasmid for gene disruption> The gene disruption and insertion in E. coli was performed by homologous recombination using pHAK1 (Deposited on March 18, 2019 at the National Institute of Technology and Evaluation, National Patent Microorganism Depository (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the 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 levansucrase gene sacB derived from Bacillus subtilis. The levansucrase gene is lethal to the host microorganism in the presence of sucrose. PCR fragments were amplified using PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio) or KOD FX Neo (product name, manufactured by Toyobo), and plasmids were prepared using E. coli HST08 strain.

[0200] Using the genomic DNA of E. coli BL21(DE3) strain as a template, PCR products containing the 5' homologous region, coding region, and 3' homologous region of the disruption target gene were obtained. The combinations of the target gene and primer sequences are shown in Table A-7 below.

[0201] [Table A-7]

[0202] Next, this PCR product was inserted into a pHAK1 plasmid fragment amplified using primers of SEQ ID NOs: 115 and 116 using an In-Fusion HD cloning kit (product name, manufactured by Clontech) and circularized. The resulting fragment was transformed into Escherichia coli HST08 strain, and a plasmid was extracted from the resulting transformant.

[0203] Using the pHAK1 plasmid extracted above into which DNA fragments of the 5' homology region, coding region, and 3' homology region of the disruption target gene were inserted, PCR was performed using the primers listed in Table A-8 below as a template to obtain a pHAK1 plasmid fragment in which a portion or the entire coding region of the disruption target gene had been removed and which contained the 5' homology region and 3' homology region.

[0204] [Table A-8]

[0205] The resulting plasmid fragment was cyclized by terminal phosphorylation and self-ligation, transformed into Escherichia coli HST08, and the plasmid was extracted from the transformant to obtain a plasmid for gene disruption.

[0206] <Construction of a plasmid for inserting hexamethylenediamine production pathway enzyme genes> A polynucleotide (SEQ ID NO: 33) encoding Eshcherichia coli PaaJ (Ec) (SEQ ID NO: 1) was cloned from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). A polynucleotide (SEQ ID NO: 38) encoding Eshcherichia coli PaaH (Ec) (SEQ ID NO: 2) was obtained by cloning from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713).

[0207] A plasmid for gene insertion was designed so that the paaJ(Ec) and paaH(Ec) genes were inserted as an expression cassette into the pflB gene region. An expression cassette was inserted between the 5' and 3' homologous regions of the pflB gene coding region contained in the previously constructed plasmid for disrupting the pflB gene, in which the promoter region (SEQ ID NO: 133), paaJ(Ec) gene coding region, linker sequence (SEQ ID NO: 134), paaH gene coding region, and terminator region (SEQ ID NO: 135) were arranged in this order.

[0208] The nucleotide sequence encoding the PaaF (L3) of the L3 strain, which is encoded by the nucleotide sequence of the PCR amplified product using the nucleotides shown in SEQ ID NO: 4 and SEQ ID NO: 46 as primers and the chromosomal DNA of the L3 strain as a template, was optimized for expression in E. coli and obtained using the artificial gene synthesis service of Eurofins Genomics, Inc. The polynucleotide (1155 bp) encoding the MmgC (L3) of the L3 strain, which is encoded by the nucleotide sequence of the PCR amplified product using the nucleotides shown in SEQ ID NO: 162 and SEQ ID NO: 163 as primers and the chromosomal DNA of the L3 strain as a template, was optimized for expression in E. coli and obtained using the artificial gene synthesis service of Eurofins Genomics, Inc.

[0209] A plasmid for gene insertion was designed so that the optimized sequences of the coding regions of the paaF (L3) and mmgC (L3) genes were inserted as an expression cassette into the yahK gene region. An expression cassette in which the promoter region (SEQ ID NO: 136), the paaF (L3) gene coding region, the linker sequence (SEQ ID NO: 137), the mmgC (L3) gene coding region, and the terminator region (SEQ ID NO: 138) were arranged in this order was inserted between the 5' and 3' homologous regions of the yahK gene coding region contained in the previously constructed plasmid for disrupting the yahK gene.

[0210] For Mycobacterium abscessus carboxylate reductase MaCar (SEQ ID NO: 20), a mutant MaCar(m) was used in which the 283rd tryptophan residue in the amino acid sequence was replaced with an arginine residue and the 303rd alanine residue was replaced with a methionine residue (SEQ ID NO: 22). The polynucleotide (SEQ ID NO: 86) encoding MaCar(m) was obtained by optimizing the base sequence for expression in E. coli and using the artificial gene synthesis service of Eurofins Genomics. The polynucleotide (SEQ ID NO: 89) encoding Nocardia iowensis Npt (SEQ ID NO: 24) was obtained by cloning the genomic DNA of Nocardia iowensis JCM18299 strain (provided by RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National Bioresource Project).

[0211] A plasmid for gene insertion was designed so that the macar(m) and npt genes were inserted as an expression cassette into the ldhA gene region. An expression cassette was inserted between the 5' and 3' homologous regions of the ldhA gene coding region contained in the previously constructed plasmid for disrupting the ldhA gene, in which the promoter region (SEQ ID NO: 139), macar(m) gene coding region, linker sequence (SEQ ID NO: 140), npt gene coding region, and terminator region (SEQ ID NO: 141) were arranged in this order.

[0212] <Construction of modified E. coli strains> Using the electroporation method (see Genetic Engineering Experiment Notes, by Takaaki Tamura, published by Yodosha), a plasmid for disrupting or inserting a desired gene was transformed into E. coli BL21(DE3) strain, and then the resulting mixture was spread 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, incubated overnight at 30°C to obtain a single colony, thus obtaining a transformant. A loopful of this 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 spread onto LB agar medium containing 100 mg / L kanamycin sulfate, incubated overnight at 42°C, and obtained a colony. A loopful of the resulting colony was inoculated into 1 mL of LB liquid medium, and cultured with shaking at 30°C. The obtained culture solution was spread on LB agar medium containing 20% ​​sucrose and incubated for 2 days at 30° C. For the obtained colonies, it was confirmed by colony direct PCR using the primer set shown in Table A-9 that the desired gene was disrupted or inserted.

[0213] The above procedure was repeated to construct the E. coli strain "No. 060" containing multiple gene disruptions and gene insertions, as shown in Table A-10. In the table, Δ indicates that the enzyme gene is deleted, and "gene name A::gene name B" indicates that the gene A region has been replaced with a region containing gene B.

[0214] [Table A-9]

[0215] [Table A-10]

[0216] <Construction of plasmid expressing hexamethylenediamine synthesis pathway enzyme genes> PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio) or KOD FX Neo (product name, manufactured by Toyobo) was used for amplifying PCR fragments, and Escherichia coli JM109 strain was used for preparing plasmids.

[0217] For the YgjG (SEQ ID NO: 27) of Eshcherichia coli, a mutant YgjG(m) (SEQ ID NO: 158) in which a part of the amino acid sequence was replaced was used. The polynucleotide encoding YgjG(m) was PCRed using oligonucleotides of SEQ ID NO: 160 and 161 as primers to obtain a PCR product containing the coding region of the ygjG(m) gene (SEQ ID NO: 159). Next, this PCR product was inserted between the restriction enzyme NcoI and HindIII cleavage sites of pACYCDuet-1 (product name, manufactured by Merck) using In-Fusion HD cloning kit (product name, manufactured by Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "ygjG(m)-pACYCDuet" was obtained as a MaCar(m) expression plasmid.

[0218] <Production test of hexamethylenediamine by recombinant E. coli culture> By electroporation, E. coli modified strain No. 060 was transformed with "paaF (L3) -ter (mtm)-pETDuet" and "ygjG (m)-pACYCDuet", and cultured at 37 ° C for one day on LB agar medium containing 50 mg / L sodium ampicillin and 30 mg / L chloramphenicol to form colonies, and transformant HMD1 was obtained. One platinum loop of colonies of transformant HMD1 was inoculated into 2 mL of LB liquid medium (14 mL round bottom tube) containing 50 mg / L sodium ampicillin and 30 mg / L chloramphenicol, and cultured with shaking at 37 ° C for 3 to 5 hours to obtain a preculture solution. 0.5 mL of preculture solution was added to synthetic medium (table below) containing 50 mg / L sodium carbenicillin, 30 mg / L chloramphenicol, and 0.02 mM IPTG in a 250 mL jar culture device (model name Bio Jr. 8, Biot Co., Ltd.), and main culture was performed. The culture conditions were as follows: culture temperature 37°C; culture pH 7.0; pH adjustment 10% (w / v) ammonia water; stirring 750 rpm; aeration 1 vvm. Glucose was added to a final concentration of 35 g / L 22 hours after inoculation of the preculture solution, and culture was continued for 41 hours.

[0219] [Table A-11]

[0220] After the cultivation was completed, the culture medium was centrifuged to separate the cells and the supernatant, and the hexamethylenediamine concentration in the supernatant was analyzed using an ion chromatograph under the following conditions.

[0221] Ion chromatographic analysis conditions Equipment: ICS-3000 (Dionex) Detector: Electrical conductivity detector Column: IonPac CG19 (2 x 50 mm) / CS19 (2 x 250 mm) (Thermo Scientific) Oven temperature: 30℃ Mobile phase: 8 mM methanesulfonic acid aqueous solution (A), 70 mM methanesulfonic acid aqueous solution (B) Gradient conditions: (A: 100%, B: 0%) - (10 min) - (A: 0%, B: 100%) - (hold for 1 min) Flow rate: 0.28 mL / min Injection volume: 20 μL

[0222] The results are shown in Table A-12. The transformant HMD1 is a transformant into which a carboxylic acid reductase and an aminotransferase gene have been introduced in addition to the enzyme gene capable of proceeding with the reactions of Steps A, B, C, and D in FIG. 1. Hexamethylenediamine was produced in the culture of the transformant HMD1 (Example 11).

[0223]

Table A-12

[0224] <2> Invention B Hereinafter, Invention B will be described based on examples, but Invention B is not limited to these examples.

[0225] <Isolation and identification of Burkholderia sp. strain LEBP-3> The Burkholderia sp. strain LEBP-3 was isolated from activated sludge in Japan using a medium with adipic acid as the sole carbon source and energy source. The Burkholderia sp. strain LEBP-3 was applied for deposit on December 4, 2020 (original deposit date) at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (NPMD) (address: Room 122, 2-5-8 Kazusa Kamashidzu, Kisarazu City, Chiba Prefecture), and has been internationally deposited as "Deposit Number: NITE BP-03334". The taxonomic characteristics of the Burkholderia sp. LEBP-3 strain are as follows.

[0226] <Mycological properties of the LEBP-3 strain> Culture temperature: 37 °C Cell morphology: Bacillus (0.7 x 1.7 - 3.0 μm), Gram stain: -, Presence of spores: -, Motility: +, Colony morphology on LB agar medium after 48 h of culture: diameter 1.9 - 2.5 mm, light yellow, circular, lenticular, entire, smooth, opaque, butter-like, Growth at 30°C: +, Growth at 45°C: +, Catalase reaction: +, Oxidase reaction: +, Acid / gas production from glucose: + / -, O / F test (oxidation / fermentation): + / -, Nitrate reducibility: -, Indole production: -, Glucose acidification: -, Urease: absent, Esculin hydrolysis: +, Glucose, L-arabinose, D-mannose, N-acetyl-D-glucosamine, gluconic acid, n-capric acid, adipic acid, malic acid, citric acid and phenyl acetate: utilizable, Maltose: non-utilizable.

[0227] <Phylogenetic analysis based on the 16S rRNA gene sequence homology of the LEBP-3 strain> Identification was performed by analyzing the nucleotide sequence of the 16S rDNA gene. The nucleotide sequence of the 16S rDNA gene was amplified by PCR and subjected to sequence analysis. The databases for BLAST homology search were DB-BA11.0 (Technosuga Lab) and the International Nucleotide Sequence Database. As a result, the LEBP-3 strain was included in the cluster composed of the genus Burkholderia, but showed a different molecular phylogenetic position from any known species, and thus the species name could not be identified.

[0228] <Acquisition of enzyme genes by PCR amplification and homology evaluation> A draft analysis of the whole genome of Burkholderia sp. LEBP-3 strain was performed. The LEBP-3 strain was cultured at 37°C with shaking in 2 mL of LB medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L). After the culture was completed, the cells were collected from the culture medium and genomic DNA was extracted using Nucleo Spin Tissue (product name, manufactured by MACHEREY-NAGEL). A draft analysis and annotation of the whole genome of Burkholderia sp. LEBP-3 strain were performed, and 7679 open reading frames (ORFs) were identified. Based on this information, we identified paaJ (L3) as a succinyl-CoA:acetyl-CoA acyltransferase, paaH (L3) as a 3-hydroxyadipyl-CoA dehydrogenase, paaF (L3) as a 3-hydroxyadipyl-CoA dehydratase, and mmgC (L3) as a 2,3-dehydroadipyl-CoA reductase.

[0229] The results of a BLAST search using the PaaJ(L3) sequence as a query are shown in Table B-1, the results of a BLAST search using the PaaH(L3) sequence as a query are shown in Table B-2, the results of a BLAST search using the PaaF(L3) sequence as a query are shown in Table B-3, and the results of a BLAST search using the MmgC(L3) sequence as a query are shown in Table B-4. The database used for the sequence search was "Refseq_Protein."

[0230] [Table B-1]

[0231] [Table B-2]

[0232] [Table B-3]

[0233] [Table B-4]

[0234] From the above results, the enzymes contained in Burkholderia sp. LEBP-3 strain were identified as novel sequences. The paaJ (L3) gene can be amplified by PCR using the nucleotides shown in SEQ ID NO: 164 and SEQ ID NO: 165 as primers. The paaH (L3) gene can be amplified by PCR using the nucleotides shown in SEQ ID NO: 166 and SEQ ID NO: 167 as primers. The paaF (L3) gene can be amplified by PCR using the nucleotides shown in SEQ ID NO: 168 and SEQ ID NO: 169 as primers. The mmgC (L3) gene can be amplified by PCR using the nucleotides shown in SEQ ID NO: 170 and SEQ ID NO: 171 as primers. The primers used in PCR, the enzymes, and the base sequences encoding the enzymes are shown in Figures 17 to 20.

[0235] An example of PCR amplification conditions is shown below. The enzyme used was PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio). 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 were performed. Oligonucleotides used as primers were added at 1 μM each. The volume of the solution used was 25 μL.

[0236] <Construction of recombinant E. coli expressing the target enzyme gene> PrimeSTAR Max DNA Polymerase (product name, Takara Bio) was used to amplify PCR fragments, and E. coli JM109 strain was used to prepare plasmids. GeneArt GeneOptinizer (software name, Thermo Fisher Scientific) or the artificial gene synthesis service of Eurofins Genomics was used to optimize the codons of the base sequence.

[0237] A polynucleotide encoding PaaJ(Ec) of Eshcherichia coli was cloned from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using oligonucleotides of SEQ ID NOs: 188 and 189 (FIG. 20A) as primers to obtain a PCR amplification product containing the coding region of the paaJ(Ec) gene (SEQ ID NO: 180) (FIG. 19A). Next, PCR was performed using pRSFDuet-1 (product name, manufactured by Merck) as a template and the nucleotides shown in SEQ ID NOs: 190 and 191 (FIG. 20A) as primers to obtain a pRSFDuet-1 fragment. The DNA fragment containing the gene coding region and the pRSFDuet-1 fragment were ligated using an In-Fusion HD cloning kit (product name, manufactured by Clontech). The product was transformed into E. coli JM109 strain, and a plasmid was extracted from the obtained transformant. "prm203" was obtained as a PaaJ(Ec) expression plasmid.

[0238] A polynucleotide encoding PaaH(Ec) of Eshcherichia coli was cloned from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using the nucleotides shown in SEQ ID NOs: 192 and 193 (FIG. 20A) as primers to obtain a PCR amplification product containing the coding region of the paaH(Ec) gene (SEQ ID NO: 181) (FIG. 19A). Next, PCR was performed using prm203 as a template and the nucleotides shown in SEQ ID NOs: 194 and 195 (FIG. 20A) as primers to obtain a prm203 fragment. The DNA fragments containing the coding regions of each gene and the prm203 fragment were ligated using In-Fusion HD cloning kit (product name, manufactured by Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "prm69" was obtained as a PaaJ(Ec)-PaaH(Ec) expression plasmid.

[0239] A polynucleotide encoding PaaF (Ec) of Eshcherichia coli was cloned from the genomic DNA of Eshcherichia coli W3110 strain (NBRC12713). PCR was performed using the nucleotides shown in SEQ ID NOs: 196 and 197 (FIG. 20A) as primers to obtain a PCR amplification product containing the coding region of the paaF (Ec) gene (SEQ ID NO: 182) (FIG. 19A). A 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. PCR was performed using the nucleotides shown in SEQ ID NOs: 198 and 199 (FIG. 20A) as primers to obtain a PCR amplification product containing the coding region of the paaF (L3) gene. PCR was performed using pETDuet-1 (product name, manufactured by Merck) as a template and the nucleotides shown in SEQ ID NOs: 204 and 205 (FIG. 20A) as primers to obtain a pETDuet-1 fragment. DNA fragments containing the coding regions of each gene were ligated to the pETDuet-1 fragment using the In-Fusion HD cloning kit (product name, manufactured by Clontech). The resulting transformants were transformed with E. coli JM109 strain, and plasmids were extracted from the resulting transformants. "prm74" was obtained as the PaaF(Ec) expression plasmid, and "prm194" was obtained as the PaaF(L3) expression plasmid.

[0240] Polynucleotides encoding dcaA(Ab) of Acinetobacter baylyi, Tfu1647 of Thermobifida fusca, and MmgC(L3) of L3 strain were obtained by optimizing the base sequence for expression in E. coli using the artificial gene synthesis service of Eurofins Genomics. PCR was performed using the nucleotides shown in SEQ ID NOs: 206 and 207 (FIG. 20A) as primers for the dcaA(Ab) gene (SEQ ID NO: 185) (FIG. 19B), the nucleotides shown in SEQ ID NOs: 208 and 209 (FIG. 20A) as primers for the Tfu1647 gene of Thermobifida fusca (SEQ ID NO: 186) (FIG. 19B), and the nucleotides shown in SEQ ID NOs: 210 and 211 (FIG. 20A) as primers for the mmgC5(L3) gene, to obtain PCR amplification products containing the coding regions of the dcaA(Ab), Tfu1647, and mmgC5(L3) genes, respectively. PCR was performed using prm74 and prm194 as templates and the nucleotides shown in SEQ ID NOs: 212 and 213 (FIG. 20A) as primers to obtain prm74 fragment and prm194 fragment, respectively. DNA fragments containing the coding regions of each gene were ligated to the prm74 fragment and prm194 fragment using In-Fusion HD cloning kit (product name, manufactured by Clontech). The resulting transformants were transformed with E. coli JM109 strain, and plasmids were extracted from the transformants. "prm127" was obtained as a PaaF(Ec)-dcaA(Ab) expression plasmid, "prm234" was obtained as a PaaF(Ec)-mmgC(L3) expression plasmid, and "prm166" was obtained as a PaaF(L3)-mmgC(L3) expression plasmid.

[0241] The polynucleotide encoding Acinetobacter baylyi tesB (Ab) was obtained by PCR from the chromosomal DNA of Acinetobacter baylyi using the nucleotides shown in SEQ ID NOs: 214 and 215 (FIG. 20A) as primers, and PCR amplification products containing the coding region of the tesB (Ab) gene were obtained. PCR was performed using pCDFDuet-1 as a template and the nucleotides shown in SEQ ID NOs: 216 and 217 (FIG. 20A) as primers to obtain a pCDFDuet-1 fragment. Each DNA fragment containing the coding region of the tesB (Ab) gene and the pCDFDuet-1 fragment were ligated using In-Fusion HD cloning kit (product name, manufactured by Clontech). The E. coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "prm92" was obtained as a TesB (Ab) expression plasmid.

[0242] A gene disruption strain of E. coli BL21(DE3) strain was prepared as follows. The ldh gene encoding lactate dehydrogenase, the atoB gene encoding acetyl-CoA acetyltransferase, and the sucD gene encoding succinyl-CoA synthetase α subunit were disrupted by homologous recombination using pHAK1 (Deposited on March 18, 2019 at the National Institute of Technology and Evaluation, National Patent Microorganism Depository Center (NPMD) (Address: Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) under the accession number NITE P-02919). pHAK1 contains a temperature-sensitive mutant repA gene, a kanamycin resistance gene, and the levansucrase gene SacB derived from Bacillus subtilis. The levansucrase gene acts lethally on the host microorganism in the presence of sucrose. PCR fragments were amplified using PrimeSTAR Max DNA Polymerase (product name, Takara Bio), and plasmids were prepared using Escherichia coli HST08 strain.

[0243] Using the genomic DNA of the E. coli BL21(DE3) strain as a template, PCR amplification products containing the upstream region, coding region, and downstream region of the disruption target gene were obtained. PCR was performed using the nucleotides shown in SEQ ID NOs: 218 and 219 (FIG. 20B) as primers for the ldh gene peripheral region, PCR was performed using the nucleotides shown in SEQ ID NOs: 220 and 221 (FIG. 20B) as primers for the atoB gene peripheral region, and PCR was performed using the nucleotides shown in SEQ ID NOs: 222 and 223 (FIG. 20B) as primers for the sucD gene peripheral region, to obtain the respective fragments.

[0244] Next, this PCR amplification product was inserted into the pHAK1 plasmid fragment amplified using the primers shown in SEQ ID NOs: 224 and 225 (Figure 20B) using an In-Fusion HD cloning kit (product name, manufactured by Clontech), and circularized.

[0245] Using the pHAK1 plasmid obtained, into which DNA fragments of the upstream region, coding region, and downstream region of the disruption target gene were inserted, as a template, PCR was performed on the region surrounding the ldh gene using the nucleotides shown in SEQ ID NOs: 226 and 227 (Figure 20B) as primers, PCR was performed on the region surrounding the atoB gene using the nucleotides shown in SEQ ID NOs: 228 and 229 (Figure 20B) as primers, and PCR was performed on the region surrounding the sucD gene using the nucleotides shown in SEQ ID NOs: 230 and 231 as primers (Figure 20B), to obtain plasmid fragments in which part or all of the coding region of the disruption target gene had been removed.

[0246] The resulting plasmid fragment was terminally phosphorylated and circularized by self-ligation to obtain a plasmid for gene disruption.

[0247] Using the calcium chloride method (see Genetic Engineering Experiment Notes, by Takaaki Tamura, published by Yodosha), a plasmid for disrupting the desired gene was transformed into E. coli BL21(DE3) strain, and then the resulting transformant was plated on LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 100 mg / L kanamycin sulfate, and cultured overnight at 30°C to obtain a single colony. A loopful of the transformant was inoculated into 1 mL of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L) containing 100 mg / L kanamycin sulfate, and cultured with shaking at 30°C. The resulting culture was plated on LB agar medium containing 100 mg / L kanamycin sulfate, and cultured overnight at 42°C. The resulting colony has the plasmid inserted into its genome by single crossover. A loopful of colonies 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 10% sucrose and cultured overnight. The resulting colonies were confirmed by colony direct PCR to confirm that the desired genes had been disrupted. First, an ldh gene disruption strain was constructed, an atoB gene disruption strain was constructed based on this ldh gene disruption strain, and a sucD gene disruption strain was constructed based on the ldh and atoB double disruption strain. The triple disruption strain of ldh, atoB, and sucD genes thus constructed was designated the ASL000 strain.

[0248] The constructed plasmid was transformed into the ASL000 strain, spread onto LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) containing 50 mg / L sodium ampicillin, 30 mg / L kanamycin sulfate, and 50 mg / L streptomycin sulfate, and incubated at 37°C for 24 hours to obtain single colonies of transformants. The obtained transformants are shown in Table B-5.

[0249] [Table B-5]

[0250] <Adipic acid production test using recombinant E. coli expressing the obtained enzyme gene> A loopful of the strains shown in Table B-5 was inoculated into 2 mL (14 mL round bottom tube) of LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L) containing 50 mg / L of ampicillin sodium, 30 mg / L of kanamycin sulfate, and 50 mg / L of streptomycin sulfate, and cultured with shaking at 37°C for 24 hours to obtain a preculture solution. 10 μL of the preculture solution obtained was inoculated into 1 mL of MM medium (96-well deep well plate) containing 50 mg / L of ampicillin sodium, 30 mg / L of kanamycin sulfate, and 50 mg / L of streptomycin sulfate, and cultured with shaking at 37°C for 48 hours. The composition of MM medium is shown in Table B-6.

[0251] [Table B-6]

[0252] After the culture was completed, the concentrations of adipic acid and adipic acid analogues in the culture solution were analyzed. The analysis was performed by GC / MS analysis using trimethylsilyl derivatization. 360 μL of an extract mixed with water:methanol:chloroform = 5:2:2 (v / v / v) was added to 40 μL of the culture solution centrifuged supernatant (added with disodium sebacate as an internal standard to a final concentration of 10 mM) and mixed well. After centrifugation (16,000 × g, 5 minutes), 100 μL of the supernatant was collected in another microtube and centrifuged and concentrated for 1 hour using a centrifugal evaporator. 100 μL of methoxyamine hydrochloride 20 mg / mL pyridine solution was added to the obtained dried product and shaken at 30 ° C for 90 minutes. 50 μL of N-methyl-N-trimethylsilyl trifluoroacetamide was added and shaken at 37 ° C for 30 minutes. The reaction solution was used as a GC / MS measurement sample and analyzed under the following conditions.

[0253] 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℃, hold for 2 minutes → increase temperature at 15℃ / minute → 325℃, hold for 13 minutes Ionization method: Electron Ionization (EI) Ionization energy: 70 eV Ion source temperature: 230℃ Scan range: m / z=50~500 Injection volume: 1μL

[0254] The results are shown in Table B-7. In the table, "3OA" stands for 3-oxoadipic acid, "LEV" stands for levulinic acid, "3HA" stands for 3-hydroxyadipic acid, "23DA" stands for 2,3-dehydroadipic acid, and "ADA" stands for adipic acid.

[0255] [Table B-7]

[0256] In the inventive strains expressing both PaaF (L3) and mmgC (L3) derived from the L3 strain, i.e., the ADA500 strain and the ADA496 strain, 3-oxoadipate (3-OA), levulinic acid (LEV), 3-hydroxyadipate (3HA), 2,3-dehydroadipate (23DA), and adipic acid (ADA) were produced in greater amounts than in the control strain, with the total amounts produced being 355 mg / L and 613 mg / L, respectively. These results indicate that the enzymes encoded by these genes have high activity in promoting the adipic acid pathway reactions, and improve the production of adipic acid and adipic acid derivatives. [Industrial Applicability]

[0257] The present invention can provide, for example, an efficient production process for C6 compounds, and is expected to be applicable to industrial-scale production.

[0258] The present invention can also be suitably used in the fermentative production of adipic acids directly from biomass raw materials.

Claims

1. A recombinant microorganism comprising an exogenous gene encoding a protein having an enzymatic activity for reducing 2,3-dehydroadipyl-CoA to adipyl-CoA, The exogenous gene is (i) a DNA encoding a protein having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (ii) DNA encoding a protein consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16; (iii) DNA consisting of a polynucleotide sequence having 90% or more sequence identity to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (iv) DNA encoding a protein having an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence of the protein encoded by the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65; (v) a DNA that hybridizes under stringent conditions of 0.1xSSC, 0.1% SDS, and 60°C with a DNA consisting of a polynucleotide sequence complementary to the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64, or 65; or (vi) DNA consisting of a degenerate isomer of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65. and The recombinant microorganism has a production pathway leading to a C6 compound via adipyl-CoA, The C6 compound is at least one selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, and 6-hydroxyhexanoic acid; The recombinant microorganism is Escherichia coli; Further comprising an exogenous gene encoding 3-hydroxyadipyl CoA dehydratase; Recombinant microorganisms.

2. an exogenous gene encoding a protein having an enzyme activity for reducing 2,3-dehydroadipyl-CoA to adipyl-CoA, (xi) a DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15 or 16, or (xiii) DNA consisting of the polynucleotide sequence shown in SEQ ID NO: 57, 58, 59, 60, 61, 62, 63, 64 or 65 The recombinant microorganism of claim 1 .

3. The recombinant microorganism according to claim 1 or 2, wherein the exogenous gene encoding a protein having an enzymatic activity of reducing 2,3-dehydroadipyl-CoA to adipyl-CoA is derived from at least one selected from Candida auris, Kluyveromyces marxianus, Pichia kudriavzevii, Thermothelomyces thermophilus, Thermothielavioides terrestris, Chaetomium thermophilum, Podospora anserina, Purpureocillium lilacinum and Pyrenophora teres.

4. The recombinant microorganism has a production pathway leading to adipic acid via adipyl-CoA. The recombinant microorganism according to any one of claims 1 to 3.

5. a gene encoding 3-oxoadipyl CoA thiolase, a gene encoding 3-hydroxyadipyl CoA dehydrogenase, - a gene encoding carboxylic acid reductase, a gene encoding alcohol dehydrogenase, and - Genes that code for transaminases (aminotransferases) The recombinant microorganism according to any one of claims 1 to 4, further comprising at least one selected from the group consisting of:

6. A method for producing a C6 compound, comprising a culturing step of culturing the recombinant microorganism according to any one of claims 1 to 5, The method for producing the C6 compound is at least one selected from the group consisting of adipic acid, hexamethylenediamine, 1,6-hexanediol, 6-aminohexanoic acid, 6-amino-1-hexanol, and 6-hydroxyhexanoic acid.

7. an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase; At least one of the 3-hydroxyadipyl-CoA dehydratase and the 2,3-dehydroadipyl-CoA reductase is derived from Burkholderia sp. strain LEBP-3 (accession number: NITE BP-03334); Escherichia coli, Recombinant microorganisms.

8. an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase; The 3-hydroxyadipyl-CoA dehydratase is produced by using chromosomal DNA of Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334) as a template, (1a) a nucleotide consisting of the base sequence shown in SEQ ID NO: 168 and a nucleotide consisting of the base sequence shown in SEQ ID NO: 169, or (1b) a nucleotide comprising the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising the base sequence shown in SEQ ID NO: 169 is encoded by DNA having a sequence identity of 90% or more to the PCR amplification product using as a primer, The DNA has 783 or 784 bp, Escherichia coli, Recombinant microorganisms.

9. an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase; The 2,3-dehydroadipyl-CoA reductase is produced by using chromosomal DNA of Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334) as a template, (2a) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (2b) a nucleotide comprising the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising the base sequence shown in SEQ ID NO: 171 is encoded by DNA having a sequence identity of 90% or more to the PCR amplification product using as a primer, The DNA has 1155 or 1156 bp, Escherichia coli, Recombinant microorganisms.

10. an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase; The 3-hydroxyadipyl-CoA dehydratase is produced by using chromosomal DNA of Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334) as a template, (1a) a nucleotide consisting of the base sequence shown in SEQ ID NO: 168 and a nucleotide consisting of the base sequence shown in SEQ ID NO: 169, or (1b) a nucleotide comprising the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising the base sequence shown in SEQ ID NO: 169 is encoded by DNA that is a PCR amplified using as a primer The DNA has 783 or 784 bp, Escherichia coli, Recombinant microorganisms.

11. an exogenous gene encoding 3-hydroxyadipyl-CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl-CoA reductase; The 2,3-dehydroadipyl-CoA reductase is produced by using chromosomal DNA of Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334) as a template, (2a) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (2b) a nucleotide comprising the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising the base sequence shown in SEQ ID NO: 171 is encoded by DNA that is a PCR amplified using as a primer The DNA has 1155 or 1156 bp, Escherichia coli, Recombinant microorganisms.

12. The recombinant microorganism according to any one of claims 7 to 11, having an adipic acid production pathway.

13. A recombinant microorganism described in any one of claims 7 to 11, which has a production pathway leading to adipic acid via adipyl-CoA, as well as a production pathway leading to hexamethylenediamine via adipyl-CoA.

14. A recombinant microorganism described in any one of claims 7 to 11, which has a production pathway leading to adipic acid via adipyl-CoA, as well as a production pathway leading to 1,6-hexanediol via adipyl-CoA.

15. A recombinant microorganism described in any one of claims 7 to 11, which has a production pathway leading to 6-amino-1-hexanol via adipyl-CoA in addition to a production pathway leading to adipic acid via adipyl-CoA.

16. A method for producing a target compound, comprising a culturing step of culturing the recombinant microorganism according to any one of claims 7 to 11, the target compound is selected from the group consisting of adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol, and 6-amino-1-hexanol; the adipic acid derivative is selected from the group consisting of oxoadipic acid, levulinic acid, 3-hydroxyadipic acid and 2,3-dehydroadipic acid; The recombinant microorganism has a production pathway leading to the selected compound of interest via adipyl-CoA. Manufacturing method.

17. A method for producing adipic acid, comprising a culturing step of culturing the recombinant microorganism according to claim 12.

18. A method for producing hexamethylenediamine, comprising a culturing step of culturing the recombinant microorganism according to claim 13.

19. A method for producing 1,6-hexanediol, comprising a culturing step of culturing the recombinant microorganism according to claim 14.

20. A method for producing 6-amino-1-hexanol, comprising a culturing step of culturing the recombinant microorganism according to claim 15.

21. (A-1) Using chromosomal DNA of Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334) as a template, (a1) a nucleotide consisting of the base sequence shown in SEQ ID NO: 168 and a nucleotide consisting of the base sequence shown in SEQ ID NO: 169, or (a2) a nucleotide comprising the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising the base sequence shown in SEQ ID NO: 169 A recombinant polypeptide encoded by DNA having a sequence identity of 90% or more to a PCR amplification product using the following primers: having 3-hydroxyadipyl CoA dehydratase activity; The DNA has 783 or 784 bp; (A-2) Using chromosomal DNA of Burkholderia sp. LEBP-3 strain (Accession Number: NITE BP-03334) as a template, (a1) a nucleotide consisting of the base sequence shown in SEQ ID NO: 168 and a nucleotide consisting of the base sequence shown in SEQ ID NO: 169, or (a2) a nucleotide comprising the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising the base sequence shown in SEQ ID NO: 169 A recombinant polypeptide having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by a DNA that is a PCR amplified product using the following primers: having 3-hydroxyadipyl CoA dehydratase activity; The DNA has 783 or 784 bp; or (A-3) Using chromosomal DNA of Burkholderia sp. LEBP-3 strain (Accession Number: NITE BP-03334) as a template, (a1) a nucleotide consisting of the base sequence shown in SEQ ID NO: 168 and a nucleotide consisting of the base sequence shown in SEQ ID NO: 169, or (a2) a nucleotide comprising the base sequence shown in SEQ ID NO: 168 and a nucleotide comprising the base sequence shown in SEQ ID NO: 169 A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using as a primer having 3-hydroxyadipyl CoA dehydratase activity; The DNA has 783 or 784 bp.

22. (B-1) Using chromosomal DNA of Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334) as a template, (b1) a nucleotide consisting of the base sequence shown in SEQ ID NO: 170 and a nucleotide consisting of the base sequence shown in SEQ ID NO: 171, or (b2) a nucleotide comprising the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising the base sequence shown in SEQ ID NO: 171 A recombinant polypeptide encoded by DNA having a sequence identity of 90% or more to a PCR amplification product using the following primers: having 2,3-dehydroadipyl CoA reductase activity; The DNA has a recombinant polypeptide of 1155 or 1156 bp; (B-2) Using chromosomal DNA of Burkholderia sp. LEBP-3 strain (Accession Number: NITE BP-03334) as a template, (b1) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (b2) a nucleotide comprising the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising the base sequence shown in SEQ ID NO: 171 A recombinant polypeptide having an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by a DNA that is a PCR amplified product using the following primers: having 2,3-dehydroadipyl CoA reductase activity; The DNA has a recombinant polypeptide of 1155 or 1156 bp; or (B-3) Using chromosomal DNA of Burkholderia sp. LEBP-3 strain (Accession Number: NITE BP-03334) as a template, (b1) a nucleotide having the base sequence shown in SEQ ID NO: 170 and a nucleotide having the base sequence shown in SEQ ID NO: 171, or (b2) a nucleotide comprising the base sequence shown in SEQ ID NO: 170 and a nucleotide comprising the base sequence shown in SEQ ID NO: 171 A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using as a primer having 2,3-dehydroadipyl CoA reductase activity; The DNA has 1155 or 1156 bp.

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