JP7038241B1 - Method for producing recombinant microorganism and adipic acid or its derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a recombinant microorganism capable of efficiently producing adipic acid or an adipic acid derivative, and a method for producing adipic acid or an adipic acid derivative. 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. [Selection diagram] Fig. 1

Description

NPMD NITE BP-03334 NPMD NITE BP-02919

The present invention relates to a recombinant microorganism and a method for producing adipic acid or a derivative thereof.

In recent years, there is concern about the depletion of fossil fuel-derived raw materials that have been conventionally used in the chemical manufacturing process, and it is considered to be one of the causes of global warming. Conversion to raw materials is desired.

Adipic acid (CAS No. 124-04-9) is a monomer compound used as a polyamide raw material typified by nylon-66, urethane, and a plasticizer raw material. The current general method for producing adipic acid is a chemical synthesis method, for example, cyclohexanol alone or a mixture of cyclohexanol and cyclohexanone (K / A oil) is oxidized with nitric acid.

As a method for producing adipic acid using a biomass-derived raw material, a biomass-derived carbon source is converted to cis, cis-muconic acid using a recombinant microorganism, and then the obtained cis, cis-muconic acid is hydrogenated. A method for producing adipic acid has been proposed (Patent Document 1). Further, a method for producing adipic acid from α-ketoglutaric acid using a genetically modified organism has also been proposed (Patent Document 2).

Further, a method for directly fermenting and producing adipic acid from a biomass-derived raw material has also been proposed (Patent Document 3). In this method, "succinyl-CoA: acetyl-CoA acyltransferase" that converts succinyl-CoA and acetyl-CoA to 3-oxoadipyl-CoA as non-naturally occurring microorganisms capable of producing adipic acid; 3-oxoadipyl. -"3-Hydroxyacyl-CoA dehydrogenase" that converts CoA to 3-hydroxyadipyl-CoA; "3-hydroxyadipyl-" that converts 3-hydroxyadipyl-CoA to 5-carboxy-2-pentenoyl-CoA "CoA dehydratase"; "5-carboxy-2-pentenoyl-CoA reductase" that converts 5-carboxy-2-pentenoyl-CoA to adipyl-CoA; and "phosphotransadipylase" that converts adipyl-CoA to adipate / Non-naturally occurring microorganisms containing "adipic acid kinase" are mentioned, but for each enzyme, only the genes encoding the enzymes that may proceed the reaction are shown, and by using these genes, biomass It has not been demonstrated that adipic acid can be produced in good yield from the derived carbon raw material.

International Publication No. 1995/007996 International Publication No. 2010/104391 Japanese Unexamined Patent Publication No. 2011-515111

An object of the present invention is to provide a recombinant microorganism capable of efficiently producing adipic acid or an adipic acid derivative, and a method for producing adipic acid or an adipic acid derivative.

The present inventors have conducted extensive research to solve the above problems, and as a result, Burkholderia sp., A microorganism newly isolated from nature, is a LEBP-3 strain (accession number: NITE BP). We have found that adipic acids can be efficiently produced by culturing a recombinant microorganism expressing a predetermined enzyme possessed by -03334), and have reached the present invention.

That is, the present invention is as follows:
[1] Recombinant microorganisms comprising at least one of an exogenous gene encoding 3-hydroxyadipyl CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl CoA reductase;
[2] At least one of the 3-hydroxyadipyl CoA dehydratase and 2,3-dehydroadipyl CoA reductase is derived from Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334). , The recombinant microorganism according to [1];
[3] The 3-hydroxyadipyl CoA dehydratase uses the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(1a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (1b) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. The sequence identity with the PCR amplification product using the above as a primer is 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. Encoded in DNA
The recombinant microorganism according to [1] or [2], wherein the DNA has 626 to 940 bp;
[4] The 2,3-dehydroadipyl CoA reductase uses the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(2a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (2b) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. The sequence identity with the PCR amplification product using the above as a primer is 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. Encoded in DNA
The recombinant microorganism according to any one of [1] to [3], wherein the DNA has 924 to 1386 bp;
[5] The 3-hydroxyadipyl CoA dehydratase uses the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(1a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (1b) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. Encoded in DNA, which is a PCR amplification product using
The recombinant microorganism according to [1] or [2], wherein the DNA has 783 or 784 bp;
[6] The 2,3-dehydroadipyl CoA reductase uses the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(2a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (2b) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. Encoded in DNA, which is a PCR amplification product using
The recombinant microorganism according to any one of [1] to [3], wherein the DNA has 1155 or 1156 bp;
[7] The recombinant microorganisms include Essericia, Corinebacterium, Bacillus, Acinetobacta, Burkhorderia, Pseudomonas, Clostridium, Saccharomyces, Sizosaccalomyces, Yarrowia, Candita, Pikia. The recombinant microorganism according to any one of [1] to [6], which belongs to a genus selected from the group consisting of the genus Aspergillus and the genus.
[8] The recombinant microorganism according to any one of [1] to [7], which has an adipic acid production pathway;
[9] The recombinant microorganism according to any one of [1] to [8], which has a hexamethylenediamine production pathway;
[10] The recombinant microorganism according to any one of [1] to [8], which has a 1,6-hexanediol production pathway;
[11] The recombinant microorganism according to any one of [1] to [8], which has a 6-amino-1-hexanol production pathway;
[12] A method for producing a target compound, which comprises a culture step of culturing the recombinant microorganism according to any one of [1] to [7].
The target compound is selected from the group consisting of adipic acid, adipic acid derivative, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol.
A production method in which the adipic acid derivative is selected from the group consisting of oxoadic acid, levulinic acid, 3-hydroxyadipic acid and 2,3-dehydroadipic acid;
[13] A method for producing adipic acid, which comprises a culture step of culturing the recombinant microorganism according to [8].
[14] A method for producing hexamethylenediamine, which comprises a culture step of culturing the recombinant microorganism according to [9].
[15] A method for producing 1,6-hexanediol, which comprises a culture step of culturing the recombinant microorganism according to [10];
[16] A method for producing 6-amino-1-hexanol, which comprises a culture step of culturing the recombinant microorganism according to [11].
[17] (A-1) Using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(A1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (a2) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. The sequence identity with the PCR amplification product using the above as a primer is 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. A recombinant polypeptide encoded by DNA
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having 626-940 bp;
(A-2) Using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template,
(A1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (a2) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. A recombinant polypeptide consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by DNA, which is a PCR amplification product using the above as a primer. And
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having 626-940 bp;
Or (A-3) using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(A1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (a2) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using the above as a primer.
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having 626-940 bp;
[18] (B-1) Using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(B1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (b2) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. The sequence identity with the PCR amplification product using the above as a primer is 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. A recombinant polypeptide encoded by DNA
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having 924 to 1386 bp;
(B-2) Using the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(B1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (b2) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. A recombinant polypeptide consisting of an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by DNA, which is a PCR amplification product using the above as a primer. And
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having 924 to 1386 bp;
Or (B-3) using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(B1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (b2) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. A recombinant polypeptide encoded by DNA consisting of a degenerate isomer of a PCR amplification product using the above as a primer.
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having 924 to 1386 bp.

The present invention provides a recombinant microorganism capable of efficiently producing adipic acid or an adipic acid derivative, and a method for producing adipic acid or an adipic acid derivative.

FIG. 1 shows an example of the production pathways of adipic acid, adipic acid derivative, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol possessed by the recombinant microorganism according to the present invention. FIG. 2 shows the gene to be amplified in PCR and the primers used. FIG. 3 shows the amino acid sequence of each enzyme. FIG. 4A shows the base sequence encoding each enzyme. FIG. 4B shows the base sequence encoding each enzyme. FIG. 5A shows the amplification target and the primers used in PCR. FIG. 5B shows the amplification target and the primers used in PCR. FIG. 6 shows the amino acid sequence of the enzyme. FIG. 7A shows the amino acid sequence of the enzyme. FIG. 7B shows the amino acid sequence of the enzyme. FIG. 7C shows the amino acid sequence of the enzyme. FIG. 7D shows the amino acid sequence of the enzyme. FIG. 7E shows the amino acid sequence of the enzyme. FIG. 8A shows the amino acid sequence of the enzyme. FIG. 8B shows the amino acid sequence of the enzyme. FIG. 8C shows the amino acid sequence of the enzyme. FIG. 8D shows the amino acid sequence of the enzyme. FIG. 9A shows the amino acid sequence of the enzyme. FIG. 9B shows the amino acid sequence of the enzyme. FIG. 9C shows the amino acid sequence of the enzyme. FIG. 9D shows the amino acid sequence of the enzyme. FIG. 9E shows the amino acid sequence of the enzyme. FIG. 10 shows the amino acid sequence of the enzyme.

Hereinafter, embodiments of the present invention will be described in detail. The present invention is not limited to the following embodiments, and can be variously modified and implemented within the scope of the gist thereof. In addition, unless otherwise specified, genetic manipulations such as DNA acquisition, vector preparation and transformation described herein are Molecular Cloning 4th Edition (Cold Spring Harbor Laboratory Press, 2012), Molecular Biology in Molecular. It can be carried out by a known method described in (Greene Publishing Associates and Willy-Interscience), Genetic Engineering Experiment Note (Takaaki Tamura, Yodosha), and the like. Unless otherwise specified in the present specification, nucleotide sequences are described from the 5'direction to the 3'direction. As used herein, the terms "polypeptide" and "protein" are used interchangeably.

In the present specification, the numerical range indicated by using "-" indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively. Within the numerical range described stepwise herein, the upper or lower limit of the numerical range at one stage may be optionally combined with the upper or lower limit of the numerical range at another stage.

The recombinant microorganism according to the present invention is genetically modified with respect to the host microorganism so that adipic acid (ADA) (CAS No. 124-04-9) or an adipic acid derivative can be efficiently produced. It is a microorganism that has been carried out.

As used herein, the "azipic 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, carbon may be substituted with a substituent such as a hydroxyl group, a carboxyl group, a ketone group or an amino group. The aliphatic hydrocarbon group of the "azipic acid derivative" preferably has 3 or 4 carbon atoms. The carboxyl group of the "azipic acid derivative" is preferably 1 or 2. Specific examples of the "adipic acid derivative" include 3-oxoadipic acid (3-Oxodipic acid; 3-OA) having an aliphatic hydrocarbon group having 4 carbon atoms and two carboxylic acids, and an aliphatic group having 3 carbon atoms. Levulic acid (LEV) having a hydrocarbon group and one carboxylic acid, 3-hydroxyadipic acid (3-Hydroxyadic acid; 3HA) having an aliphatic hydrocarbon group having 4 carbon atoms and two carboxylic acids. And 2,3-dehydroadipic acid (23DA) having an aliphatic hydrocarbon group having 4 carbon atoms and two carboxylic acids (see FIG. 1).

It will be understood by those skilled in the art that the adipic acid and the adipic acid derivative in the present specification can take a neutral or ionized form including any salt form, and that this form depends on pH. Thus, "azipic acid" and "azipic acid derivatives" include the form of free acid, as well as any salt and ionized forms. In the present specification, adipic acid and adipic acid derivatives may be collectively referred to as "adipic acids" or "adipic acid analogs".

It is also possible to synthesize 6-aminocaproic acid, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol from adipic acid produced by the recombinant microorganism according to the present invention. The synthesis method may be a chemical synthesis method or a biological synthesis method using an enzyme or a microorganism containing the enzyme. Further, as long as it is a microorganism containing a metabolic pathway for converting adipic acid into the above compound, metabolic conversion from a raw material using adipic acid as an intermediate in the cell is also possible.

The recombinant microorganism according to the present invention specifically comprises at least one of an exogenous gene encoding 3-hydroxyadipyl CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl CoA reductase. include. 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. As used herein, the term "genetically modified microorganism" is also simply referred to as "recombinant microorganism".

As used herein, the term "endogenous" or "endogenous" is used by the unmodified host microorganism referred to herein as the gene or protein encoded by it. The enzyme) is used to mean that the host microorganism has it, regardless of whether it is functionally expressed to the extent that it can promote a dominant biochemical reaction in the host cell. Be done.

As used herein, the terms "foreign" or "foreign" are used when the host microorganism prior to gene recombination does not substantially express the enzyme encoded by the gene to be introduced by the present invention, and different genes. Although the amino acid sequence of the enzyme is encoded by the above, it is used to mean that the gene or nucleic acid sequence based on the present invention is introduced into the host when the endogenous enzyme activity comparable to that of the gene is not expressed after the gene recombination. ..

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

At least one of the exogenous genes encoding 3-hydroxyadipyl CoA dehydratase and the exogenous genes encoding 2,3-dehydroadipyl CoA reductase are Burkholderia sp. LEBP-3 strains (contracted). Number: Derived from NITE BP-03334). Preferably, both the exogenous gene encoding 3-hydroxyadipyl CoA dehydratase and the exogenous gene encoding 2,3-dehydroadipyl CoA reductase are Burkholderia sp. LEBP-3 strains ( It is derived from the accession number: NITE BP-03334).

The recombinant microorganism of the present invention may be introduced with at least one of the foreign genes encoding the above-mentioned enzyme. The recombinant microorganism of the present invention preferably introduces at least one of the foreign genes encoding an enzyme that catalyzes each reaction stage of the adipic acid production pathway to express the foreign enzyme. Further, when the recombinant microorganism of the present invention expresses a sufficient amount of enzyme that catalyzes the reaction stage of the adipic acid production pathway without introducing a foreign gene, the reaction is carried out by the enzyme encoded by the endogenous gene. May progress. In the present invention, "having a production pathway" with respect to a compound means that the recombinant microorganism according to the present invention expresses an enzyme in a sufficient amount for each reaction stage of the production pathway of the compound to proceed. It means that the compound can be biosynthesized.

The recombinant microorganism according to the present invention preferably expresses at least one of 3-hydroxyadipyl CoA dehydratase and 2,3-dehydroadipyl CoA reductase. When the recombinant microorganism according to the present invention expresses at least one of these enzymes, the reaction stage of the adipic acid production pathway proceeds, and the target compound can be produced.

More preferably, the recombinant microorganism according to the invention expresses both 3-hydroxyadipyl CoA dehydratase and 2,3-dehydroadipyl CoA reductase. When the recombinant microorganism according to the present invention expresses these enzymes, each of the reaction stages of the adipic acid production pathway proceeds, and the target compound can be produced.

Even more preferably, the recombinant microorganisms according to the invention express oxidoreductase and / or β-ketothiolase in addition to 3-hydroxyadipyl CoA dehydratase and 2,3-dehydroadipyl CoA reductase. When the recombinant microorganism according to the present invention expresses these enzymes, each of the reaction stages of the adipic acid production pathway proceeds, and the target compound can be efficiently produced (see FIG. 1).

Hereinafter, each of the enzymes encoded by the genes contained in the recombinant microorganism according to the present invention will be described in detail.

<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 FIG. 1).

In one embodiment, 3-hydroxyadipyl CoA dehydratase has 65 sequence identity with a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template and a predetermined nucleotide as a primer. % Or higher, 70% or higher, 75% or higher, 80% or higher, 85% or higher, 88% or higher, 90% or higher, 93% or higher, 95% or higher, 97% or higher, 98% or higher or 99% or higher. Coded. 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.

In a preferred embodiment, 3-hydroxyadipyl CoA dehydratase encodes DNA, which is a PCR amplification product using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template and a predetermined nucleotide as a primer. Will be done. 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.

In a further embodiment, 3-hydroxyadipyl CoA dehydratase is selected from:
(A-1) Sequence identity is 65% or more, 70% or more, 75 with a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain as a template and a predetermined nucleotide as a primer. % 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 in a recombinant polypeptide encoded by DNA. There,
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having the above-mentioned base pair number (bp);
(A-2) For the amino acid sequence of a polypeptide encoded by DNA, which is a PCR amplification product using the chromosomal DNA of Burkholderia sp. Strain as a template and a predetermined nucleotide as a primer. For example, a recombinant consisting of an amino acid sequence in which 1 to 10, preferably 1 to 7, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are deleted, substituted, inserted and / or added. It ’s a polypeptide,
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having the above-mentioned base pair number (bp);
And (A-3) A set encoded by DNA of degenerate isomers of a PCR amplification product using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template and a predetermined nucleotide as a primer. It ’s a replacement polypeptide,
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having the above-mentioned base pair number (bp).

In a further preferred embodiment, the 3-hydroxyadipyl CoA dehydratase is encoded by a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template and a predetermined nucleotide as a primer. It is a recombinant polypeptide and the number of base pairs of the amplified product is 783 or 784 bp.

With respect to 3-hydroxyadipyl CoA dehydratase, the primers (forward and reverse) used for PCR include:
(1a) Nucleotides consisting of the base sequence shown in SEQ ID NO: 5 and nucleotides consisting of the base sequence shown in SEQ ID NO: 6.
(1b) A nucleotide containing the base sequence shown in SEQ ID NO: 5 and a nucleotide containing the base sequence shown in SEQ ID NO: 6.
(1c) A nucleotide consisting of a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 5 and a nucleotide consisting of a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 6.
(1d) A nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 5 and a nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 6.

The "nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6" in (1b) are "base sequence shown in SEQ ID NO: 5" and "base sequence shown in SEQ ID NO: 6," respectively. In addition, 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 have additional nucleotide lengths.

The "nucleotide containing the base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 6" in (1d) are "SEQ ID NO: 5", respectively. In addition to the "codon-optimized base sequence of the base sequence shown in" and "codon-optimized base sequence of the base sequence shown in SEQ ID NO: 6," a sequence homologous to the vector terminal is included, for example, 1 to 20 bp, preferably 1. It has an additional nucleotide length of ~ 15 bp, more preferably 5 ~ 15 bp, even more preferably 10 ~ 15 bp. As an example of the above (1d), "a nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 5 and a nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 6"
(1d') Nucleotides consisting of the base sequence shown in SEQ ID NO: 35 and nucleotides consisting of the base sequence shown in SEQ ID NO: 36 can be mentioned (FIG. 5A).

For 3-hydroxyadipyl CoA dehydratase, the preferred primer is
(1a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5, nucleotide consisting of the base sequence shown in SEQ ID NO: 6, (1b) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and nucleotide containing the base sequence shown in SEQ ID NO: 6. Is.

The DNA which is a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain using the above nucleotide as a primer preferably has 783 or 784 bp. Here, as the DNA polymerase used in PCR, those known in the art are used, for example, Taq DNA polymerase, hot-start-adjusted DNA polymerase, and proofreading having 3'-5'exonuclease activity apart from the polymerase activity. Examples include DNA polymerase.

<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 FIG. 1).

In one embodiment, 2,3-dehydroadipyl CoA reductase is sequence identity with a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template and a predetermined nucleotide as a primer. Is 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. Encoded in DNA. The DNA is, 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 more preferably 1143 to 1167 bp, still more preferably 1145 to 1165 bp, most preferably. It has 1150 to 1160 bp.

In a preferred embodiment, 2,3-dehydroadipyl CoA reductase is a PCR amplification product using the chromosomal DNA of Burkholderia sp. Strain LEBP-3 as a template and a predetermined nucleotide as a primer. Coded to. The DNA is, 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 more preferably 1143 to 1167 bp, still more preferably 1145 to 1165 bp, most preferably. It has 1150 to 1160 bp.

In a further embodiment, 2,3-dehydroadipyl CoA reductase is selected from:
(B-1) Sequence identity is 65% or more, 70% or more, 75 with a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain as a template and a predetermined nucleotide as a primer. % 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 in a recombinant polypeptide encoded by DNA. There,
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having the above-mentioned base pair number (bp);
(B-2) For the amino acid sequence of a polypeptide encoded by DNA, which is a PCR amplification product using the chromosomal DNA of Burkholderia sp. Strain as a template and a predetermined nucleotide as a primer. For example, a set consisting of an amino acid sequence in which 1 to 10, preferably 1 to 7, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are deleted, substituted, inserted and / or added. It ’s a replacement polypeptide,
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having the above-mentioned base pair number (bp);
And (B-3) A set encoded by DNA of degenerate isomers of a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain as a template and a predetermined nucleotide as a primer. It ’s a replacement polypeptide,
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having the above-mentioned base pair number (bp).

In a further preferred embodiment, the exogenous 2,3-dehydroadipyl CoA reductase is PCR amplified using the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template and a predetermined nucleotide as a primer. It is a substance, and the number of base pairs of the amplified substance is 1155 or 1156 bp.

With respect to 2,3-dehydroadipyl CoA reductase, the primers (forward and reverse) used for PCR include:
(2a) Nucleotides consisting of the base sequence shown in SEQ ID NO: 7 and nucleotides consisting of the base sequence shown in SEQ ID NO: 8.
(2b) A nucleotide containing the base sequence shown in SEQ ID NO: 7 and a nucleotide containing the base sequence shown in SEQ ID NO: 8.
(2c) A nucleotide consisting of a codon-optimized base sequence shown in SEQ ID NO: 7 and a nucleotide consisting of a codon-optimized base sequence shown in SEQ ID NO: 8.
(2d) A nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 7 and a nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 8.

The "nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8" in (2b) are "base sequence shown in SEQ ID NO: 7" and "base sequence shown in SEQ ID NO: 8," respectively. In addition, 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 have additional nucleotide lengths.

The "nucleotide containing the base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 8" in (2d) are "SEQ ID NO: 7", respectively. In addition to the "nucleotide sequence obtained by codon-optimizing the base sequence shown in the above" and "the base sequence obtained by codon-optimizing the base sequence shown in SEQ ID NO: 8," a sequence homologous to the vector terminal is included, for example, 1 to 20 bp, preferably 1. It has an additional nucleotide length of ~ 15 bp, more preferably 5 ~ 15 bp, even more preferably 10 ~ 15 bp. As an example of the above (2d), "a nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 7 and a nucleotide containing a codon-optimized base sequence of the base sequence shown in SEQ ID NO: 8"
(2d') Nucleotides consisting of the base sequence shown in SEQ ID NO: 47 and nucleotides consisting of the base sequence shown in SEQ ID NO: 48 can be mentioned (FIG. 5A).

For 2,3-dehydroadipyl CoA reductase, the preferred primer is
(2a) Nucleotides consisting of the base sequence shown in SEQ ID NO: 7, nucleotides consisting of the base sequence shown in SEQ ID NO: 8, and
(2b) A nucleotide containing the base sequence shown in SEQ ID NO: 7 and a nucleotide containing the base sequence shown in SEQ ID NO: 8.

The DNA which is a PCR amplification product using the chromosomal DNA of the Burkholderia sp. Strain using the above nucleotide as a primer preferably has 1155 or 1156 bp. Here, as the DNA polymerase used in PCR, those known in the art are used, for example, Taq DNA polymerase, hot-start-adjusted DNA polymerase, and proofreading having 3'-5'exonuclease activity apart from the polymerase activity. Examples include DNA polymerase.

As used herein, the term "PCR amplification product" refers to a product obtained as a result of PCR using a specific nucleotide as a primer. Unless otherwise specified, in PCR amplification, 1 μM specific nucleotides are used as forward and reverse primers, and PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) is used as an enzyme. 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 with an amount of 25 μL of the solution are carried out.

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 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. Host microorganisms include, for example, Essericia, Corinebacterium, Bacillus, Acinetobacta, Burkhorderia, Pseudomonas, Clostridium, Saccharomyces, Sizosaccalomyces, Yarrowia, Candita, Pikia, Aspergillus. , Klebsiella, Gluconobacta, Zymomonas, Lactobacillus, Lactococcus, Streptococcus and Streptomyces. The host microorganisms of the present invention are preferably of the genus Esselicia, Corinebacterium, Bacillus, Acinetobacta, Burkhorderia, Pseudomonas, Clostridium, Saccharomyces, Sizosaccalomyces, Yarrowia, Candita, Pikia. It belongs to a genus selected from the group consisting of the genus and the genus Aspergillus. The host microorganism is more preferably Escherichia coli.

Therefore, the recombinant microorganisms according to the present invention in which an exogenous gene is introduced into the above host microorganisms include, for example, the genus Esselicia, the genus Corinebacterium, the genus Bacillus, the genus Acinetobacta, the genus Burkholderia, the genus Pseudomonas, the genus Clostridium, and the genus Saccharomyces. , Sizosaccalomyces, Jarowia, Candita, Pikia, Aspergillus, Krebsiera, Glucnovactor, Zymomonas, Lactobacils, Lactococcus, Streptococcus and Streptomyces. Genus Essericia, Corinebacterium, Bacillus, Acinetobacta, Burkhorderia, Pseudomonas, Clostridium, Saccharomyces, Sizosaccalomyces, Yarrowia, Candita, Pikia and It belongs to a genus selected from the group consisting of the genus Aspergillus. The recombinant microorganism according to the present invention is more preferably Escherichia coli.

Regarding the adipic acid, adipic acid derivative, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol production pathways and the enzymes that catalyze each reaction stage of the recombinant microorganisms according to the present invention. This will be described below with reference to FIG.

In the conversion of step A in FIG. 1, for example, succinyl CoA: acetyl CoA acyltransferase or 3-oxoadipyl CoA thiolase is involved, and succinyl-CoA and acetyl-CoA are condensed and converted to 3-oxoadipyl-CoA. To. Other examples of enzymes that can catalyze this conversion include β-ketothiolase. Further, for example, 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-). Enzymes classified into the group such as CoA thiolase) can also be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion, but in one embodiment, PaaJ derived from Escherichia coli having the amino acid sequence shown in SEQ ID NO: 9 is used. In a preferred embodiment, the enzyme is PCR amplified using the chromosomal DNA of the Burkholderia sp. Strain, using the nucleotides set forth in SEQ ID NO: 1 and the nucleotides set forth in SEQ ID NO: 2 as primers. A succinyl CoA: acetyl CoA acyltransferase encoded by the DNA sequence (1203bp) which is a substance.

The conversion in step B of FIG. 1 involves 3-hydroxyadipyl CoA dehydrogenase, which converts 3-oxoadipyl-CoA to 3-hydroxyadipyl-CoA. Examples of other enzymes that can catalyze this conversion include EC 1.1.1. Examples thereof include oxidoreductases classified into m (m is an integer). Specifically, for example, EC 1.11.35 (3-hydroxyacyl-CoA dehydrogenase), EC 1.11.36 (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), etc. Enzymes classified in the group can be exemplified as enzymes that may have activity for this conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion, but for example, PaaH derived from Escherichia coli having the amino acid sequence shown in SEQ ID NO: 10 is used (FIG. 3). In a preferred embodiment, the enzyme is PCR amplified using the chromosomal DNA of the Burkholderia sp. Strain, using the nucleotides set forth in SEQ ID NO: 3 and the nucleotides set forth in SEQ ID NO: 4 as primers. It is a 3-hydroxyadipyl CoA dehydrogenase encoded by the DNA sequence (1521bp) of the substance.

The conversion in step C of FIG. 1 involves, for example, 3-hydroxyadipyl CoA dehydratase, which converts 3-hydroxyadipyl-CoA to 2,3-dehydroadipyl-CoA. Examples of other enzymes that can catalyze this conversion include EC 4.2.1. Examples thereof include hydrolyases classified into n (n is an integer). Specifically, for example, EC 4.2.1.17 (enoyl-CoA hydratase), EC 4.2.1.55 (3-hydroxybutanoyl-CoA dehydratase) and EC 4.21.74 (long). Enzymes classified into the group such as chain enoyl-CoA hydratase) can be exemplified as enzymes that may have activity for this conversion. In a preferred embodiment, the enzyme is the aforementioned 3-hydroxyadipyl CoA dehydratase.

The conversion in step D of FIG. 1 involves, for example, 2,3-dehydroadipyl CoA reductase, which converts 2,3-dehydroadipyl-CoA to adipyl-CoA. Another example of an enzyme that can catalyze this conversion is EC 1.3.1. Examples thereof include oxidoreductases classified into o (o is an integer). 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 ⁺ )), EC 1.3.1.186 (Crotonyl) -CoA reductase), EC 1.3.1.13 (long-chain acyl-CoA reductase) and EC 1.3.1.1104 (enoyl-ACP reductase (NADPH)) It can be exemplified as an enzyme that may have activity for conversion. In a preferred embodiment, the enzyme is the aforementioned 2,3-dehydroadipyl CoA reductase.

In the conversion of step E in FIG. 1, adipic-CoA is converted to adipic acid. Examples of enzymes that can catalyze this conversion include EC 3.1.2. Examples thereof include thioester hydratase classified as p (p is an integer). For example, enzymes classified into groups such as EC 3.1.2.1 (acetyl-CoA hydratase) and EC 3.1.2.20 (acyl-CoA hydratase) have activity for this conversion. It can be exemplified as an enzyme to be obtained. In a preferred embodiment, the enzyme is not limited as long as it has activity for this conversion, but tesB (Ab) derived from the Acinetobacter ADP1 strain shown in SEQ ID NO: 16 is used. Depending on the substrate specificity of the enzyme used, the CoA of 3-oxoadipyl-CoA, 3-hydroxyadipyl CoA and 2,3-dehydroadipyl CoA is eliminated, 3-oxoadipic acid and 3-hydroxyadipine, respectively. Produces acid, 2,3-dehydroadipic acid.

Also, as an example of another enzyme that can catalyze the conversion of step E in FIG. 1, EC 2.8.3. CoA-transferases classified as q (q is an integer) can also be mentioned. For example, EC 2.8.3.5 (3-oxoacid CoA-transferase), EC 2.8.3.6 (3-oxoadipate CoA-transferase), EC 2.8.3.18 (succinyl). -CoA: Acetate CoA-transferase) can be exemplified as an enzyme that may have activity for this conversion.

Furthermore, as an example of another enzymatic conversion that can catalyze the conversion of step E in FIG. 1, EC 2.3.1. EC 2.7.2. Also exemplifying pathways undergo dephosphorylation with phosphotransferases classified as s (where s is an integer). For example, EC 2.3.1.8 (phosphate acetyltransferase) and EC 2.3.1.19 (phosphate butyryltransferase) are used as acyltransferases, and EC 2.7.2 is used as phosphotransferases. Enzymes classified into the group such as .1 (acetate kinase) and EC 2.7.2.7 (butanoic acid kinase) can be exemplified as enzymes that may have activity for this conversion.

In step T of FIG. 1, 3-oxoadipic acid is converted to levulinic acid. This conversion is catalyzed by an enzyme (3-oxoadipate decarboxylase) or occurs spontaneously.

In the conversion of step F in FIG. 1, adipic-CoA is converted to adipic acid semialdehyde. Examples of enzymes that can catalyze this conversion include EC 1.2.1. Examples thereof include enzymes classified into t (t is an integer). For example, EC 1.2.1.10 (acetaldehyde dehydrogenase (acetylation)), EC 1.2.1.17 (glyoxylate dehydrogenase (acylated)), EC 1.21.42 (hexadecanal dehydrogenase). (Acylation)), EC 1.21.44 (Cinnamoyl-CoA reductase (acylation)), EC 1.21.75 (malonyl-CoA reductase (malonate semialdehyde formation)), EC 1. Enzymes classified into the group 2.1.76 (succinate semialdehyde dehydrogenase (acylation)) catalyze the conversion reaction that desorbs CoA and produces aldehydes, as in this conversion. However, it can be exemplified as an enzyme that may have activity. The enzyme used in the present invention is not limited as long as it has activity for the present conversion, and for example, sucD derived from Clostridium lyveri consisting of the amino acid sequence set forth in SEQ ID NO: 73 is used (FIG. 6).

In the conversion of steps G, J, and Q in FIG. 1, the carboxyl group is converted to an aldehyde. Examples of the enzyme that can catalyze this conversion include carboxylic acid reductase (CAR). For example, EC 1.21.30 (carboxylic acid reductase (NADP +)), EC 1.21.31 (L-aminoadipate semialdehyde dehydrogenase), EC 1.21.95 (L-2). -Since enzymes classified into the groups such as aminoadipate reductase) and EC 1.2.99.6 (carboxylic acid reductase) catalyze the conversion reaction that produces aldehyde from carboxylic acid, as in this conversion. It can be exemplified as an enzyme that can also have activity for this conversion. Typical examples of species from which it is derived enzymes, 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, Cobacterium cinerea, Aspergillus flavus, Aspirus The enzyme used in the present invention is not limited as long as it has activity for the present conversion, and for example, at least one of the enzymes consisting of the amino acid sequences set forth in any of SEQ ID NOs: 74 to 78 may be used. Often (FIGS. 7A-7E), preferably, MaCar (m), which is a variant of MaCar consisting of the amino acid sequence set forth in SEQ ID NO: 78, the enzyme MaCar derived from Mycobacterium abssussus having the amino acid sequence set forth in SEQ ID NO: 76. ) Is used, and more preferably MaCar (m) consisting of the amino acid sequence set forth in SEQ ID NO: 78 is used.

In addition, the carboxylic acid reductase can be converted into an active holoenzyme by being phosphopantetinylated (Venkitasubramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1,478-485 (2007)). ). Phosphopantetinylation is catalyzed by Phosphopantetheinyl Transferase (PT). Examples of the enzyme that can catalyze this reaction include enzymes classified in EC 2.7.8.7. Therefore, the microorganism of the present invention may be further modified to increase the activity of the phosphopantetinyltransferase. As a method for increasing the activity of phosphopantetinyltransferase, a method for introducing a foreign phosphopantetinyltransferase gene and a method for enhancing the expression of an endogenous phosphopantetinyltransferase gene are available. However, but not limited to these. The enzyme used in the present invention is not limited to these as long as it has a phosphopantetinyl group transfer activity, but typical enzymes include, for example, EntD of Escherichia coli, Sfp of Bacillus subtilis, and Npt of Nocardia iowensis. Venkitas bramanian et al., Journal of Biological Chemistry, Vol. 282, No. 1,478-485 (2007)), Saccharomyces cerevisiae Lys5 (Ehmann. Can be mentioned. The enzyme used in the present invention is not limited as long as it has activity for the present conversion, and for example, at least one of the enzymes consisting of the amino acid sequences set forth in SEQ ID NOs: 79 to 82 may be used. (FIGS. 8A-8D), preferably Npt of Nocardia iowensis derived from Nocardia iowensis consisting of the amino acid sequence set forth in SEQ ID NO: 80 is used.

The conversion of steps M, N, S in FIG. 1 is a transamination reaction. Examples of enzymes that can catalyze this conversion include EC 2.6.1. Examples thereof include transaminase (aminotransferase) classified into u (u is an integer). For example, EC 2.6.1.19 (4-aminobutanoic acid-2-oxoglutaric acid transaminase), EC 2.6.1.29 (diamine transaminase), EC 2.6.1.148 (5-aminovaleric acid). Enzymes classified into the group such as valeric acid transaminase) can be exemplified as enzymes that can also have activity for this conversion. The enzyme used in the present invention is not particularly limited as long as it has the conversion activity of each step, but for example, YgjG, which is a putrescine aminotransferase of Escherichia coli reported to transaminate cadaverine or spermidine. (Samsonova., Et al., BMC microbiology 3.1 (2003): 2.) and SpuC (Lu et al., Journal of bacteriology 184.14 (2002): 37, which is a putrescine aminotransferase of the genus Pseudomonas. , Galman et al., Green Chemistry 19.2 (2017): 361-366.), GABA aminotransferase GabT of Escherichia coli, and Putrescine may be used. Furthermore, Ruegeria pomeroyi, Chromobacterium violaceum, Arthrobacter citreus, Sphaerobacter thermophilus, Aspergillus fischeri, Vibrio fluvialis, Agrobacterium tumefaciens, also ω- transaminase derived from species such Mesorhizobium loti, 1,8-diamino-octane and 1,10-diaminodecane It has been reported to have transamination activity to diamine compounds such as, and may be used in the present invention (Sung et al., Green Chemistry 20.20 (2018): 4591-4595., Stratler 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 the present conversion, and for example, at least one of the enzymes consisting of the amino acid sequences set forth in SEQ ID NOs: 83 to 87 may be used. (FIGS. 9A-9E). Typical amino group donors include, but are not limited to, L-glutamic acid, L-alanine, and glycine.

In the conversion of steps H, L, R in FIG. 1, the aldehyde is converted to alcohol. Examples of enzymes that can catalyze this conversion include EC 1.1.1. Examples thereof include oxidoreductases classified into v (where v is an integer). For example, EC 1.1.1.1 (alcohol dehydrogenase), EC 1.1.1.2 (alcohol dehydrogenase (NADP +)), EC 1.11.71 (alcohol dehydrogenase [NAD (P) +]] ), Since the enzyme classified into the group such as) catalyzes the conversion reaction from aldehyde to alcohol as in the present conversion, it can be exemplified as an enzyme that may have activity for the present conversion. The enzyme used in the present invention is not limited as long as it has activity for this conversion, and for example, Ahr derived from Escherichia coli described in SEQ ID NO: 88 is used (FIG. 10).

In the conversion of steps I and O in FIG. 1, CoA is added to the carboxyl group. An example of an enzyme that can catalyze this conversion is EC 2.8.3. CoA transferases classified as w (w is an integer), EC 6.2.1. Examples thereof include acid thiol ligases classified as x (where x is an integer). The enzyme used in the present invention is not limited as long as it has activity for this conversion.

The gene encoding the above enzyme that can be used in the present invention may be derived from a non-exemplified organism or artificially synthesized, and is a substantial enzyme in a host microbial cell. Anything that can express activity is sufficient.

The recombinant microorganism according to the present invention includes succinyl CoA: acetyl CoA acyltransferase and 3-, in addition to the exogenous gene encoding 3-hydroxyadipyl CoA dehydrase and / or 2,3-dehydroadipyl CoA reductase described above. It is preferable that an exogenous gene encoding at least one enzyme selected from the group consisting of hydroxyadipyl CoA dehydrogenase is introduced.

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. ..

The recombinant microorganism according to the present invention includes the above-mentioned exogenous genes, that is, other than 3-hydroxyadipyl CoA dehydratase, 2,3-dehydroadipyl CoA reductase, succinyl CoA: acetyl CoA acyltransferase and 3-hydroxyadipyl CoA dehydrogenase. Also, further exogenous genes may be introduced.

The recombinant microorganism according to the present invention may be a host microorganism in which any gene is appropriately disrupted. Destruction of the target gene may be performed according to a method known in the art.

In addition, the enzyme gene that can be used for the purpose of the present invention includes all mutations that can occur in nature or artificially introduced mutations as long as they can express substantial enzyme activity in the host microbial cell. And may have modifications. For example, it is known that there are extra codons in the various codons that encode a particular amino acid. Therefore, in the present invention as well, an alternative codon that will eventually be translated into the same amino acid may be used. That is, because the genetic code is degenerate, multiple codons can be used to encode a particular amino acid, so that the amino acid sequence can be encoded 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 hybridize at appropriate contractile conditions (eg, 3xSSC, 68 ° C., 2xSSC, 0.1). % SDS and washed at 68 ° C.) can hybridize to the native sequence, DNA encoding the native sequence can be identified and isolated, and such genes can also be utilized in the present invention. In particular, since most organisms are known to preferentially use a subset of specific codons (optimal codons) (Gene, Vol. 105, pp. 61-72, 1991, etc.), depending on the host microorganism, " Performing "codon optimization" can also be useful in the present invention.

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

In the present invention, by introducing the above-mentioned biosynthetic enzyme gene into a host microbial cell as an "expression cassette", a more stable and high level enzyme activity can be obtained. As used herein, the term "expression cassette" means a nucleotide containing a nucleic acid to be expressed or a nucleic acid sequence that regulates transcription and translation functionally bound to a gene to be expressed. Typically, the expression cassette of the invention comprises a promoter sequence 5'upstream from the coding sequence, a terminator sequence 3'downstream, and optionally additional conventional regulatory elements in a functionally bound manner. In some cases, the nucleic acid to be expressed or the gene to be expressed is introduced into the host microorganism.

As used herein, 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 also suitably used in the present invention. In Escherichia coli, lac, trp, tac or trc, major operator and promoter regions of λ phage, regulatory regions of fd-coated proteins, glycolytic enzymes (eg, 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate). Acid dehydrogenase), glutamate decarboxylase A, promoter for serine hydroxymethyltransferase, promoter region of T7 phage-derived RNA polymerase, and the like can be used. In Corynebacterium glutamicum, HCE (high-level conscious expression) promoter, cspB promoter, sodA promoter, extension factor (EF-Tu) promoter and the like can be used.

As the terminator, a T7 terminator, an rrnBT1T2 terminator, a lac terminator and the like can be used.

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

The host microorganism used in the present invention may be a microorganism in which the gene encoding the enzyme originally possessed by the microorganism is disrupted. Examples of the gene encoding the enzyme include the ldh gene encoding lactate dehydrogenase, the atoB gene encoding acetyl-CoA acetyltransferase, and the sucD gene encoding the succinyl-CoA synthesizer α-subunit. In this way, by using a host microorganism in which a part of the gene of the enzyme originally possessed by the host microorganism is destroyed, it is possible to avoid contamination with impurities and suppress the progress of an unintended reaction. Specifically, by disrupting the ldh gene encoding lactate dehydrogenase, the production of lactic acid, which is an impurity, can be reduced. By disrupting the atoB gene encoding the acetyl-CoA acetyltransferase and the sucD gene encoding the succinyl-CoA synthesizer α subunit, the yield of adipic acid can be improved.

The expression cassette described above is inserted into a host microorganism by incorporating it into a vector consisting of, for example, a plasmid, a phage, a transposon, an IS element, a fosmid, a cosmid, or a linear or circular DNA. Plasmids and phage are preferred. These vectors may be autonomously replicated in the host microorganism or may be chromosomally replicated. Suitable plasmids are, for example, E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290 Or pBdCI; bacillus pUB110, pC194 or pBD214; Corynebacterium genus pSA77 or pAJ667 and the like. Examples of bacilli such as Bacillus include pUB110, pC194 or pBD214. In addition to these, plasmids and the like that can be used are described in "Gene Cloning and DNA nailis 6th edition", Wiley-Blackwell (2016). The introduction of the expression cassette into the vector is possible by conventional methods including excision, cloning, and ligation with appropriate restriction enzymes. Each expression cassette may be located on one vector or on two or more vectors.

After the vector having the expression cassette of the present invention is constructed as described above, a conventional method can be used as a method applicable when introducing the vector into a host microorganism. For example, a calcium chloride method, an electroporation method, a junction transfer method, a protoplast fusion method and the like can be mentioned, but the method is not limited to these, and a method suitable for the host microorganism can be selected.

Another aspect of the present invention relates to a method for producing a target compound using the recombinant microorganism. More specifically, the present invention relates to a method for producing a target compound, which comprises a culturing step of culturing the recombinant microorganism.

Here, the target compound is selected from the group consisting of adipic acid, adipic acid derivative, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol. Examples of the adipic acid derivative include 3-oxoadipic acid (3-OA), levulinic acid (LEV), 3-hydroxyadipic acid (3HA) and 2,3-dehydroadipic acid (23DA) as described above. Be done.

In one aspect, the recombinant microorganism according to the invention has an adipic acid production pathway. In this case, the present invention also relates to a method for producing adipic acid, which comprises a culture step of culturing the recombinant microorganism.

In one aspect, the recombinant microorganism according to the invention has a hexamethylenediamine production pathway. In this case, the present invention also relates to a method for producing hexamethylenediamine, which comprises a culture step of culturing the recombinant microorganism.

In one aspect, the recombinant microorganism according to the invention has a 1,6-hexanediol production pathway. In this case, the present invention also relates to a method for producing 1,6-hexanediol, which comprises a culture step of culturing the recombinant microorganism.

In one aspect, the recombinant microorganism according to the 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, which comprises a culture step of culturing the recombinant microorganism.

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 cells. The recombinant microorganism of the present invention is cultured under conditions suitable for adipic acid production and growth and maintenance of the microorganism, and suitable medium composition, culture time and culture conditions can be easily set by those skilled in the art.

Carbon sources are D-glucose, sucrose, lactose, fructose, maltose, oligosaccharides, polysaccharides, starch, cellulose, rice bran, waste sugar dense, fats and oils (eg soybean oil, sunflower oil, peanut oil, palm oil, etc.), fatty acids (eg, soybean oil, sunflower oil, peanut oil, palm oil, etc.) For example, palmitic acid, linoleic acid, oleic acid, linolenic acid, etc.), alcohol (eg, glycerol, ethanol, etc.), organic acids (eg, acetic acid, lactic acid, succinic acid, etc.), corn decomposition liquid, cellulose decomposition liquid, carbon dioxide, monoxide. Examples include carbon. Preferably, it is D-glucose, sucrose or glycerol. These carbon sources can be used individually or as a mixture.

Adipic acid produced using biomass-derived raw materials can be measured, for example, by measuring the biobase carbon content based on Carbon-14 (radiocarbon) analysis specified in ISO 16620-2 or ASTM D6866, for example, petroleum, natural gas, coal. It can be clearly distinguished from synthetic raw materials derived from such substances.

The nitrogen source can be a nitrogen-containing organic compound (eg, peptone, cazaminoic acid, trypton, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour, amino acids and urea, etc.), or an inorganic compound (eg, ammonia). Examples include aqueous solution, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, sodium nitrate, ammonium nitrate, etc.). These nitrogen sources can be used individually or as a mixture.

The medium may also contain the corresponding antibiotic if the recombinant microorganism expresses useful additional traits, eg, has a marker of resistance to the antibiotic. This reduces the risk of contamination by germs during culture. Examples of the antibiotic include β-lactam antibiotics such as ampicillin, aminoglycoside antibiotics such as canamycin, macrolide antibiotics such as erythromycin, tetracycline antibiotics, chloramphenicol and the like, but are limited thereto. Not done.

The culture may be batch type or continuous type. Further, in any case, it may be in the form of supplementing the additional carbon source or the like at an appropriate time of culturing. Further, the culture may be performed while controlling conditions such as suitable temperature, oxygen concentration, and pH. Suitable culture temperatures for transformants derived from common microbial host cells are usually in the range of 15 ° C to 45 ° C, preferably 25 ° C to 37 ° C. If the host microorganism is aerobic, shaking (flask culture, etc.) and stirring / aeration (jar fermenter culture, etc.) may be performed to ensure an appropriate oxygen concentration during fermentation. Those culture conditions can be easily set by those skilled in the art.

The method for producing a 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, such as, but is not limited to, centrifugation, membrane filtration, membrane separation, crystallization, extraction, distillation, adsorption, phase separation and various chromatographies. The separation / purification step may consist of one type of step or a combination of a plurality of steps.

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, and therefore, the present invention is not limited to these examples.

Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

<Isolation and identification of Burkholderia sp. LEBP-3 strain>
Burkholderia sp. LEBP-3 strain was isolated from activated sludge in Japan using a medium containing adipic acid as the sole carbon source and energy source. The Burkholderia sp. LEBP-3 strain applied for deposit with the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center NPMD on December 4, 2020 (receipt number:: original deposit date). NITE ABP-03334), "Consignment number: NITE BP-03334" has been deposited internationally.

Burkholderia sp. The classification properties of the LEBP-3 strain are as shown below.
<Mycological properties of LEBP-3 strain>
Culture temperature: 37 ° C,
Cell morphology: Bacillus (0.7x1.7-3.0 μm),
Gram stain:-,
Presence or absence of spores:-,
Motility: +,
Colony morphology when culturing on LB agar medium 48h: diameter 1.9-2.5 mm, pale yellow, round, lenticular, all edges, smooth, opaque, butter-like,
30 ° C growth: +,
Growth at 45 ° C: +,
Catalase reaction: +,
Oxidase reaction: +,
Acid / gas production from glucose: +/-,
O / F test (oxidation / fermentation): +/-,
Nitrate reducing:-,
Indole productivity:-,
Glucose acidification:-,
Urease: I don't have it
Aesculin degradability: +,
Glucose, L-arabinose, D-mannose, N-acetyl-D-glucosamine, gluconic acid, n-capric acid, adipic acid, malic acid, citric acid and phenyl acetate: can be assimilated,
Maltose: Can't be capitalized.

<Phylogenetic analysis by 16S rRNA gene sequence homology of LEBP-3 strain>
Identification was performed by performing a base sequence analysis of the 16SrDNA gene. The base sequence of the 16SrDNA gene was amplified by PCR and sequence analysis was performed. As the database for BLAST homology search, DB-BA11.0 (Technosuruga Lab.) And the international nucleotide sequence database were used. As a result, the LEBP-3 strain was included in the cluster composed of the genus Burkhlderia, but the species name could not be identified because it showed a molecular phylogenetic position different from that of any known species.

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

Table 1 shows the results of the BLAST search using the PaaJ (L3) sequence as a query, and Table 2 shows the results of the BLAST search using the PaaH (L3) sequence as the query.
Table 3 shows the results of performing a BLAST search using the PaaF (L3) sequence as a query, and Table 4 shows the results of performing a BLAST search using the MmgC (L3) sequence as a query. In addition, "Refseq_Protein" was specified as the database at the time of sequence search.

From the above results, Burkholderia sp. The above enzyme possessed by the LEBP-3 strain was identified as a novel sequence. The paaJ (L3) gene can be PCR amplified using the nucleotides shown in SEQ ID NO: 1 and the nucleotides shown in SEQ ID NO: 2 as primers. The paaH (L3) gene can be PCR amplified using the nucleotides shown in SEQ ID NO: 3 and the nucleotides shown in SEQ ID NO: 4 as primers. The paaF (L3) gene can be PCR amplified using the nucleotides shown in SEQ ID NO: 5 and the nucleotides shown in SEQ ID NO: 6 as primers. The mmgC (L3) gene can be PCR amplified using the nucleotides shown in SEQ ID NO: 7 and the nucleotides shown in SEQ ID NO: 8 as primers. The primers used for PCR, each enzyme, and the base sequence encoding the enzyme are shown in FIGS. 2 to 5.

An example of PCR amplification conditions is shown below. As the enzyme, PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) was used. 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 carried out for 30 cycles. 1 μM each of oligonucleotides used as primers was added. The liquid volume was 25 μL.

<Construction of recombinant E. coli expressing the acquired enzyme gene>
PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) was used for amplification of PCR fragments, and Escherichia coli JM109 strain was used for plasmid preparation. For codon optimization of the base sequence, GeneArt GeneOptinizer (software name, Thermo Fisher Scientific) or an artificial gene synthesis service of Eurofin Genomics was used.

The polynucleotide encoding PaaJ (Ec) of Eshchericia coli was cloned from the genomic DNA of Eshchericia coli W3110 strain (NBRC12713). PCR was performed using the oligonucleotides of SEQ ID NOs: 25 and 26 (FIG. 5A) as primers to obtain a PCR amplification product containing the coding region of the paaJ (Ec) gene (SEQ ID NO: 17) (FIG. 4A). Next, PCR was performed using pRSFDuet-1 (product name, manufactured by Merck) as a template and the nucleotides shown in SEQ ID NOs: 27 and 28 (FIG. 5A) as primers to obtain a pRSFDuet-1 fragment. The DNA fragment containing the gene coding region and the pRSFDuet-1 fragment were connected using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "Prm203" was obtained as a PaaJ (Ec) expression plasmid.

The polynucleotide encoding PaaH (Ec) of Eshchericia coli was cloned from the genomic DNA of Eshchericia coli W3110 strain (NBRC12713). PCR was performed using the nucleotides shown in SEQ ID NOs: 29 and 30 (FIG. 5A) as primers to obtain a PCR amplification product containing the coding region of the paaH (Ec) gene (SEQ ID NO: 18) (FIG. 4A). Next, PCR was performed using prm203 as a template and the nucleotides shown in SEQ ID NOs: 31 and 32 (FIG. 5A) as primers to obtain a prm203 fragment. A DNA fragment containing each gene coding region and a prm203 fragment were connected using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia 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.

The polynucleotide encoding PaaF (Ec) of Eshchericia coli was cloned from the genomic DNA of Eshchericia coli W3110 strain (NBRC12713). PCR was performed using the nucleotides shown in SEQ ID NOs: 33 and 34 (FIG. 5A) as primers to obtain a PCR amplification product containing the coding region of the paaF (Ec) gene (SEQ ID NO: 19) (FIG. 4A). The polynucleotide encoding PaaF (L3) of the L3 strain was obtained by optimizing the base sequence for Escherichia coli expression and using an artificial gene synthesis service of Eurofin Genomics. PCR was performed using the nucleotides shown in SEQ ID NOs: 35 and 36 (FIG. 5A) 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: 41 and 42 (FIG. 5A) as primers to obtain a pETDuet-1 fragment. A DNA fragment containing the coding region of each gene and a pETDuet-1 fragment were connected using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "Prm74" was obtained as a PaaF (Ec) expression plasmid, and "prm194" was obtained as a PaaF (L3) expression plasmid.

The polynucleotide encoding dcaA (Ab) of Acinetobacter baylyi, Tfu1647 of Thermobifida fusca, and MmgC (L3) of the L3 strain optimized the base sequence for E. coli expression and used the artificial gene synthesis service of Eurofin Genomics. And got it. The decaA (Ab) gene (SEQ ID NO: 22) (FIG. 4B) uses the nucleotides shown in SEQ ID NOs: 43 and 44 (FIG. 5A) as primers, and the Tfu1647 gene (SEQ ID NO: 23) (FIG. 4B) of Thermobifida fusca is SEQ ID NO: 45. And 46, using the nucleotide shown in FIG. 5A as a primer, the mmgC5 (L3) gene was subjected to PCR using the nucleotides shown in SEQ ID NOs: 47 and 48 (FIG. 5A) as primers, and dcaA (Ab), Tfu1647, and mmgC5 (L3), respectively. PCR amplifications containing the coding region of the gene were obtained respectively. PCR was performed using prm74 and prm194 as templates and the nucleotides shown in SEQ ID NOs: 49 and 50 (FIG. 5A) as primers to obtain prm74 fragments and prm194 fragments, respectively. A DNA fragment containing the coding region of each gene and a prm74 fragment and a prm194 fragment were connected using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "Prm127" as a PaaF (Ec) -dcaA (Ab) expression plasmid, "prm234" as a PaaF (Ec) -mmgC (L3) expression plasmid, and "prm166" as a PaaF (L3) -mmgC (L3) expression plasmid. , I got each.

For the polynucleotide encoding tesB (Ab) of Acinetobacter baylyi, PCR was performed from the chromosomal DNA of Acinetobacter baylyi using the nucleotides shown in SEQ ID NOs: 51 and 52 (FIG. 5A) as primers, and PCR containing the coding region of the tesB (Ab) gene was performed. Amplified products were obtained respectively. PCR was performed using pCDFDuet-1 as a template and the nucleotides shown in SEQ ID NOs: 53 and 54 (FIG. 5A) as primers to obtain a pCDFDuet-1 fragment. A DNA fragment containing each tesB (Ab) gene coding region and a pCDFDuet-1 fragment were connected using an In-Fusion HD cloning kit (product name, manufactured by Clontech). Escherichia coli JM109 strain was transformed, and a plasmid was extracted from the obtained transformant. "Prm92" was obtained as a TesB (Ab) expression plasmid.

A gene-disrupted strain of Escherichia coli BL21 (DE3) strain was prepared as follows. Disruption of the ldh gene encoding lactate dehydrogenase, the attoB gene encoding acetyl-CoA acetyltransferase, and the sucD gene encoding the succinyl CoA synthesizer α subunit is called pHAK1 (receipt number NITE P-02919, an independent administrative agency product evaluation technology. Infrastructure Organization Biotechnology Center Deposited with the Patented Microbial Depositary Center (NPMD) on March 18, 2019. The homologous recombination method using the international deposit number: NITE BP-02919) was performed. pHAK1 contains a temperature-sensitive mutant repA gene, a kanamycin resistance gene, and a levansculase gene SacB derived from Bacillus subtilis. The levansucrase gene acts lethal to host microorganisms in the presence of sucrose. PrimeSTAR Max DNA Polymerase (product name, manufactured by Takara Bio Inc.) was used for amplification of the PCR fragment, and Escherichia coli HST08 strain was used for plasmid preparation.

Using the genomic DNA of the Escherichia coli BL21 (DE3) strain as a template, a PCR amplification product containing an upstream region, a coding region, and a downstream region of the disruption target gene was obtained. The ldh gene peripheral region was PCRed using the nucleotides shown in SEQ ID NOs: 55 and 56 (FIG. 5B) as primers, and the atoB gene peripheral region was PCRed using the nucleotides shown in SEQ ID NOs: 57 and 58 (FIG. 5B) as primers. For the peripheral region, PCR was performed using the nucleotides shown in SEQ ID NOs: 59 and 60 (FIG. 5B) as primers to obtain respective fragments.

Next, this PCR amplification product was inserted into the pHAK1 plasmid fragment amplified using the primers shown in SEQ ID NOs: 61 and 62 (FIG. 5B) using an In-Fusion HD cloning kit (product name, manufactured by Clontech), and circular. It became.

PCR using the pHAK1 plasmid into which the DNA fragments of the upstream, coding, and downstream regions of the obtained disruption target gene were inserted as a template, and the nucleotides shown in SEQ ID NOs: 63 and 64 (FIG. 5B) as primers for the region around the ldh gene. The area around the atoB gene was PCRed using the nucleotides shown in SEQ ID NOs: 65 and 66 as primers (FIG. 5B), and the region around the sucD gene was subjected to PCR using the nucleotides shown in SEQ ID NOs: 67 and 68 as primers (FIG. 5B). , A plasmid fragment was obtained in which a part or all of the coding region of the disruption target gene was removed.

The obtained plasmid fragment was cyclized by terminal phosphorylation and self-ligation to obtain a plasmid for gene disruption.

Kanamycin sulfate 100 mg / L is contained after transforming Escherichia coli BL21 (DE3) strain with a plasmid for disruption of a desired gene by the calcium chloride method (see Yeast Co., Ltd. Genetic Engineering Experiment Note, Takaaki Tamura). It was applied to LB agar medium (trypton 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, agar powder 15 g / L) and cultured at 30 ° C. overnight to obtain a single colony to obtain a transformant. rice field. This 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 of canamycin sulfate, and cultured at 30 ° C. with shaking. went. The obtained culture solution was applied to LB agar medium containing 100 mg / L of kanamycin sulfate and cultured at 42 ° C. overnight. The resulting colony has a plasmid inserted into the genome by a single crossover. The colonies were inoculated in 1 mL of LB liquid medium with a single platinum loop and cultured at 30 ° C. with shaking. The obtained culture solution was applied to LB agar medium containing 10% sucrose and cultured overnight. It was confirmed by colony direct PCR that the desired gene was disrupted in the obtained colonies. First, an ldh gene-disrupted strain was constructed, an attoB gene-disrupted strain was constructed based on this ldh gene-disrupted strain, and a sucD gene-disrupted strain was further constructed based on the ldh and atoB double-disrupted strains. The prepared triple-disrupted strain of the ldh, atoB, and sucD genes was designated as the ASL000 strain.

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

<Adipic acid production test using recombinant Escherichia coli expressing the acquired enzyme gene>
The strains shown in Table 5 were LB liquid medium (trypton 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L) containing ampicillin sodium 50 mg / L, canamycin sulfate 30 mg / L, and streptomycin sulfate 50 mg / L. Ampicillin ear was inoculated into 2 mL (14 mL round bottom tube) and shake-cultured at 37 ° C. for 24 hours to obtain a preculture solution. 10 μL of the previously obtained preculture solution was inoculated into 1 mL (96-well deepwell plate) of MM medium containing 50 mg / L of ampicillin sodium, 30 mg / L of kanamycin sulfate, and 50 mg / L of streptomycin sulfate, and at 37 ° C. Shaking culture was performed for 48 hours. The composition of the MM medium is shown in Table 6.

After completion of the culture, the concentrations of adipic acid and adipic acid analogs in the culture solution were analyzed. The analysis was performed by GC / MS analysis using trimethylsilyl derivatization. 360 μL of the extract mixed with water: methanol: chloroform = 5: 2: 2 (v / v / v) in 40 μL of the culture solution centrifuge supernatant (added disodium sebacate as an internal standard to a final concentration of 10 mM). Added and mixed well. After centrifugation (16,000 × g, 5 minutes), 100 μL of the supernatant was collected in another microtube and concentrated by centrifugation for 1 hour on a centrifugal evaporator. To the obtained dry matter, 100 μL of a 20 mg / mL pyridine solution of methoxyamine hydrochloride was added, and the mixture was shaken at 30 ° C. for 90 minutes. 50 μL of N-methyl-N-trimethylsilyltrifluoroacetamide 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.

GC / MS analysis condition device: GCMS-QP-2020NX (manufactured by Shimadzu Corporation)
Column: Fused silica capillary tube Inactive treatment tube (length 1 m, outer diameter 0.35 mm, inner diameter 0.25 mm, manufactured by GL Science), InertCap 5MS / NP (length 30 m, inner diameter 0.25 mm, film thickness 0) .25 μm, manufactured by GL Science Co., Ltd.)
Sample injection volume: 1 μL
Sample introduction method: Split (split ratio 25: 1)
Vaporization chamber temperature: 230 ° C
Carrier gas: Helium Carrier gas ray velocity: 39.0 cm / sec Oven temperature: 80 ° C, 2 minutes holding → 15 ° C / min temperature rise → 325 ° C, 13 minutes holding Ionization method: Electron ionization method (EI)
Ionization energy: 70eV
Ion source temperature: 230 ° C
Scan range: m / z = 50-500
Injection volume: 1 μL

The results are shown in Table 7. In the table, "3OA" is 3-oxoadipic acid, "LEV" is levulinic acid, "3HA" is 3-hydroxyadipic acid, "23DA" is 2,3-dehydroadipic acid, and "ADA" is. Shows adipic acid.

In the invention strains expressing both PaaF (L3) and mmgC (L3) derived from the L3 strain, that is, the ADA500 strain and the ADA496 strain, 3-oxoadipic acid (3-OA), levulinic acid (LEV), 3- Hydroxyadipic acid (3HA), 2,3-dehydroadipic acid (23DA), and adipic acid (ADA) were produced in a larger amount than in the control strain, and the total value of these was 355 mg / L and 613 mg / L, respectively. From the above results, it was shown that the enzymes encoded by these genes have high activity to promote the adipic acid pathway reaction and improve the production amount of adipic acid and adipic acid derivatives.

The genetically modified microorganism according to the present invention can be suitably used in the direct fermentation production of adipic acids from a biomass raw material.

Claims (17)

Includes an exogenous gene encoding 3-hydroxyadipyl CoA dehydratase and an exogenous gene encoding 2,3-dehydroadipyl CoA reductase.
The 3-hydroxyadipyl CoA dehydratase and 2,3-dehydroadipyl CoA reductase are derived from the Burkholderia sp. LEBP-3 strain (accession number: NITE BP-03334).
A recombinant microorganism having a pathway to produce adipic acid, adipic acid derivatives, hexamethylenediamine, 1,6-hexanediol or 6-amino-1-hexanol . The 3-hydroxyadipyl CoA dehydratase uses the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(1a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (1b) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. Encoded in DNA having 90% or more sequence identity with the PCR amplification product using as a primer.
The recombinant microorganism according to claim 1 , wherein the DNA has 783 or 784 bp. The 2,3-dehydroadipyl CoA reductase uses the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(2a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (2b) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. Encoded in DNA having 90% or more sequence identity with the PCR amplification product using as a primer.
The recombinant microorganism according to claim 1 or 2 , wherein the DNA has 1155 or 1156 bp. The 3-hydroxyadipyl CoA dehydratase uses the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(1a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (1b) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. Encoded in DNA, which is a PCR amplification product using
The recombinant microorganism according to claim 1 , wherein the DNA has 783 or 784 bp. The 2,3-dehydroadipyl CoA reductase uses the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(2a) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (2b) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. Encoded in DNA, which is a PCR amplification product using
The recombinant microorganism according to claim 1 or 4 , wherein the DNA has 1155 or 1156 bp. The recombinant microorganisms include Essericia, Corinebacterium, Bacillus, Acinetobacta, Burkholderia, Pseudomonas, Clostridium, Saccharomyces, Sizocaccalomyces, Yarrowia, Candita, Pikia and Aspergillus. The recombinant microorganism according to any one of claims 1 to 5 , which belongs to a genus selected from the group consisting of. The recombinant microorganism according to any one of claims 1 to 6 , which has an adipic acid production pathway. The recombinant microorganism according to any one of claims 1 to 7 , which has a hexamethylenediamine production route. The recombinant microorganism according to any one of claims 1 to 7 , which has a 1,6-hexanediol production route. The recombinant microorganism according to any one of claims 1 to 7 , which has a 6-amino-1-hexanol production pathway. A method for producing a target compound, which comprises a culture step of culturing the recombinant microorganism according to any one of claims 1 to 6 .
The target compound is selected from the group consisting of adipic acid, adipic acid derivative, hexamethylenediamine, 1,6-hexanediol and 6-amino-1-hexanol.
A production method, wherein the adipic acid derivative is selected from the group consisting of oxoadic acid, levulinic acid, 3-hydroxyadipic acid and 2,3-dehydroadipic acid. A method for producing adipic acid, which comprises a culturing step of culturing the recombinant microorganism according to claim 7 . A method for producing hexamethylenediamine, which comprises a culturing step of culturing the recombinant microorganism according to claim 8 . A method for producing 1,6-hexanediol, which comprises a culturing step of culturing the recombinant microorganism according to claim 9 . A method for producing 6-amino-1-hexanol, which comprises a culture step of culturing the recombinant microorganism according to claim 10 . (A-1) Using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template,
(A1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (a2) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. A recombinant polypeptide encoded by DNA having a sequence identity of 90% or more with a PCR amplification product using the same as a primer.
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having 783 or 784 bp;
(A-2) Using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template,
(A1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (a2) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. A recombinant polypeptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by DNA, which is a PCR amplification product using the above as a primer. And
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having 783 or 784 bp;
Or (A-3) using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(A1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 5 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 6, or (a2) Nucleotide containing the base sequence shown in SEQ ID NO: 5 and the nucleotide containing the base sequence shown in SEQ ID NO: 6. A recombinant polypeptide encoded by DNA consisting of a PCR amplification product or its degenerate isomer using the above as a primer.
Has 3-hydroxyadipyl CoA dehydratase activity and
The DNA is a recombinant polypeptide having 783 or 784 bp. (B-1) Using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template,
(B1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (b2) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. A recombinant polypeptide encoded by DNA having a sequence identity of 90% or more with a PCR amplification product using the same as a primer.
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having 1155 or 1156 bp;
(B-2) Using the chromosomal DNA of the Burkholderia sp. Strain LEBP-3 as a template.
(B1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (b2) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. A recombinant polypeptide consisting of an amino acid sequence in which 1 to 3 amino acids are deleted, substituted, inserted and / or added to the amino acid sequence of a polypeptide encoded by DNA, which is a PCR amplification product using the above as a primer. And
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having 1155 or 1156 bp;
Or (B-3) using the chromosomal DNA of the Burkholderia sp. LEBP-3 strain as a template.
(B1) Nucleotide consisting of the base sequence shown in SEQ ID NO: 7 and the nucleotide consisting of the base sequence shown in SEQ ID NO: 8, or (b2) Nucleotide containing the base sequence shown in SEQ ID NO: 7 and the nucleotide containing the base sequence shown in SEQ ID NO: 8. A recombinant polypeptide encoded by DNA consisting of a PCR amplification product or its degenerate isomer using the above as a primer.
Has 2,3-dehydroadipyl CoA reductase activity and
The DNA is a recombinant polypeptide having 1155 or 1156 bp.

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