JP2024109093A - Acetovanillone converting enzyme gene and production of useful substances using the same - Google Patents

Abstract

[Problem] An object of the present invention is to provide a gene cluster which is composed of fewer genes than the acetovanillone conversion enzyme gene cluster possessed by Sphingobium species SYK-6 and which is capable of converting acetovanillone to vanillic acid with high efficiency, a microorganism possessing said gene cluster, and a method for producing vanillic acid using them. [Solution] The above object is achieved by the acpA gene and acpB gene which contain a nucleotide sequence encoding the amino acid sequence of an enzyme having the activity of catalyzing the reaction of converting acetovanillone to α-hydroxyacetovanillone, the acpC gene which contains a nucleotide sequence encoding the amino acid sequence of an enzyme having the activity of catalyzing the reaction of converting α-hydroxyacetovanillone to vanillic acid, a microorganism and transformed microorganism possessing these genes, and a production method using them. [Selected Figure] Figure 13B

Description

Application for application of Article 30, Paragraph 2 of the Patent Act has been filed. 68th Lignin Symposium, 5th Annual Meeting of the Lignin Society, November 10, 2023, presentation materials. 68th Lignin Symposium, 5th Annual Meeting of the Lignin Society (online), cover, table of contents, abstract, colophon, published on November 6, 2023.

The present invention relates to an acetovanillone converting enzyme that catalyzes the reaction of converting acetovanillone to vanillic acid via a vanillic acid intermediate, and a production system for useful substances such as vanillic acid that uses the enzyme.

In order to realize a carbon-neutral society, there is a demand to produce functional materials from non-edible biomass instead of petroleum-based raw materials. As part of this, development of cellulose derivatives and aliphatic polymer raw materials using non-edible biomass as raw materials is progressing. However, to date, almost no polymers are known that have satisfactory rigidity, heat resistance, etc.

Lignin is one type of non-edible biomass. Lignin is an amorphous polymeric substance that exists as a component of the vascular cell walls of plants, and is a complex condensation of phenylpropane-based structural units. Its major chemical structural feature is that it contains a methoxy group. Lignin functions to bond woody plant cells together and strengthen the tissue, and is present in wood at about 18% to 36% and in herbaceous plants at about 15% to 25%. Therefore, in order to make effective use of wood, attempts have been made to decompose lignin and obtain useful compounds. It is known that there are three types of lignin derived from biomass such as wood: p -hydroxyphenyl lignin (H-type lignin), guaiacyl lignin (G-type lignin), and syringyl lignin (S-type lignin).

Among the compounds obtained by decomposing lignin, there are some that can be used as monomers that have aromatic rings in the molecule. Polymers made from such aromatic monomers can have excellent functionality, such as rigidity and heat resistance.

One of the candidates for aromatic monomers derived from lignin is vanillic acid. Polymers obtained by polymerizing vanillic acid have a very high melting point. For example, highly functional polymers obtained from monomers obtained by modifying vanillic acid are known to have high melting points and high functionality. However, vanillic acid cannot be obtained at all by decomposing lignin.

Acetovanillone is one of the compounds obtained by decomposing lignin. Acetovanillone is the main aromatic monomer produced by the oxidative decomposition of lignin. Acetovanillone is produced from any plant biomass, regardless of whether it is coniferous, broadleaf, or herbaceous. For example, acetovanillone is also found in the waste liquid (black liquor) from kraft cooking, a wood pulp manufacturing process carried out at the industrial level. Therefore, if acetovanillone could be converted to vanillic acid, it would lead to an improvement in the yield of vanillic acid in the production of vanillic acid from lignin and an improvement in the purity of the target product.

For example, by using a microorganism capable of assimilating acetovanillone or an enzyme thereof, it may be possible to microbiologically or biochemically convert acetovanillone into vanillic acid. Microorganisms capable of assimilating acetovanillone that have been known thus far include Sphingobium sp. SYK-6 strain (e.g., Non-Patent Documents 1 and 2), Rhodococcus rhodochrous GD01 strain (e.g., Non-Patent Documents 3 and 4), Rhodococcus rhodochrous GD02 strain (e.g., Non-Patent Documents 3 and 4), and Arthrobacter sp. TGJ4 strain (e.g., Non-Patent Document 5). However, the details of the enzymatic mechanism involved in the conversion of acetovanillone have only been clarified for Sphingobium species SYK-6 strain (hereinafter also referred to as SYK-6 strain) and Rhodococcus rhodochrous GD02 strain (hereinafter also referred to as GD02 strain).

The SYK-6 strain is known to carboxylate acetovanillone and acetosyringone to vanilloyl acetic acid and 3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid, respectively, using an enzyme system encoded by six genes, namely, acvA gene, acvB gene , acvC gene , acvD gene, acvE gene, and acvF gene (e.g., Non-Patent Document 1). Furthermore, the SYK-6 strain can convert vanilloyl acetic acid and 3-(4-hydroxy-3,5-dimethoxyphenyl)-3-oxopropanoic acid to vanillic acid and syringic acid, respectively, by an enzyme system encoded by two genes, the vceA gene and the vceB gene (e.g., Non-Patent Document 2). Thus, the SYK-6 strain can produce vanillic acid from acetovanillone by the enzyme system encoded by the acetovanillone converting enzyme gene group consisting of a total of eight genes.

By introducing the above-mentioned eight genes of the SYK-6 strain into another host microorganism, vanillic acid can be synthesized from acetovanillone in a microorganism other than the SYK-6 strain. An example of such a host microorganism is the Pseudomonas sp. NGC7 strain (deposit number: NITE BP-03043; hereinafter, also referred to as the NGC7 strain) (for example, Patent Document 1). The NGC7 strain can degrade p -hydroxybenzoic acid and syringic acid, which are aromatic compounds derived from lignin. In addition, the NGC7 strain can degrade vanillic acid by possessing the vanillate O -demethylase gene. Therefore, in order to produce vanillic acid using the NGC7 strain, it is necessary to inactivate the vanillate O -demethylase gene on the chromosome.

Therefore, the present inventors attempted to introduce the above-mentioned eight genes of the SYK-6 strain into the NGC7 strain in which the vanillate O -demethylase gene on the chromosome had been inactivated, and to produce vanillic acid from acetovanillone using the resulting transformed microorganism (see, for example, Patent Document 2).

International Publication No. 2020/080467 International Publication No. 2022/210236

Higuchi et al., 08 Aug 2022, Applied and Environmental Microbiology, Vol. 88, No. 16, e0072422. Higuchi et al., Sep 2019, Metabolic Engineering, Vol. 55, 258-267. Navas et al., 10 Sep 2021, Frontiers in Microbiology, Vol. 12, 735000. Dexter et al., 18 Oct 2022, Proceedings of the National Academy of Sciences, Vol. 119, No.43, e2213450119 Tanihata et al., 23 Apr 2012, Bioscience, Biotechnology, and Biochemistry, Vol. 76, No. 4, 838-840. Abe et al., 15 Mar 2005, Journal of Bacteriology, Vol. 187, No. 6, 2030-2037.

However, according to the inventors' investigations, there is a problem in that the conversion reaction from acetovanillone to vanillic acid using the acetovanillone converting enzyme gene group of the SYK-6 strain is very slow. In addition, the SYK-6ΔligMΔdesA strain described in Non-Patent Document 6, which is a vanillic acid accumulating strain derived from the SYK-6 strain, does not decompose vanillic acid, but has lost the ability to decompose syringic acid, an analog of vanillic acid, and cannot grow using aromatic compounds derived from H-type lignin as a carbon source in the absence of methionine. Therefore, when trying to produce vanillic acid using the SYK-6 strain, broad-leaved trees containing a large amount of S-type lignin cannot be used, and the type of biomass that can be used is limited.

The transformed NGC7 strain, which is obtained by inactivating the vanillate O -demethylase gene described in Patent Document 2 and introducing the acetovanillone enzyme gene group of the SYK-6 strain, can produce vanillic acid from acetovanillone. The transformed NGC7 strain thus obtained maintains the assimilation properties of the NGC7 strain, can decompose syringic acid, and can grow using aromatic compounds derived from H-type lignin as a carbon source.

However, in the above-mentioned transformed NGC7 strain, the activity of the enzymes encoded by the acetovanillone conversion enzyme gene group of the SYK-6 strain is low, so the conversion reaction from acetovanillone to vanillic acid is still slow. Furthermore, the acetovanillone enzyme gene group of the SYK-6 strain is composed of as many as eight genes, and there is a problem that transforming other microorganisms with this acetovanillone enzyme gene group is time-consuming and complicated.

In addition, to date, no gene group capable of converting acetovanillone to vanillic acid with a smaller number of genes than the acetovanillone conversion enzyme gene group possessed by the SYK-6 strain has been known, nor has any microorganism possessing such a gene group been known.

The problem that the present invention aims to solve is to provide a gene group that is composed of fewer genes than the acetovanillone conversion enzyme gene group possessed by Sphingobium species SYK-6 strain and that can convert acetovanillone to vanillic acid with high efficiency, a microorganism that possesses the gene group, and a method for producing vanillic acid from acetovanillone using the same.

In an attempt to solve the above problems, the present inventors collected soil samples from all over Japan and conducted extensive research into isolating microorganisms that have the potential to assimilate acetovanillone. As a result, they obtained several strains of Pseudomonas microorganisms that can grow using acetovanillone as the sole carbon source, and among these they succeeded in selecting and isolating strains with good growth potential.

The isolated Pseudomonas microorganism was examined for the presence of an acetovanillone conversion enzyme gene cluster similar to that of the SYK-6 strain, and surprisingly, no genes with high sequence identity to the individual genes of the acetovanillone conversion enzyme gene cluster were found. In other words, it was presumed that the isolated Pseudomonas microorganism metabolizes acetovanillone using an enzyme system different from that of the SYK-6 strain.

After further trial and error, the present inventors finally succeeded in discovering the presence of an acetovanillone conversion enzyme gene cluster in the isolated Pseudomonas microorganism. Surprisingly, this acetovanillone conversion enzyme gene cluster was comprised of a total of three types of genes: two types of genes encoding enzymes with the activity of catalyzing the reaction of converting acetovanillone to α-hydroxyacetovanillone, and one type of gene encoding an enzyme with the activity of catalyzing the reaction of converting α-hydroxyacetovanillone to vanillic acid.

When this acetovanillone converting enzyme gene group was introduced into the transformed NGC7 strain in which the vanillate O -demethylase gene had been inactivated, it was surprisingly possible to produce vanillic acid from acetovanillone with a higher efficiency than in the case where the transformed NGC7 strain described in Patent Document 2 was used.

Furthermore, the isolated Pseudomonas microorganism had the property of assimilating vanillic acid. Thus, the present inventors, through repeated trial and error, discovered the presence of a vanillate O 2 -demethylase gene in the isolated Pseudomonas microorganism, and when the vanillate O 2 -demethylase gene was inactivated, vanillic acid could be produced from acetovanillone.

Based on the above findings and success stories, the present inventors have finally succeeded in creating a gene group capable of converting acetovanillone to vanillic acid with high efficiency and with a small number of associated genes, a microorganism possessing said gene group, and a method for producing vanillic acid from acetovanillone using them, as a solution to the problems of the present invention. The present invention was completed based on the findings and success stories first obtained by the present inventors.

Therefore, according to one aspect of the present invention, the following embodiments are provided.
[1] A transformed microorganism into which the acpA gene and the acpB gene have been introduced as foreign genes and which expresses the introduced genes.
[2] The transformed microorganism according to [1], wherein the host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043).
[3] A method for producing α-hydroxyacetovanillone, comprising the step of allowing acetovanillone to act on an expression product of the acpA gene and the acpB gene, or on the transformed microorganism according to [1] or [2], to obtain α-hydroxyacetovanillone.
[4] A transformed microorganism into which the acpA gene, the acpB gene, and the acpC gene have been introduced as foreign genes, and which expresses the introduced genes.
[5] The transformed microorganism according to [4], wherein the host microorganism is a transformed microorganism of Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043) in which the vanillate O -demethylase gene on the chromosome is deleted.
[6] A method for producing vanillic acid, comprising a step of allowing acetovanillone to act on an expression product of the acpA gene, the acpB gene and the acpC gene, or on the transformed microorganism according to [4] or [5], to obtain vanillic acid.
[7] The transformed microorganism according to [4] or [5], wherein the transformed microorganism is a transformed microorganism of Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043) in which the pobA gene on the chromosome of the host microorganism is deleted.
[8] A method for producing 4-hydroxybenzoic acid, comprising the step of allowing 4'-hydroxyacetophenone to act on an expression product of the acpA gene, the acpB gene and the acpC gene, or allowing it to act on the transformed microorganism according to [7], to obtain 4-hydroxybenzoic acid.
[9] An acpA gene comprising any one of the nucleotide sequences (1) to (4) below, the nucleotide sequence encoding the amino acid sequence of an enzyme having an activity of catalyzing a reaction of converting acetovanillone to α- hydroxyacetovanillone and/or an activity of catalyzing a reaction of converting 4'-hydroxyacetophenone to 2,4'-dihydroxyacetophenone:
(1) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 1 or 4 in the sequence listing or a nucleotide sequence complementary to said nucleotide sequence, and which has 1 to 10 base deletions, substitutions and/or additions per unit of 100 bases in said nucleotide sequence set forth in SEQ ID NO: 1 or 4. (2) A nucleotide sequence which has 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or 4. (3) A nucleotide sequence which encodes an amino acid sequence which has 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 50. Nucleotide sequence (4) A nucleotide sequence encoding an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and/or added per unit consisting of 100 amino acids in the amino acid sequence set forth in SEQ ID NO:50. [10] An acpB gene, which is any of the nucleotide sequences of (1) to (4) below, and which contains a nucleotide sequence encoding the amino acid sequence of an enzyme having an activity of catalyzing a reaction of converting acetovanillone to α- hydroxyacetovanillone and/or an activity of catalyzing a reaction of converting 4'-hydroxyacetophenone to 2,4'-dihydroxyacetophenone:
(1) A nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 2 or 5 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence, and has 1 to 10 base deletions, substitutions and/or additions per unit of 100 bases in the nucleotide sequence set forth in SEQ ID NO: 2 or 5. (2) A nucleotide sequence having 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2 or 5. (3) A nucleotide sequence encoding an amino acid sequence having 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 51. (4) A nucleotide sequence encoding an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and/or added per unit of 100 amino acids in the amino acid sequence set forth in SEQ ID NO: 51. [11] Pseudomonas sp. MHK4 strain (Accession No.: NITE P-03794).
[12] The host microorganism is Pseudomonas sp. MHK4 strain (accession number: NITE P-03794), and the vanillate O -demethylase gene on the chromosome is deleted.
Transformed microorganisms.
[13] The transformed microorganism according to [12], wherein the vanillate O 2 -demethylase gene is at least one vanillate O 2 -demethylase gene selected from the group consisting of a vanA3 gene (SEQ ID NO: 23) and a vanB3 gene (SEQ ID NO: 24).
[14] A method for producing vanillic acid, comprising a step of obtaining vanillic acid by allowing acetovanillone to act on the transformed microorganism according to [12] or [13].
[15] The host microorganism is Pseudomonas sp. MHK4 strain (accession number: NITE P-03794), and the 4-hydroxybenzoate monooxygenase gene on the chromosome is deleted.
Transformed microorganisms.
[16] The transformed microorganism according to [15], wherein the 4-hydroxybenzoate monooxygenase gene is at least one 4-hydroxybenzoate monooxygenase gene selected from the group consisting of a PobA1 gene (SEQ ID NO: 61) and a pobA2 gene (SEQ ID NO: 62).
[17] A method for producing 4-hydroxybenzoic acid, comprising a step of obtaining 4-hydroxybenzoic acid by allowing 4'-hydroxyacetophenone to act on the transformed microorganism according to claim 15 or 16.

According to another aspect of the present invention, the following embodiments are provided.
[1] An acpA gene comprising any one of the following nucleotide sequences (1) to (4), the nucleotide sequence encoding the amino acid sequence of an enzyme having an activity for catalyzing a reaction for converting acetovanillone to α - hydroxyacetovanillone:
(1) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 1 or 4 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence, and in the nucleotide sequence set forth in SEQ ID NO: 1 or 4, has deletion, substitution and/or addition of 1 to 10 bases per unit consisting of 100 bases. (2) A nucleotide sequence which has 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or 4. (3) A nucleotide sequence which encodes an amino acid sequence which has 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 50. (4) A nucleotide sequence which encodes an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and/or added per unit consisting of 100 amino acids in the amino acid sequence set forth in SEQ ID NO: 50. [2] An acpB gene, which is any of the nucleotide sequences set forth in (1) to (4) below, and which comprises a nucleotide sequence which encodes the amino acid sequence of an enzyme having the activity of catalyzing a reaction for converting acetovanillone to α - hydroxyacetovanillone.
(1) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 2 or 5 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence, and in the nucleotide sequence set forth in SEQ ID NO: 2 or 5, has deletion, substitution and/or addition of 1 to 10 bases per unit consisting of 100 bases. (2) A nucleotide sequence which has 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2 or 5. (3) A nucleotide sequence which encodes an amino acid sequence which has 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 51. (4) A nucleotide sequence which encodes an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and/or added per unit consisting of 100 amino acids in the amino acid sequence set forth in SEQ ID NO: 51. [3] A transformed microorganism into which the acpA gene and the acpB gene set forth in [1] to [2] have been introduced as foreign genes and which expresses the introduced genes.
[4] The transformed microorganism according to [3], wherein the host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043).
[5] A method for producing α-hydroxyacetovanillone, comprising the step of allowing acetovanillone to act on the expression products of the acpA gene and the acpB gene described in [1] and [2], or on the transformed microorganism described in [3], to obtain α-hydroxyacetovanillone.
[6] The transformed microorganism according to [3], further comprising an acpC gene, which is any one of the nucleotide sequences (1) to (4) below and which encodes an amino acid sequence of an enzyme having an activity for catalyzing a reaction for converting α-hydroxyacetovanillone to vanillic acid, introduced as a foreign gene, and which expresses the introduced gene:
(1) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 3 or 6 in the sequence listing or a nucleotide sequence complementary to the nucleotide sequence, and in the nucleotide sequence set forth in SEQ ID NO: 3 or 6, has deletion, substitution and/or addition of 1 to 10 bases per unit consisting of 100 bases. (2) A nucleotide sequence which has 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 3 or 6. (3) A nucleotide sequence which encodes an amino acid sequence which has 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 52. (4) A nucleotide sequence which encodes an amino acid sequence in which 1 to 10 amino acids have been deleted, substituted and/or added per unit consisting of 100 amino acids in the amino acid sequence set forth in SEQ ID NO: 52. [7] The transformed microorganism according to [6], wherein the host microorganism is Pseudomonas sp. NGC7 strain (Accession No.: NITE BP-03043).
[8] The transformed microorganism according to [6], wherein the host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043) lacking a vanillate O -demethylase gene on its chromosome.
[9] A method for producing vanillic acid, comprising a step of allowing acetovanillone to act on an expression product of the acpA gene, the acpB gene, and the acpC gene according to [1], [2], and [6], or on the transformed microorganism according to [6], to obtain vanillic acid.
[10] Pseudomonas sp. MHK4 strain (accession number: NITE P-03794).
[11] The host microorganism is Pseudomonas sp. MHK4 strain (accession number: NITE P-03794), and the vanillate O -demethylase gene on the chromosome is deleted.
Transformed microorganisms.
[12] The transformed microorganism according to [11], wherein the vanillate O 2 -demethylase gene is at least one vanillate O 2 -demethylase gene selected from the group consisting of vanA3 gene (SEQ ID NO: 23) and vanB3 gene (SEQ ID NO: 24).
[13] A method for producing vanillic acid, comprising a step of allowing acetovanillone to act on the transformed microorganism according to [10] or [11] to obtain vanillic acid.

According to the present invention, acetovanillone can be converted to vanillic acid with higher efficiency than when the acetovanillone conversion enzyme gene group possessed by the Sphingobium species SYK-6 strain is used. Furthermore, according to the present invention, since fewer genes are used than the acetovanillone conversion enzyme gene group possessed by the SYK-6 strain, acetovanillone can be easily converted to vanillic acid.

Acetovanillone is a major aromatic compound contained in the waste liquid (black liquor) after kraft cooking and soda cooking, which are lignin decomposition methods currently used at the industrial level in wood pulp manufacturing processes. However, black liquor generated during wood pulp manufacturing processes is concentrated and then incinerated in a recovery boiler, and is only used as a heat source. According to the present invention, it is expected that acetovanillone, which has been treated as an industrial by-product until now, can be turned into a resource through conversion to vanillic acid, a useful substance. Furthermore, according to the present invention, vanillic acid can be produced easily and efficiently from acetovanillone obtained on an industrial scale, making it possible to produce vanillic acid on an industrial scale.

FIG. 1A is a copy of the accession certificate for accession number NITE P-03794 (Pseudomonas sp. strain MHK4). FIG. 1B is a copy of the certificate of viability of the accession number NITE P-03794 (Pseudomonas sp. strain MHK4). 2 shows the results of conversion of acetovanillone (AV) to α-hydroxyacetovanillone using the NGC7Δ aph (pSEVA acpAB ) strain (cell suspension adjusted to an OD ₆₀₀ of 10.0), as described in the Examples below. (A) HPLC chromatogram immediately after the start of the reaction. (B) HPLC chromatogram 8 hours after the start of the reaction. (C) HPLC-MS (ESI-MS) analysis results of α-hydroxyacetovanillone produced 8 hours after the start of the reaction. 3 shows the results of conversion of acetovanillone (AV) to vanillic acid using acetovanillone (AV) and the NGC7Δ aph [pSEVA acpCAB ] strain (cell suspension adjusted to an OD ₆₀₀ of 10.0), as described in the Examples below. (A) HPLC chromatogram immediately after the start of the reaction. (B) HPLC chromatogram 2 hours after the start of the reaction. 4 shows the results of a reaction using an AcpA crude enzyme solution and acetovanillone (AV) as described in the Examples below. (A) HPLC chromatogram immediately after the start of the reaction. (B) HPLC chromatogram 30 minutes after the start of the reaction. 5 shows the results of a reaction using an AcpB crude enzyme solution and acetovanillone (AV) as described in the Examples below. (A) HPLC chromatogram immediately after the start of the reaction. (B) HPLC chromatogram 30 minutes after the start of the reaction. 6 shows the results of the reaction using the AcpA crude enzyme solution, the AcpB crude enzyme solution, and acetovanillone (AV) as described in the Examples below. (A) HPLC chromatogram immediately after the start of the reaction. (B) HPLC chromatogram 30 minutes after the start of the reaction. 7 shows the results of reactions using AcpA crude enzyme solution, AcpB crude enzyme solution, AcpC crude enzyme solution, and acetovanillone (AV) as described in the Examples below. (A) HPLC chromatogram immediately after the start of the reaction. (B) HPLC chromatogram 30 minutes after the start of the reaction. FIG. 8A is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC7Δ aph [pSEVA acv +pTS093 vce ] strain, as described in the Examples below. FIG. 8B is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC7Δ aph [pSEVA acpCAB ^opt ] strain, as described in the Examples below. FIG. 9 is a photograph of an agar medium in which the NGC7Δ aph [pSEVA acv +pTS093 vce ] strain and the NGC7Δ aph [pSEVA acpCAB ^opt ] strain were cultured in a medium containing acetovanillone as a carbon source, as described in the Examples below. FIG. 10A is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC729[pSEVA acpCAB ^opt ] strain (cell suspension adjusted to an OD ₆₀₀ of 5.0), as described in the Examples below. FIG. 10B is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC729::P _tac : acpCAB ^opt strain (cell suspension adjusted to an OD ₆₀₀ of 5.0), as described in the Examples below. FIG. 11A is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC729[pSEVA acpCAB ^opt ] strain (cell suspension adjusted to an OD ₆₀₀ of 1.0), as described in the Examples below. FIG. 11B is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC729::P _tac : acpCAB ^opt strain (cell suspension adjusted to an OD ₆₀₀ of 1.0), as described in the Examples below. FIG. 12A is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC729 [pSEVA acv +pTS093 vce ] strain (cell suspension adjusted to an OD ₆₀₀ of 5.0), as described in the Examples below. FIG. 12B is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the NGC729 [pSEVA acv ^opt +pTS093 vce ^opt ] strain (cell suspension adjusted to an OD ₆₀₀ of 5.0), as described in the Examples below. FIG. 13A is a graph showing the results of evaluating the decomposition ability of lignin decomposition products by the NGC729 [pSEVA acv +pTS093 vce ] strain and measuring the acetovanillone (AV) concentration, vanillin (VN) concentration, vanillic acid (VA) concentration, glucose concentration, and OD ₆₀₀ over time, as described in the Examples below. FIG. 13B is a graph showing the results of evaluating the decomposition ability of lignin decomposition products by the NGC729::P _tac : acpCAB ^opt strain and measuring the acetovanillone (AV) concentration, vanillin (VN) concentration, vanillic acid (VA) concentration, glucose concentration, and OD ₆₀₀ over time, as described in the Examples below. FIG. 14 is a diagram showing the results of vanillic acid (VA) decomposition using the MHK4 strain and the MHK4Δ vanA3B3 strain (cell suspensions adjusted to give an OD ₆₀₀ of 5.0), as described in the Examples below. FIG. 15 is a diagram showing the results of conversion of acetovanillone (AV) to vanillic acid (VA) using the MHK4Δ vanA3B3 strain (a cell suspension adjusted to give an OD ₆₀₀ of 5.0), as described in the Examples below. FIG. 16 is a diagram showing the results of decomposition of acetovanillone (AV) using the MHK4 strain and the SYK-6 strain, as described in the Examples below. FIG. 17ABCDEFGH shows the results of enzyme reaction using acetovanillone (AV), acetophenone, 4'-hydroxyacetophenone (HAP), 3',5'-dimethoxy-4'-hydroxyacetophenone, 3',4'-dihydroxyacetophenone, 3',4'-dimethoxyacetophenone, 3'-hydroxy-4'-methoxyacetophenone and 3'-methoxyacetophenone as substrates by the AcpAB enzyme, as described in the Examples below. FIG. 17IJKLMNOP shows the results of enzyme reaction using 3'-hydroxyacetophenone, 3',4',5'-trimethoxyacetophenone, 4'-hydroxypropiophenone, 4'-hydroxybutyrophenone, 2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, vanillic acid (VA), and 4'-hydroxybenzoic acid (HBA) as substrates with the AcpAB enzyme, as described in the Examples below. As described in the Examples below, Fig. 18 shows the results of an enzyme reaction using α-hydroxyacetovanillone as a substrate and an AcpC crude enzyme solution. Fig. 18A shows the results immediately after the start of the reaction, and Fig. 18B shows the results one hour after the start of the reaction. As described in the Examples below, Fig. 19 shows the results of an enzyme reaction using 2,4'-dihydroxyacetophenone as a substrate and an AcpC crude enzyme solution. Fig. 19A shows the results immediately after the start of the reaction, and Fig. 19B shows the results one hour after the start of the reaction. 20A and 20B show the results of an enzyme reaction using an AcpC crude enzyme solution with α-hydroxyacetosyringone as a substrate, as described in the Examples below. Fig. 20A shows the results immediately after the start of the reaction, and Fig. 20B shows the results one hour after the start of the reaction. FIG. 21 is a diagram showing the results of a conversion reaction from 4'-hydroxyacetophenone (HAP) to 4-hydroxybenzoic acid (HBA) using the NGC7Δ pobA [pSEVA acpCAB ^opt ] strain, as described in the Examples below. FIG. 22 is a diagram showing the results of a conversion reaction from a mixture of 4'-hydroxyacetophenone (HAP) and acetovanillone (AV) to 4'-hydroxybenzoic acid (HBA) using the NGC7Δ pobA [pSEVA acpCAB ^opt ] strain, as described in the Examples below.

Each aspect of the present invention will be described in detail below, but the present invention is not limited to the items in this section, and various aspects may be adopted as long as the object of the present invention is achieved.

Unless otherwise specified, each term in this specification is used in the sense commonly used by those skilled in the art in technical fields such as microbiology, and should not be construed as having an unduly restrictive meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' knowledge and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

"Comprising" means that elements other than the elements explicitly stated as being included can be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." That is, "comprising" can mean including the elements explicitly stated and any one or more elements, consisting of the elements explicitly stated, or consisting essentially of the elements explicitly stated. Elements include limitations such as ingredients, steps, conditions, parameters, etc.
The term "and/or" means any one or any or all combinations of two or more of the associated listed items.
In a numerical range, "to" means a range including the numerical values before and after it, for example, "0% to 100%" means a range equal to or greater than 0% and equal to or less than 100%. "More than" and "less than" mean a lower limit and an upper limit, respectively, without including the numerical value before it, for example, "more than 1" means a numerical value greater than 1, and "less than 100" means a numerical value less than 100.
The number of digits in an integer value is the same as the number of significant digits. For example, 1 has one significant digit, and 10 has two significant digits. Also, the number of digits after the decimal point in a decimal value is the same as the number of significant digits. For example, 0.1 has one significant digit, and 0.10 has two significant digits.

"Gene deletion" means that a gene does not function normally, preventing expression of the gene, such as when the gene is not normally transcribed or when an enzyme (protein) that should be produced by expression of the gene is not normally translated. Gene deletion can occur, for example, when the structure of a gene is changed by destruction, deletion, replacement, introduction, or the like of all or part of the gene. However, gene deletion can also occur when gene expression is suppressed without any change in the structure of the gene, for example, by blocking the control region of the gene.
"Gene expression" means that an enzyme having an amino acid sequence encoded by the nucleotide sequence of a gene (an enzyme encoded by a gene) is produced with its original structure and activity through transcription, translation, etc. "Gene expression product" means an enzyme produced corresponding to a gene.
"Overexpression of a gene" means that, as a result of the introduction of a gene, an enzyme encoded by the gene is produced in an amount exceeding that which is naturally expressed by the host microorganism.
"Wild-type microorganism" refers to a naturally occurring microorganism that has not been artificially genetically modified. "Wild-type gene" refers to a gene that is naturally present on the genomic DNA of a wild-type microorganism. "Wild-type enzyme" refers to an enzyme encoded by a wild-type gene. In this specification, genome and chromosome are synonymous.
The term "foreign gene" refers to a gene that is not originally present in the genomic DNA of the microorganism to be introduced, and is also called a heterologous gene.
"High efficiency" conversion means rapid conversion of one substance to another and/or substantially complete conversion of one substance to another.

(Characteristics of the acetovanillone converting enzyme gene cluster)
The present inventors have isolated Pseudomonas sp. MHK4 strain (accession number: NITE P-03794) (hereinafter, also referred to as MHK4 strain) as a microorganism having a new acetovanillone converting enzyme gene cluster. Furthermore, the present inventors have elucidated that the acetovanillone converting enzyme gene cluster possessed by the MHK4 strain consists of three genes, the acpA gene, the acpB gene, and the acpC gene.

The acetovanillone converting enzyme gene cluster possessed by the MHK4 strain has the following characteristics in producing vanillic acid from acetovanillone, compared to the acetovanillone converting enzyme gene cluster consisting of the acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, and acvF gene, as well as the vceA gene and vceB gene derived from Sphingobium sp. SYK-6 strain (hereinafter also referred to as SYK-6 strain):

When the acetovanillone converting enzyme gene group of the SYK-6 strain is used, acetovanillone is converted to vanillic acid through at least five reaction steps: (1) phosphorylation of acetovanillone, (2) dephosphorylation of the phosphorylated product, (3) carboxylation of the dephosphorylated product, (4) Cα-Cβ cleavage accompanied by CoA addition to the carboxylated product, and (5) hydrolysis of the CoA thioester. In contrast, when the acetovanillone converting enzyme gene group of the MHK4 strain is used, acetovanillone can be converted to vanillic acid through only two reaction steps: (1) hydroxylation of the acetyl group of acetovanillone and (2) oxidative cleavage of the hydroxymethyl group of α-hydroxyacetovanillone. Therefore, it is expected that acetovanillone can be converted to vanillic acid more quickly by using the acetovanillone converting enzyme gene group of the MHK4 strain.

When the acetovanillone conversion enzyme gene group of the SYK-6 strain is used, a total of two molecules of ATP are required to convert acetovanillone to vanillic acid. In contrast, when the acetovanillone conversion enzyme gene group of the MHK4 strain is used, no ATP is required to convert acetovanillone to vanillic acid. Therefore, by using the acetovanillone conversion enzyme gene group of the MHK4 strain, it is expected that the microorganism will be able to metabolize and decompose acetovanillone while reducing intracellular ATP consumption.

When the acetovanillone converting enzyme gene group of the SYK-6 strain is used, a total of eight enzyme genes are introduced into the host microorganism to produce a transformed microorganism. In contrast, when the acetovanillone converting enzyme gene group of the MHK4 strain is used, a total of three enzyme genes are introduced into the host microorganism to produce a transformed microorganism. Therefore, when a plasmid incorporating the acetovanillone converting enzyme gene group is introduced into a host microorganism, the acetovanillone converting enzyme gene group of the SYK-6 strain cannot be incorporated entirely into one type of plasmid, whereas the acetovanillone converting enzyme gene group of the MHK4 strain can be incorporated entirely into one type of plasmid.

Due to the difference in the mechanism of action of the acetovanillone converting enzyme gene cluster between the SYK-6 strain and the MHK4 strain as described above, when acetovanillone is converted to vanillic acid by a transformed microorganism obtained by introducing a plasmid incorporating the acetovanillone converting enzyme gene cluster into a host microorganism, Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043), the acetovanillone converting enzyme gene cluster of the MHK4 strain can produce vanillic acid from acetovanillone simply and efficiently, compared to the acetovanillone converting enzyme gene cluster of the SYK-6 strain.

The only microbial metabolic systems for acetovanillone produced by the oxidative decomposition of lignin at the industrial level that had been elucidated were SYK-6 and GD02 strains, which have an acetovanillone conversion enzyme gene cluster similar to that of SYK-6. When using the acetovanillone conversion enzyme gene cluster of SYK-6 and other strains, the number of genes is large and complex, and the conversion rate of acetovanillone is slow, so that it has not been possible to contribute to the production of useful substances from acetovanillone. However, when using the acetovanillone conversion enzyme gene cluster of the MHK4 strain, acetovanillone can be converted to vanillic acid quickly with fewer genes compared to the acetovanillone conversion enzyme gene cluster of SYK-6 and other strains, and it is expected that useful substances can be produced from acetovanillone easily and efficiently.

( acpA gene and acpB gene)
Conversion of acetovanillone to vanillic acid by the acetovanillone converting enzyme gene cluster possessed by the MHK4 strain is presumed to be carried out as shown in the following scheme (I).

Scheme (I)

First, acetovanillone is converted to α-hydroxyacetovanillone by AcpA enzyme and AcpB enzyme, which are the gene products of the acpA gene and the acpB gene. Next, α-hydroxyacetovanillone is converted to vanillic acid by AcpC enzyme, which is the gene product of the acpC gene. In addition, 4'-hydroxyacetophenone (HAP) is converted to 2,4'-dihydroxyacetophenone (DHAP) by AcpA enzyme and AcpB enzyme. Next, DHAP is converted to 4-hydroxybenzoic acid (HBA) by AcpC enzyme.

It is highly likely that the AcpA enzyme and the AcpB enzyme do not function independently, but rather function as a single enzyme (AcpAB enzyme) by forming a complex as a subcomponent. For convenience, the gene products of the acpA gene and the acpB gene are referred to as the AcpA enzyme and the AcpB enzyme, respectively, in this specification, but in fact they may be a complex of the AcpAB enzyme, and this possibility is not excluded.

In one embodiment of the present invention, the acpA gene is a gene comprising a nucleotide sequence encoding the amino acid sequence of the AcpA enzyme.In one embodiment of the present invention, the acpB gene is a gene comprising a nucleotide sequence encoding the amino acid sequence of the AcpB enzyme.

The AcpA enzyme and the AcpB enzyme are enzymes that have the activity of catalyzing the reaction of converting acetovanillone to α-hydroxyacetovanillone. In addition, the AcpA enzyme and the AcpB enzyme have the activity of decomposing HAP, 3',4'-dihydroxyacetophenone, 4'-hydroxypropiophenone, and 4'-hydroxybutyrophenone. For example, the AcpA enzyme and the AcpB enzyme have the activity of catalyzing the reaction of converting HAP to DHAP. As long as the AcpA enzyme and the AcpB enzyme have the activity, there are no particular limitations on the amino acid sequences of the enzymes.

The amino acid sequence of the AcpA enzyme encoded by the acpA gene of the MHK4 strain is the amino acid sequence set forth in SEQ ID NO: 50. The amino acid sequence of the AcpB enzyme encoded by the acpB gene of the MHK4 strain is the amino acid sequence set forth in SEQ ID NO: 51.

The amino acid sequences of the AcpA enzyme and the AcpB enzyme may be those of the wild-type enzymes, which have one to several amino acid deletions, substitutions, additions, etc. Here, the range of "one to several" in "one to several amino acid deletions, substitutions, additions" of the amino acid sequence is not particularly limited, but for example, if 100 amino acids in the amino acid sequence are one unit, the number of amino acids per unit is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and even more preferably 1, 2, 3, 4, or 5. In addition, "amino acid deletion" means the absence or disappearance of an amino acid residue in the sequence, "amino acid substitution" means that an amino acid residue in the sequence is replaced with another amino acid residue, and "amino acid addition" means that a new amino acid residue is added to the sequence so as to be inserted.

Specific examples of "deletion, substitution, or addition of one or several amino acids" include replacement of one or several amino acids with other chemically similar amino acids. For example, a hydrophobic amino acid may be replaced with another hydrophobic amino acid, or a polar amino acid may be replaced with another polar amino acid having the same charge. Such chemically similar amino acids are known in the art for each amino acid. Specific examples include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

Examples of amino acid sequences having one or several amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the wild-type enzyme include amino acid sequences having a certain level of sequence identity with the amino acid sequence of the wild-type enzyme, such as amino acid sequences having a sequence identity of 50% or more, preferably 60% or more or 65% or more, more preferably 70% or more or 75% or more, even more preferably 80% or more or 85% or more, and even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of the wild-type enzyme. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

As described in the Examples below, the acpA gene and the acpB gene encoding the AcpA enzyme and the AcpB enzyme, respectively, possessed by the MHK4 strain exhibited sequence identities of 33.6% and 44.1% with the gene encoding the oxygenase component ( VanA ) and the gene encoding the oxidoreductase component (VanB) of the oxygen-adding vanillate O -demethylase derived from the Pseudomonas putida KT2440 strain. From these facts, it is presumed that the AcpA enzyme and the AcpB enzyme possessed by the MHK4 strain are monooxygenases, specifically, the AcpA enzyme is the oxygenase component and the AcpB enzyme is the oxidoreductase component. Therefore, an enzyme having an amino acid sequence that has 50% or more sequence identity with the amino acid sequence of the AcpA enzyme possessed by the MHK4 strain and that is presumed to be the oxygenase component of a monooxygenase may be used as the AcpA enzyme; an enzyme having an amino acid sequence that has 50% or more sequence identity with the amino acid sequence of the AcpB enzyme possessed by the MHK4 strain and that is presumed to be the oxidoreductase component of a monooxygenase may be used as the AcpB enzyme.

The method for obtaining the AcpA enzyme and the AcpB enzyme is not particularly limited, but examples thereof include a method including culturing a wild-type microorganism such as the MHK4 strain in which expression of the AcpA enzyme and the AcpB enzyme is observed, or a transformed microorganism into which genes encoding the amino acid sequences of the AcpA enzyme and the AcpB enzyme have been introduced, and then recovering the AcpA enzyme and the AcpB enzyme as a culture supernatant, as a microbial disruptant after culture, or by purifying them from these. For example, when the AcpA enzyme and the AcpB enzyme are present in the culture supernatant, an enzyme concentrate containing the AcpA enzyme and the AcpB enzyme can be obtained from the culture supernatant by means such as ammonium sulfate precipitation, and then the AcpA enzyme and the AcpB enzyme can be separated by means such as gel filtration chromatography and SDS-PAGE using the molecular weight as an indicator. The theoretical molecular weights calculated from the constituent elements of the AcpA enzyme and the AcpB enzyme having the amino acid sequences shown in SEQ ID NOs: 50 and 51 are approximately 39,000 and approximately 35,000, respectively.

The acpA gene and the acpB gene in one embodiment of the present invention are not particularly limited as long as they contain nucleotide sequences that code for the amino acid sequences of the AcpA enzyme and the AcpB enzyme, respectively. The AcpA enzyme and the AcpB enzyme are produced by expressing the acpA gene and the acpB gene in an organism.

The acpA gene and the acpB gene may be wild-type genes, or may be genes having a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene.

"A nucleotide sequence that hybridizes under stringent conditions" refers to a nucleotide sequence of DNA obtained by using a DNA having the nucleotide sequence of a wild-type gene as a probe and employing a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like.

"Stringent conditions" are conditions under which the signal of a specific hybrid is clearly distinguished from that of a nonspecific hybrid, and vary depending on the hybridization system used and the type, sequence, and length of the probe. Such conditions can be determined by changing the hybridization temperature and the washing temperature and salt concentration. For example, if even nonspecific hybrid signals are strongly detected, specificity can be increased by raising the hybridization and washing temperatures and, if necessary, lowering the washing salt concentration. Also, if no specific hybrid signals are detected, the hybrids can be stabilized by lowering the hybridization and washing temperatures and, if necessary, increasing the washing salt concentration.

Specific examples of stringent conditions include using a DNA probe as the probe, hybridization using 5xSSC, 1.0% (w/v) blocking reagent for nucleic acid hybridization (Roche Diagnostics), 0.1% (w/v) N-lauroyl sarcosine, and 0.02% (w/v) SDS overnight (approximately 8 to 16 hours). Washing is performed twice for 15 minutes using 0.1 to 0.5xSSC, 0.1% (w/v) SDS, preferably 0.1xSSC, 0.1% (w/v) SDS. The temperature for hybridization and washing is 65°C or higher, preferably 68°C or higher.

Examples of genes having a nucleotide sequence that hybridizes under stringent conditions include DNA obtained by hybridizing under the above-mentioned stringent conditions using a filter on which DNA having the nucleotide sequence of a wild-type gene derived from a colony or plaque or a fragment of said DNA is immobilized, and DNA that can be identified by hybridizing at 40°C to 75°C in the presence of 0.5M to 2.0M NaCl, preferably at 65°C in the presence of 0.7M to 1.0M NaCl, and then washing the filter at 65°C using 0.1 to 1x SSC solution (1x SSC solution is 150 mM sodium chloride, 15 mM sodium citrate). Methods for preparing probes and hybridization are described in Molecular Cloning: A Laboratory Manual, 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 (hereinafter, these documents may be referred to as reference technical documents). In addition to such conditions as the salt concentration and temperature of the buffer, a person skilled in the art can appropriately set conditions for obtaining a gene having a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene, taking into account other conditions such as probe concentration, probe length, and reaction time.

Examples of genes containing a nucleotide sequence that hybridizes under stringent conditions include genes that have a certain level of sequence identity with the nucleotide sequence of a gene having the nucleotide sequence of a wild-type gene used as a probe, such as genes having a nucleotide sequence that is 50% or more, preferably 60% or more or 65% or more, more preferably 70% or more or 75% or more, even more preferably 80% or more or 85% or more, and even more preferably 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the nucleotide sequence of a wild-type gene. The upper limit of the sequence identity is not particularly limited, and is typically 100%.

Nucleotide sequences that hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of a wild-type gene include, for example, nucleotide sequences having one to several, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, more preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and even more preferably 1, 2, 3, 4 or 5 nucleotide deletions, substitutions, additions, etc., per unit of the nucleotide sequence of the wild-type gene, where 100 nucleotides in the nucleotide sequence are taken as one unit. Here, "nucleotide deletion" means that a nucleotide in the sequence is missing or lost, "nucleotide substitution" means that a nucleotide in the sequence is replaced with another nucleotide, and "nucleotide addition" means that a new nucleotide is added so as to be inserted.

An enzyme encoded by a gene having a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of a wild-type gene is likely to be an enzyme having an amino acid sequence that has one or several amino acid deletions, substitutions, additions, etc. in the amino acid sequence encoded by the nucleotide sequence of the wild-type gene, but has the same activity as the enzyme encoded by the nucleotide sequence of the wild-type gene.

Specific embodiments of the acpA gene and the acpB gene are the acpA gene and the acpB gene derived from the MHK4 strain, which have the nucleotide sequences set forth in SEQ ID NOs:1 and 2.

The acpA gene and the acpB gene may be nucleotide sequences that code for the same amino acid sequence as the nucleotide sequence of the wild-type gene by utilizing the fact that there are several types of codons corresponding to one amino acid, and may include a nucleotide sequence different from that of the wild-type gene, for example, a nucleotide sequence in which the codons, secondary structure, GC content, etc. are optimized. For example, examples of nucleotide sequences in which codons have been modified from the nucleotide sequences of the acpA gene and the acpB gene derived from the MHK4 strain include the nucleotide sequences set forth in SEQ ID NOs: 4 and 5. The nucleotide sequence in which the codons have been modified is preferably a nucleotide sequence in which the codons have been modified so as to be easily expressed in a host microorganism, for example.

The method for determining the sequence identity of a nucleotide sequence and an amino acid sequence is not particularly limited, but for example, the sequence identity of a nucleotide sequence and an amino acid sequence can be determined by using a commonly known method to align the nucleotide sequence of a wild-type gene and the amino acid sequence of a wild-type enzyme expressed by the wild-type gene with the target nucleotide sequence and amino acid sequence, and to use a program for calculating the degree of identity between the two sequences. Specifically, the determination can be performed by referring to the section of Patent Document 2 (Means for calculating sequence identity).

( acpC gene)
The acpC gene is a gene that contains a nucleotide sequence that codes for the amino acid sequence of the AcpC enzyme.

The AcpC enzyme is an enzyme that has the activity of catalyzing the reaction of converting α-hydroxyacetovanillone to vanillic acid. The AcpC enzyme also has the activity of catalyzing the reaction of converting DHAP to HBA. There are no particular limitations on the amino acid sequence of the AcpC enzyme, so long as it has this activity.

The amino acid sequence of the AcpC enzyme encoded by the acpC gene of the MHK4 strain is the amino acid sequence set forth in SEQ ID NO:52.

The amino acid sequence of the AcpC enzyme may be an amino acid sequence having one or several amino acid deletions, substitutions, additions, etc. in the amino acid sequence of the wild-type enzyme.

As described in the Examples below, the acpC gene encoding the AcpC enzyme of the MHK4 strain showed 81% and 99% amino acid sequence identity with 2,4'-dihydroxyacetophenone dioxygenase (DAD) of Burkholderia sp. AZ11 strain and Alcaligenes sp. 4HAP strain, respectively. From this, the AcpC enzyme of the MHK4 strain is presumed to be 2,4'-dihydroxyacetophenone dioxygenase. Therefore, an enzyme having an amino acid sequence having 50% or more sequence identity with the amino acid sequence of the AcpC enzyme of the MHK4 strain and presumed to be 2,4'-dihydroxyacetophenone dioxygenase may be used as the AcpC enzyme.

The AcpC enzyme can be obtained in the same manner as the AcpA enzyme and the AcpB enzyme. The theoretical molecular weight calculated from the constituent elements of the AcpC enzyme having the amino acid sequence shown in SEQ ID NO:52 is approximately 20,000.

The acpC gene is not particularly limited as long as it contains a nucleotide sequence encoding the amino acid sequence of the AcpC enzyme. The AcpC enzyme is produced by expressing the acpC gene in an organism.

The acpC gene may be a wild-type gene, or may be a gene having a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of the wild-type gene.

A specific embodiment of the acpC gene is the acpC gene from the MHK4 strain, having the nucleotide sequence set forth in SEQ ID NO:3.

The acpC gene may contain a nucleotide sequence in which codons, secondary structure, GC content, etc. have been optimized relative to the nucleotide sequence of the wild-type gene. For example, an example of a nucleotide sequence in which codons have been modified relative to the nucleotide sequence of the acpC gene derived from the MHK4 strain is the nucleotide sequence set forth in SEQ ID NO:6.

(Origin of the gene)
The acpA gene, the acpB gene, and the acpC gene are derived from, for example, a microorganism having the ability to decompose acetovanillone and a microorganism in which the expression of these enzymes is observed. Examples of the microorganisms from which the acpA gene, the acpB gene, and the acpC gene are derived include microorganisms of the genus Pseudomonas including the MHK4 strain, microorganisms of the genus Burkholderia , microorganisms of the genus Alcaligenes , microorganisms of the genus Sphingobium , microorganisms of the genus Rhodococcus , and microorganisms of the genus Arthrobacter , and the like, with the MHK4 strain and its closely related species being preferred. A closely related species of the MHK4 strain refers to a Pseudomonas microorganism having 99.0% to 99.9% identity in the nucleotide sequence of the 16S rRNA gene with the MHK4 strain.

In addition, since the nucleotide sequences of the acpA gene and the acpB gene of the MHK4 strain have a certain degree of sequence identity with the nucleotide sequences of the genes possessed by Pseudomonas putida, the origin of the acpA gene and the acpB gene is preferably a microorganism of the genus Pseudomonas.

Since the nucleotide sequence of the acpC gene of the MHK4 strain has a certain degree of sequence identity with the nucleotide sequences of genes possessed by Burkholderia sp. AZ11 strain and Alcaligenes sp. 4HAP strain, the origin of the acpC gene is preferably a microorganism belonging to the genus Burkholderia or Alcaligenes. Note that the 2,4'-dihydroxyacetophenone dioxygenase gene possessed by Burkholderia sp. AZ11 strain and Alcaligenes sp. 4HAP strain can be used as the acpC gene.

The microorganisms from which the acpA gene, acpB gene, and acpC gene are derived are not particularly limited and include those listed above. However, since it is preferable that the AcpA enzyme, AcpB enzyme, and AcpC enzyme expressed in a transformed microorganism into which these genes have been introduced are not inactivated by the growth conditions of the host microorganism and exhibit activity, it is preferable that the microorganisms have growth conditions similar to those of the host microorganism to be transformed by introducing the acpA gene, acpB gene, and acpC gene.

(Transformed microorganisms)
Another aspect of the present invention is a transformed microorganism into which an acetovanillone converting enzyme gene group has been introduced as a foreign gene. The transformed microorganism of the first aspect of the present invention is a transformed microorganism into which the acpA gene and the acpB gene have been introduced as foreign genes and which expresses the introduced genes. The transformed microorganism of the second aspect of the present invention is a transformed microorganism into which the acpA gene, the acpB gene and the acpC gene have been introduced as foreign genes and which expresses the introduced genes. Hereinafter, the combination of the acpA gene and the acpB gene, and the combination of the acpA gene, the acpB gene and the acpC gene are collectively referred to as the "acetovanillone converting enzyme gene group".

Transformed microorganisms are produced by introducing the acetovanillone converting enzyme gene group as foreign genes into a host microorganism.

The host microorganism is not particularly limited as long as it is a microorganism into which the acetovanillone conversion enzyme gene group can be introduced as a foreign gene and into which the introduced acetovanillone conversion enzyme gene group can be expressed. Examples of host microorganisms include microorganisms of the genus Pseudomonas, Burkholderia, Alcaligenes, Sphingobium, Rhodococcus, Arthrobacter, Escherichia, and Corynebacterium. However, microorganisms of the genus Pseudomonas, Burkholderia, Alcaligenes, Sphingobium, Rhodococcus, and Arthrobacter, which include acetovanillone-degrading strains, are preferred, and microorganisms of the genus Pseudomonas are more preferred because the acetovanillone conversion enzyme gene group of the MHK4 strain can be introduced and expressed.

Pseudomonas sp. NGC7 strain (Accession No.: NITE BP-03043) has assimilation properties for H-type lignin-derived aromatic compounds, S-type lignin-derived aromatic compounds, and G-type lignin-derived aromatic compounds, and is excellent in producing useful substances from lignin. Therefore, the host microorganism is preferably Pseudomonas sp. NGC7 strain.

The host microorganism may be either a wild-type microorganism or a transformed microorganism obtained by genetically recombining a wild-type microorganism. For example, when the host microorganism has a gene for an enzyme having an activity of catalyzing a reaction to decompose vanillic acid (e.g., vanillate O -demethylase gene), a transformed microorganism obtained by introducing an acetovanillone converting enzyme gene group into the host microorganism not only produces vanillic acid but also decomposes it, so that it is difficult to accumulate vanillic acid. In addition, when drug selection is used to select a transformed strain, a drug resistance gene that functions to show resistance to a drug to be used makes drug selection difficult. Therefore, the host microorganism is preferably a microorganism lacking a vanillic acid decomposing enzyme gene and/or a microorganism lacking a drug resistance gene. However, a transformed microorganism may be obtained by deleting a vanillic acid decomposing enzyme gene or the like after introducing an acetovanillone converting enzyme gene group into a wild-type microorganism.

Vanillate O -demethylase is essentially composed of a vanillate O -demethylase oxygenase component and a vanillate O -demethylase oxidoreductase component. The gene encoding the vanillate O -demethylase oxygenase component is called the vanA gene, and the gene encoding the vanillate O -demethylase oxidoreductase component is called the vanB gene.

The vanillate O -demethylase oxygenase component (EC 1.14.13.82; hereinafter, also referred to as VanA enzyme) expressed by the vanA gene cleaves the methyl ether bond of vanillic acid using electrons derived from NADH or NADPH supplied via the oxygen reductase component and oxygen atoms supplied from molecular oxygen, producing protocatechuic acid, formaldehyde, and water.

The vanillate O 2 -demethylase oxygenase component has a Rieske[2Fe-2S] iron-sulfur domain (W7-V107, PROSITE entry no. PS51296) in its amino acid sequence, and C and H (C47, H49, C66, H69) in the domain are involved in Fe—S binding.

The vanillate O -demethylase oxidoreductase component (EC 1.14.13.82; hereinafter, also referred to as VanB enzyme) expressed by the vanB gene is known as one of the oxidoreductases that extracts electrons from NADH or NADPH and transfers them to oxygenase (oxygenase). The vanillate O -demethylase oxidoreductase component transfers electrons derived from NADH or NADPH to the VanA enzyme, which is the vanillate O -demethylase oxygenase component.

The vanillate O 2 -demethylase oxidoreductase component has a 2Fe-2S ferredoxin type iron-sulfer binding domain (G229-I316, PROSITE entry no. PS51085) in its amino acid sequence, and Cs (C265, C270, C273, C303) in the amino acid sequence are involved in Fe—S binding. In addition, the vanillate O 2 -demethylase oxidoreductase component has an NAD-binding domain (L109-D201, Pfam entry no. PF00175) and a Ferredoxin reductase type FAD-binding domain (M1-A101, PROSITE entry no. PS51384) in its amino acid sequence.

The expression product of the vanA gene (VanA enzyme) and the expression product of the vanB gene (VanB enzyme) function in cooperation to form vanillate O -demethylase (VanAB enzyme). VanAB enzyme is responsible for demethylating vanillic acid, but can also function as syringic acid O -demethylase and 3- O -methylgallic acid demethylase, since it can convert syringic acid to 3- O -methylgallic acid and 3- O -methylgallic acid to gallic acid, respectively. The SYK-6 strain has the desA gene as a gene encoding syringic acid O -demethylase, and the ligM gene as a gene encoding vanillic acid/3-O-methylgallic acid O -demethylase.

The enzymes encoded by the desA and ligM genes of the SYK-6 strain catalyze a methyl transfer reaction to tetrahydrofolic acid, and tetrahydrofolic acid is required for the progress of the reaction. Since the regeneration of tetrahydrofolic acid requires C1 metabolism to supply C1 compounds required for methionine, thymidine, purine biosynthesis, etc., these enzymes must function in conjunction with C1 metabolism. In contrast, the vanillate O -demethylase expressed by the NGC7 and MHK4 strains is an oxygen-adding type and is composed of an oxygenase component and an oxidoreductase component, so that it does not require coupling with C1 metabolism and has a reduced complexity.

As described above, the vanA gene and the vanB gene are considered to function as a gene set. As described in Patent Document 2, the NGC7 strain is assumed to have the following gene sets of vanA gene and vanB gene: a gene set of vanA1 gene and vanB1 gene, a gene set of vanA2 gene and vanB2 gene, a gene set of vanA3 gene and vanB3 gene, and a gene set of vanA4 gene and vanB4 gene. However, among these gene sets, the set of vanA4 gene and vanB4 gene is considered to be deeply involved in the degradation of vanillic acid.

Among the genes encoding the VanAB enzyme possessed by the NGC7 strain, the vanA4 gene, which is an oxygenase component, acts directly on vanillic acid. Therefore, when the host microorganism is the NGC7 strain and vanillic acid is to be produced from acetovanillone, the transformed microorganism of one embodiment of the present invention is preferably deleted of the vanA4 gene, and more preferably deleted of both the vanA4 gene and the vanB4 gene in order to highly reduce the action on vanillic acid. However, since it is expected that the yield of vanillic acid can be increased and the decomposition of vanillic acid can be further reduced, the transformed microorganism of one embodiment of the present invention is preferably deleted of other vanA genes and/or vanB genes in addition to the vanA4 gene and the vanB4 gene. The other vanA genes and vanB genes to be deleted may be appropriately selected with reference to Patent Document 2, taking into account the assimilation and other properties of the resulting transformed microorganism.

On the other hand, the pobA gene of the NGC7 strain expresses the PobA enzyme having the activity of converting HBA to protocatechuic acid. The PobA enzyme is a type of 4-hydroxybenzoate monooxygenase (EC 1.14.13.2 or EC 1.14.13.33). Therefore, when the final product is HBA, it is preferable that the transformed microorganism of one embodiment of the present invention lacks the pobA gene.

For details of a method for producing a transformed microorganism, such as a method for introducing a gene into a host microorganism or a method for deleting a gene from a host microorganism, refer to the items (cloning of genes by genetic engineering techniques), (construction of a recombinant vector containing a gene), and (method for producing a transformed microorganism) described in Patent Document 2. When a recombinant vector is used, the recombinant vector incorporating the acetovanillone converting enzyme gene group may contain other foreign genes (heterologous genes). The foreign genes are not particularly limited as long as they are genes that do not naturally occur in the host microorganism, and examples thereof include DNA fragments derived from pUC118, specifically lactose promoter regions (P _lac ), drug resistance genes, and RBS sequences.

The method of introducing the acetovanillone converting enzyme gene group into the host microorganism is not particularly limited, and examples of the method include a method in which a DNA construct such as a plasmid vector incorporating the acetovanillone converting enzyme gene group is introduced into the host microorganism so that it grows autonomously and expresses the genes, and a method in which the acetovanillone converting enzyme gene group is incorporated into the chromosome of the host microorganism by using homologous recombination. However, the method of incorporating the acetovanillone converting enzyme gene group into the chromosome of the host microorganism is preferred because it increases the amount of acetovanillone decomposed per bacterial cell.

(Specific embodiment of transformed microorganism)
A specific embodiment of the transformed microorganism is a transformed microorganism (1) in which the host microorganism is Escherichia coli, the acpA gene, the acpB gene, and/or the acpC gene are introduced as foreign genes, and the introduced genes are expressed. By using the transformed microorganism (1), the AcpA enzyme, the AcpB enzyme, and the AcpC enzyme can be obtained. Specific examples of the transformed microorganism (1) are the BL21(DE3)[ pE16acpA ] strain, the BL21(DE3)[ pE16acpB ] strain, and the BL21(DE3)[ pE16acpC ] strain, which are described in the Examples below.

A specific embodiment of the transformed microorganism is a transformed microorganism (2) in which the host microorganism is an NGC7 strain, the acpA gene and the acpB gene are introduced as foreign genes, and the introduced genes are expressed. The transformed microorganism (2) can convert acetovanillone to α-hydroxyacetovanillone. The transformed microorganism (2) can also convert HAP to DHAP.

Another specific embodiment of the transformed microorganism is a transformed microorganism (3) in which the host microorganism is the NGC7 strain, the acpA gene, the acpB gene, and the acpC gene are introduced as foreign genes, and the introduced genes are expressed. The transformed microorganism (3) can convert acetovanillone to vanillic acid via α-hydroxyacetovanillone. However, since the NGC7 strain has the ability to assimilate vanillic acid, it is difficult for the transformed microorganism (3) to accumulate vanillic acid. A specific example of the transformed microorganism (3) is the NGC7Δ aph [pSEVA acpCAB ^opt ] strain described in the Examples below.

Another specific embodiment of the transformed microorganism is a transformed microorganism (4) in which the host microorganism is an NGC7 strain lacking the vanA gene and the vanB gene, preferably the vanA4 gene and the vanB4 gene, and in which the acpA gene, the acpB gene and the acpC gene have been introduced as foreign genes and which express the introduced genes. The transformed microorganism (4) can convert acetovanillone to vanillic acid via α-hydroxyacetovanillone. The transformed microorganism (4) can grow using aromatic compounds derived from H-type lignin, such as 4-hydroxybenzoic acid, as a carbon source, and can produce vanillic acid from aromatic compounds derived from G-type lignin, such as ferulic acid and vanillin, and acetovanillone or a mixture of aromatic compounds derived from G-type lignin containing acetovanillone. Specific examples of the transformed microorganism (4) include the NGC729[pSEVA acpCAB ^opt ] strain and the NGC729::P _tac : acpCAB ^opt strain described in the Examples below.

Another specific embodiment of the transformed microorganism is a transformed microorganism (5) in which the host microorganism is an NGC7 strain lacking the pobA gene, the acpA gene, the acpB gene, and the acpC gene are introduced as foreign genes, and the introduced genes are expressed. The transformed microorganism (5) can convert HAP to HBA via DHAP. The transformed microorganism (5) can produce HBA from aromatic compounds derived from H-type lignin such as HAP while growing using aromatic compounds derived from G-type lignin such as acetovanillone as a carbon source. A specific example of the transformed microorganism (5) is the NGC7Δ pobA [pSEVA acpCAB ^opt ] strain described in the Examples below.

(Production method)
By using an appropriate combination of the AcpA enzyme, AcpB enzyme, and AcpC enzyme (hereinafter sometimes collectively referred to as AcpABC enzyme) obtained by the transformed microorganism (1), it is possible to produce α-hydroxyacetovanillone from acetovanillone, to produce vanillic acid from α-hydroxyacetovanillone, and to produce vanillic acid from acetovanillone via α-hydroxyacetovanillone. A production method according to one aspect of the present invention is a production method for α-hydroxyacetovanillone, comprising the step of obtaining α-hydroxyacetovanillone by allowing acetovanillone to act on the AcpA enzyme, which is an expression product of the acpA gene, and the AcpB enzyme, which is an expression product of the acpB gene. A production method according to another aspect of the present invention is a production method for vanillic acid, comprising the step of obtaining vanillic acid by allowing acetovanillone to act on the AcpA enzyme, which is an expression product of the acpA gene, the AcpB enzyme, which is an expression product of the acpB gene, and the AcpC enzyme, which is an expression product of the acpC gene. The AcpA enzyme, the AcpB enzyme and the AcpC enzyme may be synthesized using a cell-free protein synthesis system by utilizing the acpA gene, the acpB gene and the acpC gene.

By using the transformed microorganism (2), α-hydroxyacetovanillone can be produced from acetovanillone. Another embodiment of the production method of the present invention is a method for producing α-hydroxyacetovanillone, comprising the step of allowing acetovanillone to act on a transformed microorganism into which acpA gene and acpB gene have been introduced as foreign genes and which expresses the introduced genes, thereby obtaining α-hydroxyacetovanillone.

Vanillic acid can be produced from acetovanillone by using the transformed microorganism (3) and the transformed microorganism (4). Another embodiment of the production method of the present invention is a method for producing vanillic acid, comprising the step of obtaining vanillic acid by allowing acetovanillone to act on a transformed microorganism into which the acpA gene, the acpB gene, and the acpC gene have been introduced as foreign genes and which expresses the introduced genes. Another embodiment of the production method of the present invention is a method for producing vanillic acid, comprising the step of obtaining vanillic acid by allowing acetovanillone to act on a transformed microorganism into which the acpA gene, the acpB gene, and the acpC gene have been introduced as foreign genes, which expresses the introduced genes, and which further lacks the vanillate O -demethylase gene on the chromosome.

By using the transformed microorganism (5), HBA can be produced from HAP. Another embodiment of the production method of the present invention is a method for producing HBA, comprising a step of allowing HAP to act on a transformed microorganism into which acpA gene, acpB gene, and acpC gene have been introduced as foreign genes, which express the introduced genes, and which further lacks the pobA gene on its chromosome, to obtain HBA.

Acetovanillone is contained in the decomposition products obtained by alkaline oxidative decomposition of lignin, such as kraft cooking and soda cooking, so the source of acetovanillone may be a mixture of aromatic compounds, such as alkaline oxidative decomposition products of softwood lignin, which is mainly composed of G-type lignin.

The method of allowing acetovanillone to act on the AcpABC enzyme is not particularly limited as long as it allows contact between acetovanillone and the AcpABC enzyme and allows the AcpABC enzyme to produce and/or accumulate α-hydroxyacetovanillone and/or vanillic acid (hereinafter sometimes collectively referred to as the target product). For example, there is a method of allowing acetovanillone to act on the AcpABC enzyme under conditions of a pH of 6.5 to 8.5, preferably 7.0 to 8.0, a temperature of 20°C to 40°C, preferably 25°C to 35°C, and a time of several minutes to several hours, preferably 10 minutes to 60 minutes.

The method of making acetovanillone act on a transformed microorganism is not particularly limited as long as it is a method in which acetovanillone comes into contact with the transformed microorganism and the target product can be produced and/or accumulated by the AcpABC enzyme possessed by the transformed microorganism. For example, a method of producing a target product by culturing a transformed microorganism under various culture conditions suitable for the transformed microorganism using a medium containing acetovanillone and suitable for the growth of the transformed microorganism is included. The culture method is not particularly limited, and examples include a solid culture method or a liquid culture method performed under aeration conditions.

When the host microorganism is the NGC7 strain, the culture method can be carried out by referring to the item (production method) in Patent Document 2. An excerpt is described below.

The medium may be any of synthetic and natural media, as long as it contains a carbon source, a nitrogen source, inorganic substances, and other nutrients in an appropriate ratio, as is the case with conventional media for culturing Pseudomonas microorganisms. For example, MMx-3 medium, Wx minimal medium, and the like, as described in the Examples below, may be used, but are not limited thereto. The carbon source may be acetovanillone alone, or a combination of acetovanillone and other carbon sources, such as other lignin-derived aromatic compounds, sugars, and organic acids. The medium components preferably include components necessary for cell growth and enzyme activation, such as Mg ²⁺ and Fe ²⁺ . Iron ions, magnesium ions, and the like may be added to the medium as compounds, but may also be added as mineral-containing substances.

The culture conditions may be those for Pseudomonas microorganisms generally known to those skilled in the art. For example, the initial pH of the medium may be adjusted to 5-10, the culture temperature may be 20°C-40°C, and the culture time may be set appropriately to several hours to several days, preferably 1-7 days, more preferably 2-5 days. There are no particular limitations on the culture method, and aeration and stirring submerged culture, shaking culture, stationary culture, etc. may be used, but it is preferable to culture under conditions where the dissolved oxygen concentration is sufficient by aeration, etc. Furthermore, fed-batch culture may be used in which acetovanillone or other carbon sources are added depending on the decrease in carbon sources such as acetovanillone and the increase in the target product.

For example, examples of culture media and culture conditions include shaking culture and agitation culture in which the carbon source is MMx-3 medium or Wx medium containing only acetovanillone or acetovanillone and glucose, the temperature is 25°C to 35°C, the shaking speed (agitation speed) is 100 rpm to 2,000 rpm, and the time is 1 hour to 5 days. Another example of culture media and culture conditions includes shaking culture and agitation culture using LB medium containing acetovanillone, with a dissolved oxygen concentration of 5% to 20%.

There are no particular limitations on the method for obtaining the target product from the culture after the culture is completed. Since the target products, α-hydroxyacetovanillone and vanillic acid, accumulate in the culture medium, the culture is separated into the bacterial cells and the culture supernatant by ordinary solid-liquid separation procedures such as filtration and centrifugation, and the target product is extracted from the collected culture supernatant by solid-phase extraction using a column or solvent extraction using a solvent in which the target product is soluble.

The extraction solvent is not particularly limited as long as it dissolves the target product, and examples include ethyl acetate and diethyl ether.

The target product can be separated and purified from the culture solution by known methods such as precipitation, extraction, distillation, recrystallization, and adsorbents, or by methods partially modified from the above methods. As a specific embodiment of the purification method, for example, hydrochloric acid is added to the culture supernatant to adjust the pH to 2 to 5, and then diethyl ether is added and mixed. The diethyl ether layer is dried under reduced pressure using an evaporator, and the resulting precipitate is washed with ion-exchanged water, recovered by suction filtration, and dried under reduced pressure. The dried solid is dissolved in glacial acetic acid and recrystallized to obtain purified vanillic acid. In addition, purified vanillic acid can also be obtained by a method using a synthetic adsorbent, such as the method described in the document by Gomes et al. (Separation and Purification Technology, vol. 216, p. 92-101, 2019), or by a method of reduced pressure distillation described in JP-A-2017-171591.

Qualitative or quantitative analysis of the target product may be performed by HPLC as described in the examples below.

By using the transformed microorganism according to one embodiment of the present invention, it is possible to selectively obtain a target product without decomposing it.
For example, when the transformed microorganism (4) is used, when the host microorganism is the NGC7 strain and the initial OD ₆₀₀ is 5.0, 1.0 mM acetovanillone can be converted to vanillic acid within 1 hour so that acetovanillone is at the detection limit.
Furthermore, when the transformed microorganism (4) is used, when the host microorganism is the NGC7 strain and the initial OD ₆₀₀ is 0.1, 5.7 mM vanillin, 1.6 mM acetovanillone, and 2.7 mM vanillic acid can be converted from acetovanillone to vanillic acid within 35 hours, so that acetovanillone is at the lower limit of detection.

In the manufacturing method of the present invention, various steps or operations can be added before or after the above steps, or between the steps, as long as the object of the present invention can be achieved. Also, in the same manner as the above method, HBA can be produced from HAP by using HAP as a substrate instead of acetovanillone.

(Pseudomonas species MHK4 strain)
Another embodiment of the present invention is the Pseudomonas sp. MHK4 strain (Accession No.: NITE P-03794). The MHK4 strain has the acpA gene, the acpB gene, and the acpC gene set forth in SEQ ID NOs: 1 to 3, and can convert acetovanillone to vanillic acid via α-hydroxyacetovanillone. In addition, it can convert HAP to HBA via DHAP.

On the other hand, the MHK4 strain assimilates vanillic acid because it also has the vanA3 gene and the vanB3 gene shown in SEQ ID NOs: 23 and 24, which are vanillate O -demethylase genes. Therefore, when using the MHK4 strain to produce vanillic acid from acetovanillone, it is preferable to delete the vanA3 gene and/or the vanB3 gene of the MHK4 strain.

Another aspect of the present invention is a transformed microorganism in which a host microorganism is Pseudomonas sp. strain MHK4 (accession number: NITE P-03794) and which lacks at least one vanillate O -demethylase gene, preferably at least one vanillate O -demethylase gene selected from the group consisting of the vanA3 gene (SEQ ID NO: 23) and the vanB3 gene (SEQ ID NO: 24), located on a chromosome. Another aspect of the present invention is a method for producing vanillic acid, comprising the step of allowing acetovanillone to act on such a transformed microorganism to obtain vanillic acid.

Furthermore, the MHK4 strain assimilates HBA because it also has the pobA1 gene and the pobA2 gene shown in SEQ ID NOs:61 and 62, which are 4-hydroxybenzoate monooxygenase genes. Therefore, when producing HBA from HAP using the MHK4 strain, it is preferable to delete the pobA1 gene and/or the pobA2 gene of the MHK4 strain.

Another aspect of the present invention is a transformed microorganism in which a host microorganism is Pseudomonas sp. MHK4 strain (accession number: NITE P-03794) and at least one 4-hydroxybenzoate monooxygenase gene, preferably selected from the group consisting of the pobA1 gene (SEQ ID NO: 61) and the pobA2 gene (SEQ ID NO: 62), located on a chromosome is deleted. Another aspect of the present invention is a method for producing HBA, comprising the step of allowing HAP to act on such a transformed microorganism to obtain HBA.

(Uses of vanillic acid)
Vanillic acid and HBA obtained by utilizing the gene, microorganism, transformed microorganism, and production method according to one embodiment of the present invention can be converted into various industrially useful compounds. Vanillic acid has an aromatic ring structure that imparts heat resistance and rigidity to a polymer, and is expected to be used in the synthesis of functional polymers such as liquid crystal polyesters. Vanillic acid and vanillic acid derivatives obtained by modifying vanillic acid are expected to be used, for example, by themselves or together with other components, as heat-resistant plastics, tracking-resistant plastics, liquid crystal copolymer polyester resins, and the like. α-Hydroxyacetovanillone obtained by utilizing the transformed microorganism and production method according to one embodiment of the present invention can be used as a substrate for vanillic acid to produce vanillic acid. HBA and DHAP can also be used in the same manner as vanillic acid and α-hydroxyacetovanillone.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the problem of the present invention can be solved.

[1. Isolation of Pseudomonas sp. MHK4 strain]
Soil samples were collected from 278 locations in Japan, including Hokkaido, Iwate Prefecture, Miyagi Prefecture, Fukushima Prefecture, Ibaraki Prefecture, Tochigi Prefecture, Saitama Prefecture, Niigata Prefecture, Nagano Prefecture, and Shiga Prefecture. Soil samples were collected by digging up the soil to a depth of about 5 to 10 cm from the soil surface. Each sampled soil _was added to Wx buffer [9.8 g/L _{Na2HPO4.12H2O ,} 1.7 g/ _{L KH2PO4 ,} 1.0 g/L ( _NH4 ) _2SO4 ] and suspended to obtain a slurry suspension (10 to 30 mg). The suspension was then transferred to _0.5 mL of Wx liquid medium [9.8 g/L _{Na2HPO4.12H2O} , 1.7 g/L _KH2PO4 , 1.0 g/L ( _NH4 ) _{2SO4 ,} 100 mg/L _MgSO4.7H2O , 9.5 mg/ _{L FeSO4.7H2O} , _10.75 mg/L _{ZnSO4 .} MgO, 2 mg/L CaCO ₃ , 1.44 mg/L ZnSO 4.7H ₂ O, 1.12 _mg /L MnSO _{4.4H 2} O, 0.25 mg/L CuSO _{4.5H 2} O, 0.28 mg/L CoSO _{4.7H 2} O, 0.06 mg/L H ₃ BO ₃ , 51.3 μL 12N HCl] and cultured with shaking at 25° C. for 72 hours. Subsequently, 5 μL of the obtained culture solution was added to 0.5 mL of Wx liquid medium and cultured with shaking at 25° C. for 48 hours. 0.1 mL of the culture solution of the sample in which the growth of microorganisms was confirmed by visually checking the turbidity of the culture solution was inoculated into 5 mL of Wx liquid medium containing 5 mM AV, and cultured with shaking at 25 ° C. for 48 to 96 hours. Next, 0.1 mL of the culture solution of the sample in which the growth of microorganisms was confirmed in the same manner was inoculated into 5 mL of Wx liquid medium containing 10 mM AV, and cultured with shaking at 25 ° C. for 48 to 96 hours. Next, the culture solution of the sample in which the growth of microorganisms was confirmed in the same manner was smeared on Wx agar medium containing 10 mM AV, and cultured stationarily. After the culture, the colonies formed on the agar medium were isolated and cultured in Wx liquid medium containing 5 mM AV as the only carbon source, and those that were recognized as strains that grew well were selected as MHK4 strains.

[2. Biological classification of Pseudomonas sp. MHK4 strain]
Pseudomonas sp. MHK4 strain (hereinafter simply referred to as MHK4 strain) has been deposited at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on December 9, 2022, with the identification of the microorganism as "MHK4" and the accession number as "Accession Number: NITE P-03794". A copy of the deposit receipt for MHK4 is shown in FIG. 1A, and a certificate of viability is shown in FIG. 1B.

The test results examining the properties of the MHK4 strain are shown in Tables 1 to 3.

Phylogenetic analysis of the 16S rRNA gene sequence showed that the partial sequence of the 16S rRNA gene of the MHK4 strain showed 97.6% to 99.6% homology with the 16S rRNA sequences of the Pseudomonas vancouverensis DhA-51 strain, Pseudomonas umsongensis Ps 3-10 strain, Pseudomonas mohnii IpA-2 strain, and Pseudomonas moorei RW10 strain.

We commissioned Techno Suruga Lab to carry out the first and second bacterial tests on the MHK4 strain, and conducted physiological and biochemical property tests. As a result, it was found that the MHK4 strain is a motile gram-negative bacillus, that the catalase reaction and oxidase reaction were positive, and that it oxidized glucose (Table 1, Table 2). These results showed that the MHK4 strain was consistent with the properties of Pseudomonas microorganisms [Barrow et al. (1993). "Cowan and Steel's Manual for the identification of medical bacteria. 3rd edition." Cambridge: University Press. ].

The MHK4 strain was subjected to a second stage bacterial test using API20NE kit (bioMerieux Japan). As a result, it was found that the MHK4 strain reduced nitrate, did not show arginine dihydrolase activity, and hydrolyzed esculin. The MHK4 strain also assimilated L-arabinose, D-mannose, and D-mannitol, but did not assimilate N-acetyl-D-glucosamine. In addition, additional tests showed that the MHK4 strain produced fluorescent dyes on King's agar medium, but showed negative results in levan formation, Tween 80 hydrolysis, growth in the presence of 5% NaCl, and lecithinase activity (Table 3). These properties of the MHK4 strain were all different from those of the four strains that are phylogenetically closely related to the MHK4 strain [Camara et al. (2007) . “ Pseudomonas reinekei sp. nov., Pseudomonas moorei sp. nov. and Pseudomonas mohnii sp. nov., novel species capable of degr "Ading chlorosalicylates or isopimaric acid" International Journal of Systematic and Evolutionary Microbiology 57:923-931; n et al. (1999). “Ｐｈｙｓｉｏｌｏｇｉｃａｌ ａｎｄ ｐｈｙｌｏｇｅｎｅｔｉｃ ｄｉｖｅｒｓｉｔｙ ｏｆ ｂａｃｔｅｒｉａ ｇｒｏｗｉｎｇ ｏｎ ｒｅｓｉｎ ａｃｉｄｓ” Ｓｙｓｔｅｍａｔｉｃ ａｎｄ Ａｐｐｌｉｅｄ Ｍｉｃｒｏｂｉｏｌｏｇｙ ２２：６８－７８． ].

In particular, the MHK4 strain differs from P. moorei and P. mohnii in that it produces a fluorescent pigment, reduces nitrate, and does not exhibit arginine dihydrolase activity; differs from P. vancouverensis in that it utilizes L-arabinose and does not utilize N-acetyl-D-glucosamine; and differs from P. umsongensis in that it does not exhibit arginine dihydrolase activity and utilizes D-mannitol.

From the results of the above physiological and biochemical property tests, it was presumed that the MHK4 strain was a species of Pseudomonas sp. that had not been isolated so far, and it was named Pseudomonas sp. MHK4 strain.

[3. AV conversion using a kanamycin resistance gene ( aph )-deficient strain of Pseudomonas sp. NGC7 strain into which PSMH_1607 ( acpC ), PSMH_1608 ( acpA ) and PSMH_1609 ( acpB ) have been introduced]
Genomic DNA of the MHK4 strain was prepared by a conventional method, and the entire nucleotide sequence was analyzed. Genes showing amino acid sequence identity with the AV converting enzyme gene group of the Sphingobium sp. SYK-6 strain were searched from the genome sequence of the MHK4 strain. Table 4 shows genes derived from Pseudomonas sp. MHK4 strain that showed amino acid sequence identity with the enzymes encoded by the AV converting enzyme gene group derived from the Sphingobium sp. SYK-6 strain.

As shown in Table 4, the MHK4 strain genomic DNA contained a gene (PSMH_5054) that showed 50% identity with the acvD gene of the SYK-6 strain and a gene (PSMH_2308) that showed 38% identity with the vceA gene of the SYK-6 strain. However, there were no genes that showed 30% or more identity with the acvA gene, acvB gene, acvC gene, acvE gene, acvF gene, and vceB gene of the SYK-6 strain. From the above, it was presumed that the MHK4 strain metabolizes AV by an enzyme system different from that of the SYK-6 strain.

Since it was estimated that the MHK4 strain metabolizes AV using an enzyme system different from that of the SYK-6 strain, the genes whose expression was induced when the MHK4 strain was cultured in Wx liquid medium containing 5 mM AV and 20 mM glucose were examined by RNA-Seq analysis, compared to when the MHK4 strain was cultured in 10 mL of Wx liquid medium containing 20 mM glucose. Table 5 shows the top 10 genes whose expression was induced when the strain was cultured in Wx liquid medium containing 5 mM AV and 20 mM glucose.

Among the genes listed in Table 5, the gene cluster consisting of PSMH_1607 ( acpC ), PSMH_1608 ( acpA ), and PSMH_1609 ( acpB ) was the most highly induced. The acpA and acpB genes showed 33.6% and 44.1% sequence identity with the genes encoding the oxygenase component (VanA) and the oxidoreductase component (VanB) of oxygen-additive vanillate O -demethylase derived from Pseudomonas putida KT2440 strain, respectively, suggesting that the gene products of the acpA and acpB genes function as monooxygenases.

The acpC gene showed 81% and 99% amino acid sequence identity with 2,4'-dihydroxyacetophenone dioxygenase (DAD) from Burkholderia sp. strain AZ11 (GenBank accession number: AB636681.1 (version: AB636681.1)) and DAD from Alcaligenes sp. strain 4HAP (GenBank accession number: AJ133820 (version: AJ133820.1)), respectively. DAD of the AZ11 and 4HAP strains catalyzes the reaction of oxidatively cleaving the hydroxymethyl group of 2,4'-dihydroxyacetophenone to convert it to 4-hydroxybenzoic acid and formic acid, respectively. Since the enzyme encoded by the acpC gene shows high amino acid sequence identity with DAD of the AZ11 and 4HAP strains, it is possible that α-hydroxyacetovanillone, which is structurally similar to 2,4'-dihydroxyacetophenone, is converted to VA and formic acid.

From the above, it was predicted that in the MHK4 strain, AV would be hydroxylated by the gene products of the acpA gene and the acpB gene and converted to α-hydroxyacetovanillone, and then converted and metabolized by the gene product of the acpC gene to vanillic acid (hereinafter also referred to as VA) and formic acid.

An expression plasmid for the acpA/acpB gene set was prepared by the following procedure.
A DNA fragment of about 2.1 kbp containing the acpA / acpB gene set was amplified by PCR using the genomic DNA of Pseudomonas sp. MHK4 strain as a template and a primer set consisting of primers 1 and 2 of SEQ ID NOs: 25 and 26.

The obtained fragment was linked to pSEVA241_P _lac (see Applied and Environmental Microbiology, Vol. 88, e0072422, 2022; carrying a kanamycin resistance gene) previously digested with EcoRI and BamHI by seamless cloning using NEBuilder HiFi DNA Assembly (New England Biolabs) to prepare a plasmid pSEVA acpAB for expressing the acpA / acpB gene set.

The prepared plasmid pSEVA acpAB was introduced into a kanamycin resistance gene ( aph )-deficient strain (hereinafter also referred to as NGC7Δ aph strain) of Pseudomonas sp. NGC7 strain (Accession No.: NITE BP-03043; Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation) by electroporation as follows. The NGC7Δ aph strain was prepared with reference to Example 10 of International Publication No. WO2022/210236.

The NGC7Δ aph strain obtained by shaking culture in 10 mL of LB liquid medium was washed with 5 mL of 0.5 M glucose aqueous solution and then suspended in 0.2 mL of 0.5 M glucose aqueous solution. The cell suspension and the plasmid pSEVA acpAB were mixed and applied with 10 kV using a Micro Pulser (Bio-Rad Laboratories). Immediately after application of the voltage _, 1 mL of SOC liquid medium (20 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 2.5 mM KCl, ₁₀ mM MgCl2.6H2O, ₁₀ mM _MgSO4.7H2O , 20 mM glucose) was added and the cells were cultured with shaking at 30°C for 1 hour, and strains capable of growing on LB agar medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 15 g/L agar) containing 25 mg/L kanamycin (Km) were selected.

The obtained NGC7Δ aph (pSEVA acpAB ) strain was inoculated into 10 mL of LB containing 25 mg/L Km and cultured overnight at 30° C. with shaking. 0.1 mL of the obtained culture was inoculated into 10 mL of fresh LB containing 25 mg/L Km and cultured at 30° C. with shaking for 16 hours. The cells derived from the obtained culture were washed with MMx-3 buffer [34.2 g/L Na ₂ HPO ₄ ·12H ₂ O, 6.0 g/L KH ₂ PO ₄ , 2.5 g/L (NH ₄ ) ₂ SO ₄ , 1.0 g/L NaCl] and then suspended again in MMx-3 buffer to prepare 1 mL of cell suspension with OD ₆₀₀ = 10. AV was added to the cell suspension to a final concentration of 0.2 mM, and the resulting mixture was subjected to an AV conversion reaction by shaking at 1,500 rpm at 30° C. Eight hours after the start of the reaction, a portion of the reaction solution was centrifuged, and the obtained supernatant was subjected to HPLC analysis or HPLC-MS analysis.

HPLC analysis was performed using a high performance liquid chromatograph (Agilent 1200 series, Agilent Technologies). The column used was a ZORBAX Eclipse Plus C18 column (diameter 4.6 mm, length 150 mm, particle size 0.5 μm), and was kept at 40° C. Gradient elution mode (solvent A: 5% (v/v) CH ₃ OH, 1% (v/v) CH ₃ COOH, solvent B: 50% (v/v) CH ₃ OH, 1% (v/v) CH ₃ COOH) was used, and after equilibration with solvent A, the ratio of solvent B was increased to 20% over 8 minutes from the start of analysis, and then the ratio of solvent B was increased to 100% over 5 minutes. The flow rate of the mobile phase was 1.0 mL/min, and the measurement wavelength was 280 nm.

For the HPLC-MS analysis, a high-performance liquid chromatograph (Acquity ultraperformance liquid chromatography system, Nippon Waters) and a tandem quadrupole mass spectrometer (ACQUITY TQ detector, Nippon Waters) were used. The column used was a TSKgel ODS-140HTP column (diameter 2.1 mm, length 100 mm, particle size 2.3 μm, Tosoh Corporation), and was kept at 30° C. The mobile phase was isocratic elution of 90% solvent A [99.9% (v/v) _H2O , 0.1% (v/v) HCOOH] and 10% solvent B [99.9% (v/v) _CH3CN , 0.1% (v/v) HCOOH]. The flow rate of the mobile phase was 0.5 mL/min, and the measurement wavelength was 280 nm. Detection by electrospray ionization mass spectrometry (ESI-MS) was performed in negative ion mode. The detection conditions were as follows: capillary voltage, 3.0 kv; cone voltage, 10-40 V; source temperature, 120° C.; desolvation temperature, 350° C.; desolvation gas flow rate, 650 liters/h; cone gas flow rate, 50 liters/h.

The results are shown in Figure 2. Figure 2(A) shows the HPLC chromatogram immediately after the start of the reaction, and Figure 2(B) shows the HPLC chromatogram 8 hours after the start of the reaction.

As shown in Figures 2(A) and 2(B), the HPLC analysis showed that a conversion product peak was detected at a retention time of 11.3 minutes after 8 hours of reaction. Figure 2(C) shows the results of analyzing the conversion product by HPLC-MS (ESI-MS). As shown in Figure 2(C), a peak was observed at m / z 181, which corresponds to the [M-H] ^- of α-hydroxyacetovanillone (molecular weight 182), indicating that AV was converted to α-hydroxyacetovanillone by the gene product of the acpAB gene.

A plasmid for expressing the acpC gene in addition to the acpA / acpB gene set was prepared by the following procedure.

Using the genomic DNA of the MHK4 strain as a template, a PCR method using a primer set consisting of primers 3 and 2 of SEQ ID NOs: 27 and 26 was used to amplify a DNA fragment of about 2.6 kbp containing the acpC gene, the acpA gene, and the acpB gene.

The obtained fragment was ligated by seamless cloning method using pSEVA241_P _lac previously digested with EcoRI and BamHI and NEBuilder HiFi DNA assembly to prepare a plasmid pSEVAacpCAB for expressing the acpC gene, the acpA gene, and the acpB gene.

The prepared plasmid pSEVA acpCAB was introduced into the NGC7Δ aph strain by electroporation as described above.

The obtained NGC7Δ aph [pSEVA acpCAB ] strain was inoculated into 10 mL of LB containing 25 mg/L Km and cultured overnight at 30 ° C. with shaking. 0.1 mL of the obtained culture was inoculated into 10 mL of LB containing new 25 mg/L Km and cultured at 30 ° C. with shaking for 16 hours. The cells derived from the obtained culture were washed with MMx-3 buffer and then suspended again in MMx-3 buffer to prepare 1 mL of cell suspension with OD ₆₀₀ = 10. AV was added to the cell suspension to a final concentration of 0.2 mM, and the resulting mixture was subjected to AV conversion reaction by shaking at 30 ° C. and 1,500 rpm. Two hours after the start of the reaction, a portion of the reaction solution was centrifuged to obtain a supernatant, which was subjected to HPLC analysis in the same manner as above.

The results are shown in Figure 3. Figure 3(A) shows the HPLC chromatogram immediately after the start of the reaction, and Figure 3(B) shows the HPLC chromatogram two hours after the start of the reaction.

As shown in Figures 3(A) and 3(B), a peak of VA was detected as a conversion product at a retention time of 12.8 minutes after 2 hours of reaction, indicating that α-hydroxyacetovanillone produced from AV by the gene products of the acpAB genes was converted to VA by the gene product of the acpC gene. From the above, it was demonstrated that AV can be converted to VA by combining the acpA gene, acpB gene, and acpC gene of the MHK4 strain.

[4. AV conversion using AcpA enzyme, AcpB enzyme, and AcpC enzyme]
Plasmids for expressing each of the acpA gene, the acpB gene and the acpC gene in E. coli were prepared by the following procedure.

Using the genomic DNA of the MHK4 strain as a template, a PCR method using a primer set consisting of primers 4 and 5 of SEQ ID NOs: 28 and 29 was used to amplify a DNA fragment of about 1.1 kbp containing the acpA gene.

Using the genomic DNA of the MHK4 strain as a template, a PCR method using a primer set consisting of primers 6 and 7 of SEQ ID NOs: 30 and 31 was used to amplify a DNA fragment of about 1.0 kbp containing the acpB gene.

Using the genomic DNA of the MHK4 strain as a template, a PCR method using a primer set consisting of primers 8 and 9 of SEQ ID NOs: 32 and 33 was used to amplify a DNA fragment of about 0.6 kbp containing the acpC gene.

The resulting fragments were each ligated by seamless cloning using pET-16b plasmid (Merck Millipore) previously digested with NdeI and BamHI and NEBuilder HiFi DNA assembly to prepare acpA gene expression plasmid pE16acpA , acpB gene expression plasmid pE16acpB , and acpC gene expression plasmid pE16acpC , respectively.

The constructed plasmids pE16acpA , pE16acpB , and pE16acpC were each introduced into Escherichia coli BL21 (DE3) strain.

The obtained BL21(DE3)[pE16 acpA ] strain, BL21(DE3)[pE16 acpB ] strain, and BL21(DE3)[pE16 acpC ] strain were inoculated into 10 mL of LB containing 100 mg/L of ampicillin (Amp) and cultured overnight with shaking at 37°C. 0.1 mL of the obtained culture was inoculated into 10 mL of fresh LB containing 100 mg/L of Amp and cultured with shaking at 30°C until OD ₆₀₀ reached 0.5. When OD ₆₀₀ reached 0.5, isopropyl β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM. In addition, _FeSO4 was further added to the BL21(DE3)[ pE16acpA ] and BL21(DE3)[ pE16acpB ] strains to a final concentration of 0.1 mM. After the addition, each strain was subjected to shaking culture at 15°C for 20 hours.

The cells from each culture medium were washed with 50 mM Tris-HCl buffer (pH 7.5) and then suspended again in 0.5 mL of the above buffer. The cells in the cell suspension were disrupted using an ultrasonic homogenizer, and the cell suspension after disruption was centrifuged to obtain supernatants, which were used as AcpA crude enzyme solution, AcpB crude enzyme solution, and AcpC crude enzyme solution, respectively. The protein concentration of each crude enzyme solution was measured by the Bradford method.

The crude enzyme solutions were mixed in 50 mM Tris-HCl buffer (pH 7.5) so that the final concentration of each was 0.5 mg/mL, the final concentration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) was 0.4 mM, and the final concentration of AV was 0.2 mM, and the mixture was reacted at 30°C for 30 minutes. After the reaction, a portion of the reaction solution was taken and mixed with an equal amount of acetonitrile to stop the reaction. The resulting reaction solution was centrifuged, and the AV and reaction products contained in the supernatant were subjected to HPLC analysis in the same manner as above.

The results are shown in Figures 4 to 7. Figure 4 (A) shows the HPLC chromatogram immediately after the start of the reaction using the AcpA crude enzyme solution and AV, and Figure 4 (B) shows the HPLC chromatogram 30 minutes after the start of the reaction. Figure 5 (A) shows the HPLC chromatogram immediately after the start of the reaction using the AcpB crude enzyme solution and AV, and Figure 5 (B) shows the HPLC chromatogram 30 minutes after the start of the reaction. Figure 6 (A) shows the HPLC chromatogram immediately after the start of the reaction using the AcpA crude enzyme solution, the AcpB crude enzyme solution, and AV, and Figure 6 (B) shows the HPLC chromatogram 30 minutes after the start of the reaction. Figure 7 (A) shows the HPLC chromatogram immediately after the start of the reaction using the AcpA crude enzyme solution, the AcpB crude enzyme solution, the AcpC crude enzyme solution, and AV, and Figure 7 (B) shows the HPLC chromatogram 30 minutes after the start of the reaction.

As shown in Figures 4 to 6, unlike when either the AcpA crude enzyme solution or the AcpB crude enzyme solution was used, when both the AcpA crude enzyme solution and the AcpB crude enzyme solution were used, a peak of α-hydroxyacetovanillone was detected as the conversion product at a retention time of 10.4 minutes 30 minutes after the reaction, indicating that AV was converted to α-hydroxyacetovanillone by the AcpA and AcpB enzymes.

As shown in Figure 7, when the AcpA crude enzyme solution, AcpB crude enzyme solution, and AcpC crude enzyme solution were used, a VA peak was detected at a retention time of 12.2 minutes after 30 minutes of reaction, indicating that AV was converted to α-hydroxyacetovanillone by the AcpA and AcpB enzymes, and then α-hydroxyacetovanillone was converted to VA by the AcpC enzyme. From the above, it was found that AV can be converted to VA by combining the AcpA, AcpB, and AcpC enzymes.

[5. Introduction of codon-optimized acpA , acpB and acpC genes and DNA sequences with optimal ribosome binding sites (RBS) added into NGC7Δaph strain and AV conversion]
Analysis of the codons of the acpA gene, acpB gene, and acpC gene revealed that the proportion of codons whose usage frequency in the genome of Pseudomonas putida , a species closely related to the NGC7 strain, was 10% or less of the total was 11.4% in the acpA gene, 13.5% in the acpB gene, and 5.6% in the acpC gene, respectively, which was a high proportion. Therefore, the base sequences of the acpA gene, acpB gene, and acpC gene were modified using GENEius software (Eurofins Genomics LLC) so that the codon usage frequency was equivalent to that in the genome of Pseudomonas putida . In addition, using RBS Calculator (https://salislab.net/software/predict_rbs_calculator), Ribosome binding site (RBS) sequences containing Shine-Dalgarno sequences were designed upstream of the start codons of the acpA gene, acpB gene, and acpC gene so that the translation efficiency in Pseudomonas putida would be maximized. The synthesis of the designed DNA base sequence was entrusted to Twist Bioscience.

The plasmid into which the synthesized DNA sequence was inserted was digested with EcoRI to obtain a DNA fragment of about 2.7 kbp containing the acpC ^opt gene, the acpA ^opt gene and the acpB ^opt gene.

The resulting fragment was ligated to pSEVA241_P _lac previously digested with EcoRI to prepare a plasmid pSEVA acpCAB ^opt for expressing the acpC ^opt gene, the acpA ^opt gene, and the acpB ^opt gene.

The prepared plasmid pSEVA acpCAB ^opt was introduced into the NGC7Δ aph strain by electroporation as described above.

Sphingobium sp. The plasmid pSEVA acv expressing the acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, and acvF gene from the AV convertase gene group derived from the SYK-6 strain (Applied and Environmental Microbiology, Vol. 88, e0072422, 2022) (see Applied and Environmental Microbiology, Vol. 88, e0072422, 2022) expressing the vceA gene and the vceB gene. .

The obtained NGC7Δ aph [pSEVA acpCAB ^opt ] strain and NGC7Δ aph [pSEVA acv + pTS093 vce ] strain were inoculated into 10 mL of LB containing 25 mg / L Km or 25 mg / L Km + 15 mg / L tetracycline (Tc), respectively, and cultured overnight with shaking at 30 ° C. 0.1 mL of each of the obtained cultures was inoculated into a new 10 mL of LB containing 25 mg / L Km or 25 mg / L Km + 15 mg / L Tc, and cultured with shaking at 30 ° C. for 16 hours. Each of the obtained cultures was subjected to centrifugation to recover the bacterial cells, and the obtained bacterial cells were suspended in MMx-3 buffer, and 1.2 mL of the cell suspension was prepared so that the OD ₆₀₀ was 5.0. AV was added to each of the obtained cell suspensions to a final concentration of 1.0 mM, and the resulting mixture was subjected to an AV conversion reaction by shaking at 30° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a supernatant, and the AV and VA concentrations were measured by HPLC analysis in the same manner as above.

The results for the NGC7Δ aph [pSEVA acv +pTS093 vce ] strain are shown in FIG. 8A, and the results for the NGC7Δ aph [pSEVA acpCAB ^opt ] strain are shown in FIG. 8B.

8A and 8B, when the NGC7Δ aph [pSEVA acv +pTS093 vce ] strain was used, only about 20% of AV was converted in a 24-hour reaction, whereas when the NGC7Δ aph (pSEVA acpCAB ^opt ) strain was used, AV was almost completely converted to VA in a 4-hour reaction. From the above, it was shown that by using the AV convertase gene group of the MHK4 strain, AV can be converted to VA more quickly than when the known AV convertase gene group derived from the SYK-6 strain is used.

[6. Growth ability of Pseudomonas sp. NGC7Δaph strain with acpCAB ^opt introduced in AV]
The NGC7Δ aph [pSEVA acpCAB ^opt ] strain and the NGC7Δ aph [pSEVA acv +pTS093 vce ] strain were inoculated into 10 mL of LB containing 25 mg/L Km or 25 mg/L Km + 15 mg/L Tc, respectively, and subjected to shaking culture at 30° C. overnight. 0.1 mL of each of the obtained cultures was inoculated into 10 mL of fresh LB (containing 25 mg/L Km or 25 mg/L Km + 15 mg/L Tc), and subjected to shaking culture at 30° C. for 16 hours. Cells derived from the obtained cultures were suspended in 0.9% (w/v) NaCl to prepare 0.1 mL of a cell suspension with an OD ₆₀₀ of 20.0. 20 μL of the obtained cell suspension was spread on MMx-3 agar medium [34.2 g/L Na ₂ HPO ₄ ·12H ₂ O, 6.0 g/L KH ₂ PO ₄ , 1.0 g/L NaCl, 2.5 g/L (NH ₄ ) ₂ SO ₄ , 49.3 mg/L MgSO ₄ ·7H ₂ O, 15 mg/L CaCl ₂ ·2H ₂ O, 5 mg/L FeSO ₄ ·7H ₂ O, 15 g/L agar] containing 5 mM AV and 25 mg/L Km or 25 mg/L Km + 15 mg/L Tc, and cultured at 30 ° C. for 72 hours. The state of the medium after culture is shown in Figure 9.

As shown in Fig. 9, the NGC7Δaph [pSEVA acv + pTS093 vce ] strain showed almost no growth, whereas the NGC7Δaph [pSEVA acpCAB ^opt ] strain showed remarkable growth. From the above, it was found that the acpA gene, acpB gene, and acpC gene of the MHK4 strain can be used to impart the ability to grow using AV as a carbon source to a host microbial strain.

[ 7. Evaluation of VA accumulation from AV using NGC7ΔvanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 ΔvanA3B3 ΔareA strain]
The plasmid pSEVA acpCAB ^opt was used to transform the NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 Δ areA strain (hereinafter also referred to as NGC729 strain), which is a VA production host of the NGC7 strain, to obtain the NGC729[pSEVA acpCAB ^opt ] strain.

The NGC7Δ vanA1B1 Δ vanA4B4 Δ aph Δ vanA2B2 Δ vanA3B3 strain used was that described in Example 10 of International Publication No. WO2022/210236.

On the other hand, it was confirmed that the genome of the NGC7 strain contains PSN_2384 ( areA gene; SEQ ID NO: 53), a gene encoding an amino acid sequence that showed 96.9% sequence identity with the vanillin reductase gene ( PP_2426 ) derived from the Pseudomonas putida KT2440 strain. The function of the areA gene reduces vanillin contained in the chemical decomposition product of lignin and converts it to vanillyl alcohol, so when vanillic acid is produced from the chemical decomposition product of lignin, it is assumed that the vanillin contained in the decomposition product is converted to vanillyl alcohol, resulting in a decrease in the production amount of vanillic acid. Therefore, a mutant strain in which the areA gene, which is presumed to function as vanillin reductase, was destroyed was prepared.

Using the genomic DNA of the NGC7 strain as a template, a primer set consisting of primers 26 and 27 of SEQ ID NOs: 54 and 55, and a primer set consisting of primers 28 and 29 of SEQ ID NOs: 56 and 57, respectively, was used to amplify approximately 1.2 kbp DNA fragments upstream of the 5' end of the areA gene and downstream of the 3' end of the areA gene, respectively. Each fragment was ligated to pK18 mobsacB previously digested with HindIII and BamHI by seamless cloning using NEBuilder HiFi DNA assembly to prepare a plasmid pK18ΔareA for preparing a strain lacking the areA gene region.

The plasmid pK18ΔareA was introduced into the NGC7ΔvanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 ΔvanA3B3 strain to prepare the NGC7ΔvanA1B1 ΔvanA4B4 Δaph ΔvanA2B2 ΔvanA3B3 ΔareA strain ( NGC729 strain) in which the internal region of the areA gene on the genomic DNA was deleted.

A plasmid for inserting the acpC ^opt / acpA ^opt / acpB ^opt gene set into the genome of the NGC729 strain was prepared by the following procedure.

Using the genomic DNA of Pseudomonas sp. NGC7 strain as a template, a PCR method was performed using a primer set consisting of primers 10 and 11 of SEQ ID NOs: 34 and 35, and a primer set consisting of primers 12 and 13 of SEQ ID NOs: 36 and 37, respectively, to amplify DNA fragments of approximately 1.2 kbp upstream of the 5'-terminus of the vanA4 gene and downstream of the 3'-terminus of the vanB4 gene of the NGC7 strain.

Similarly, a ^DNA fragment of about 2.7 kbp in which the acpC ^opt / acpA ^opt / acpB ^opt gene set was linked downstream of the tac promoter was amplified by PCR using a primer set consisting of primers 14 and 15 of SEQ ID NOs: 38 and 39, using the plasmid pSEVA acpCAB opt as a template. Each amplified fragment was linked by seamless cloning method using pK18 mobsacB (see Gene, Vol. 145, pp. 69-73, 1994) previously digested with HindIII and BamHI and NEBuilder HiFi DNA assembly to prepare a plasmid (pK18Δ vanA4B4 ::P _tac : acpCAB ^opt ) for inserting the acpC ^opt / acpA ^opt / acpB ^opt gene set into the genome of NGC729 strain.

According to a conventional method, the prepared plasmid pK18Δ vanA4B4 ::P _tac : acpCAB ^opt was introduced into the NGC729 strain by electroporation, and a strain capable of growing on LB agar medium containing 25 mg/L Km was selected.

The obtained Km-resistant strain was inoculated into LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) containing 25% sucrose and subjected to shaking culture at 30° C. for 24 hours. The obtained culture solution was smeared on LB agar medium containing 25% sucrose and subjected to stationary culture at 30° C. The transformant in which it was confirmed by colony direct PCR that the acpC ^opt / acpA ^opt / acpB ^opt gene set was inserted into the genomic DNA was designated as NGC729::P _tac : acpCAB ^opt strain.

The obtained NGC729 [pSEVA acpCAB ^opt ] strain and NGC729:: P _tac : acpCAB ^opt strain were inoculated into 10 mL of LB containing or not containing 25 mg / L Km, and subjected to shaking culture at 30 ° C. overnight. 0.1 mL of the obtained culture was inoculated into 10 mL of LB containing or not containing fresh 25 mg / L Km, and subjected to shaking culture at 30 ° C. for 16 hours. The obtained culture was subjected to centrifugation to recover the cells, and the obtained cells were suspended in MMx-3 buffer to prepare 1.2 mL of cell suspension so that OD ₆₀₀ was 5.0. AV was added to the obtained cell suspension so that the final concentration was 1.0 mM, and the obtained mixture was subjected to AV conversion reaction by shaking at 30 ° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a reaction supernatant, and the AV and VA concentrations were measured by HPLC analysis in the same manner as above.

The reaction results using the NGC729 [pSEVA acpCAB ^opt ] strain are shown in FIG. 10A, and the reaction results using the NGC729::P _tac : acpCAB ^opt strain are shown in FIG. 10B. As shown in FIG. 10A and FIG. 10B, in the reactions using the NGC729 [pSEVA acpCAB ^opt ] strain and the NGC729::P _tac : acpCAB ^opt strain, AV was completely converted to VA in 1 hour of reaction. From the above, it was found that VA production from AV is possible by introducing the acpC ^opt / acpA ^opt / acpB ^opt gene set into the NGC729 strain, which is a VA production host of the NGC7 strain, regardless of gene introduction by plasmid or direct introduction into genomic DNA.

The AV conversion reaction was carried out using 1.2 mL of cell suspension prepared so that OD ₆₀₀ was 1.0 for each of the NGC729[pSEVA acpCAB ^opt ] strain and the NGC729::P _tac : acpCAB ^opt strain.

The results for the NGC729[pSEVA acpCAB ^opt ] strain are shown in FIG. 11A, and the results for the NGC729::P _tac : acpCAB ^opt strain are shown in FIG. 11B.

11A and 11B, the rate of VA production from AV was faster when the NGC729::P _tac : acpCAB ^opt strain was used than when the NGC729[pSEVA acpCAB ^opt ] strain was used. From the above, it was found that the NGC729::P _tac : acpCAB ^opt strain in which the gene was directly introduced into the genomic DNA is more suitable for VA production from AV.

[8. Introduction of codon-optimized acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene and vceB gene, and DNA sequence with optimal RBS added, into NGC729 strain and AV conversion]
Using GENEius software, the base sequences were designed so that the codons of the acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene, and vceB gene have the same codon usage frequency as that in the Pseudomonas putida genome. In addition, using RBS Calculator, RBS sequences containing Shine- Dalgarno sequences were designed upstream of the start codons of the acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene, and vceB gene, respectively, so that the translation efficiency in Pseudomonas putida is maximized. The synthesis of the designed DNA base sequence was outsourced to Twist Bioscience.

Using the plasmid into which the synthesized DNA base sequence was inserted as a template, a PCR method using a primer set consisting of primers 16 and 17 of SEQ ID NOs: 40 and 41 was performed to amplify a DNA fragment of about 3.0 kbp in which the acvA ^opt gene and the acvB ^opt gene were linked.

The resulting fragment was ligated to pSEVA241_P _lac previously digested with EcoRI and KpnI by seamless cloning using NEBuilder HiFi DNA assembly to obtain pSEVA acvAB ^opt .

Similarly, a DNA fragment of about 4.5 kbp in which the acvC opt gene, acvD opt gene, acvE ^opt gene and acvF ^opt gene were linked downstream of the E. coli-derived lactose promoter was amplified by PCR using a primer set consisting of primers 18 and 19 of SEQ ^ID NOs: 42 and 43, using the ^plasmid into which the synthesized DNA base sequence was inserted as a template.

The resulting fragment was ligated to pSEVA acvAB ^opt , which had been previously digested with HindIII and BamHI, by seamless cloning using NEBuilder HiFi DNA assembly to obtain pSEVA acv ^opt .

Similarly, a DNA fragment of about 1.5 kbp in which the vceA ^opt gene and the vceB ^opt gene were linked was amplified by PCR using a primer set consisting of primers 20 and 21 of SEQ ID NOs: 44 and 45, using the plasmid into which the synthetic gene had been inserted as a template.

The resulting fragment was ligated to pTS093 previously digested with BamHI and EcoRI and NEBuilder HiFi DNA assembly by seamless cloning method to obtain pTS093 vce ^opt .

The resulting plasmids pSEVA acv ^opt and pTS093 vce ^opt were used to transform the NGC729 strain to obtain the NGC729 [pSEVA acv ^opt +pTS093 vce ^opt ] strain.

Similarly, the NGC729 strain was transformed with the plasmids pSEVA acv and pTS093 vce to obtain the NGC729 [pSEVA acv + pTS093 vce ] strain.

Each of the obtained NGC729 [pSEVA acv ^opt + pTS093 vce ^opt ] strain and NGC729 [pSEVA acv + pTS093 vce ] strain was inoculated into 10 mL of LB liquid medium containing 25 mg / L Km + 15 mg / L Tc, and subjected to shaking culture (180 rpm) at 30 ° C. overnight. 0.1 mL of each of the obtained cultures was inoculated into 10 mL of fresh LB containing 25 mg / L Km + 15 mg / L Tc, and subjected to shaking culture at 30 ° C. for 16 hours. The cells derived from each of the obtained cultures were washed with MMx-3 buffer, and then suspended again in MMx-3 buffer, and 1.2 mL of cell suspension was prepared so that the OD ₆₀₀ was 5.0. AV was added to the obtained cell suspension to a final concentration of 1.0 mM, and the resulting mixture was then subjected to an AV conversion reaction by shaking at 30° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a reaction supernatant, and the AV and VA concentrations were measured by HPLC analysis in the same manner as above.

The reaction results using the NGC729 [pSEVA acv +pTS093 vce ] strain are shown in FIG. 12A, and the reaction results using the NGC729 [pSEVA acv ^opt +pTS093 vce ^opt ] strain are shown in FIG. 12B.

As shown in Figures 12A and 12B, the rate of VA production from AV was slower when the NGC729[pSEVA acv opt ⁺ pTS093 vce opt] strain was used compared to when the NGC729[pSEVA acv +pTS093 vce ^opt ] strain was used, and no effect was obtained by optimizing the codons of the acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, vceA gene, and vceB gene and by adding an optimal RBS.

[9. Evaluation of VA productivity from a coniferous sulfite lignin decomposition product model using NGC729 [pSEVA acv + pTS093 vce ] strain and NGC729::P _tac : acpCAB ^opt strain]
NGC729 [pSEVA acv + pTS093 vce ] strain and NGC729:: P _tac : acpCAB ^opt strain were inoculated into 10 mL of LB liquid medium containing or not containing 25 mg / L Km + 15 mg / L Tc, and subjected to shaking culture (180 rpm) at 30 ° C. overnight. 0.1 mL of the obtained culture was inoculated into 10 mL of fresh LB liquid medium containing or not containing 25 mg / L Km + 15 mg / L Tc, and subjected to shaking culture at 30 ° C. for 16 hours. The obtained culture was subjected to centrifugation to recover the bacterial cells, and the obtained bacterial cells were washed with MMx-3 medium and then suspended again in MMx-3 medium to prepare 1 mL of cell suspension so that OD ₆₀₀ was 10.

MMx-3 liquid medium [34.2 g/L Na ₂ HPO ₄ ·12H 2 O, 6.0 g/L KH 2 PO ₄ , 1.0 g/L NaCl, 2.5 g/L (NH 4 ) 2 SO 4 , 49.3 mg/L MgSO ₄ ·7H ₂ O, 15 mg/L CaCl ₂ ·2H 2 O, 5 mg/L FeSO ₄ ·7H ₂ O] (25 mg/L Km and 15 mg/L CaCl ₂ ·2H ₂ O, 5 mg/L FeSO ₄ ·7H ₂ O)] containing 0.75 _mL of 200 g/L glucose solution and 0.2 mL of a coniferous sulfite lignin decomposition product model [292 mM vanillin (hereinafter also referred to as VN), 81.5 mM AV, 126.5 mM VA)] was used _. The cell suspension was added to 10 mL of medium (with or without Tc) to give an initial OD ₆₀₀ of 0.1. The obtained medium containing the cells was subjected to shaking culture at 30°C.

After the start of the culture, the culture was sampled at regular intervals to measure the OD ₆₀₀ of the culture medium, and the culture medium was centrifuged to obtain a culture supernatant, and the concentrations of AV, VN, VA, and glucose were measured. OD ₆₀₀ was measured using a spectrophotometer ("BIOmaster XB-10", Tomy Seiko). The concentrations of AV, VN, and VA were measured using the above-mentioned HPLC analysis. The glucose concentration was measured using "Biosensor BF-5" (Oji Scientific Instruments).

The results of the culture using the NGC729 [pSEVA acv + pTS093 vce ] strain are shown in FIG. 13A, and the results of the culture using the NGC729::P _tac : acpCAB ^opt strain are shown in FIG. 13B.

As shown in Figures 13A and 13B, in the reaction using the NGC729 [pSEVA acv + pTS093 vce ] strain, AV remained even after 68 hours of culture, whereas in the reaction using the NGC729::P _tac : acpCAB ^opt strain, AV was completely converted to VA after 30 hours of culture.

These results show that even in cultures using the lignin degradation product model, when the AV converting enzyme gene group of the MHK4 strain is used, AV can be converted to VA more quickly than when the AV converting enzyme gene group derived from the SYK-6 strain is used.

The VA yield after 68 hours of culture was 95.7% when the NGC729 [pSEVA acv + pTS093 vce ] strain was used, and 99.4% when the NGC729::P _tac : acpCAB ^opt strain was used. The VA yield was calculated by [VA amount (mol) after 68 hours of culture]/[AV amount (mol) at the start of culture + VN amount (mol) + VA amount (mol)] x 100 (%).

[10. Preparation of MHK4Δ vanA3B3 strain]
The MHK4Δ vanA3B3 strain, which is a mutant strain in which the vanillate O -demethylase gene ( vanAB ) was disrupted from the MHK4 strain, was prepared by the following procedure.

Oxygenation-type vanillate O -demethylase is composed of an oxygenase component (VanA) and an oxidoreductase component (VanB). Genes encoding proteins having amino acid sequences with high sequence identity to the deduced amino acid sequences of vanillate O -demethylase oxygenase component (VanA) and vanillate O -demethylase oxidoreductase component (VanB) derived from P. putida KT2440 strain were searched from the genome sequence of the MHK4 strain. As a result of the search, it was confirmed that the genomic DNA of the MHK4 strain contains a gene vanA3 (PSMH_1445) encoding an amino acid sequence that showed 87.9% sequence identity with VanA, and a gene vanB3 (PSMH_1446) encoding an amino acid sequence that showed 74.7% sequence identity with VanB.

An MHK4Δ vanA3B3 strain in which each of the vanA3 gene and vanB3 gene sets was deleted was prepared by the following procedure: The gene deletion was performed by deleting the entire region of the gene.

Using the genomic DNA of the MHK4 strain as a template, a primer set consisting of primers 22 and 23 of SEQ ID NOs: 46 and 47, and a primer set consisting of primers 24 and 25 of SEQ ID NOs: 48 and 49, respectively, was used to amplify approximately 1.2 kbp DNA fragments upstream of the 5' end of the vanA3 gene and downstream of the 3' end of the vanB3 gene, respectively. Each fragment was ligated by seamless cloning using pK18 mobsacB previously digested with HindIII and BamHI and NEBuilder HiFi DNA Assembly to prepare a plasmid pK18ΔvanA3B3 for preparing strains lacking the gene regions of the vanA3 gene and vanB3 gene.

The prepared plasmid pK18Δ vanA3B3 was introduced into the MHK4 strain by electroporation as described above, and a strain capable of growing on LB agar medium containing 25 mg/L Km was selected.

The obtained Km resistant strain was inoculated into LB liquid medium containing 10% sucrose and subjected to shaking culture at 30°C for 16 to 24 hours. The obtained culture solution was inoculated again into the same medium and cultured three times. After culture, the culture solution was smeared on LB agar medium and cultured stationarily at 30°C. The transformant in which it was confirmed by colony direct PCR that the internal region of the vanA3B3 gene on the genomic DNA had been deleted was designated as the MHK4Δ vanA3B3 strain.

[11. Evaluation of VA degradability of MHK4Δ vanA3B3 strain]
The MHK4 strain and the MHK4Δ vanA3B3 strain were each inoculated into 10 mL of LB liquid medium and subjected to shaking culture for 24 hours at 30° C. 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium and subjected to shaking culture at 30° C. for 16 hours.

The resulting culture solution was subjected to centrifugation to recover the cells, and the cells were suspended in Wx buffer to prepare 1.2 mL of a cell suspension so that the OD ₆₀₀ was 5.0. VA was added to the resulting cell suspension to a final concentration of 1.0 mM, and the resulting mixture was subjected to a VA conversion reaction by shaking at 30° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a supernatant, which was used to measure the VA concentration by HPLC analysis in the same manner as above. The measurement results are shown in Figure 14.

As shown in Fig. 14, the MHK4 strain degraded all of the added VA, whereas the MHK4Δ vanA3B3 strain did not degrade VA. These results demonstrated that vanillate O -demethylase encoded by the vanA3 gene and vanB3 gene is an oxygen-adding type vanillate O -demethylase involved in VA degradation in the MHK4 strain.

[12. Evaluation of VA accumulation from AV using MHK4Δ vanA3B3 strain]
The MHK4Δ vanA3B3 strain was inoculated into 10 mL of LB liquid medium and subjected to shaking culture for 24 hours at 30° C. 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium and subjected to shaking culture at 30° C. for 16 hours.

The resulting culture solution was subjected to centrifugation to recover the cells, and the cells were suspended in Wx buffer to prepare 1.2 mL of cell suspension so that the OD ₆₀₀ was 5.0. AV was added to the resulting cell suspension to a final concentration of 1.0 mM, and the resulting mixture was subjected to an AV conversion reaction by shaking at 30° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a supernatant, which was used to measure the AV and VA concentrations by HPLC analysis in the same manner as above. The measurement results are shown in Figure 15.

15, AV was completely converted to VA after 2 hours of reaction, and VA was accumulated. From the above, it was found that VA production from AV is possible by using the MHK4Δ vanA3B3 strain.

[13. Evaluation of AV degradation ability of MHK4 and SYK-6 strains]
The MHK4 strain and the SYK-6 strain were inoculated into 10 mL of LB liquid medium and subjected to shaking culture at 30° C. for 16 or 24 hours. 0.1 mL of the obtained culture solution was inoculated into 10 mL of fresh LB liquid medium and subjected to shaking culture at 30° C. for 16 or 24 hours.

The resulting culture solution was subjected to centrifugation to recover the cells, and the cells were suspended in Wx buffer to prepare 1.4 mL of a cell suspension so that the OD ₆₀₀ was 5.0. AV was added to the resulting cell suspension to a final concentration of 1.0 mM, and the resulting mixture was subjected to an AV conversion reaction by shaking at 30° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a supernatant, and the AV concentration was measured. The AV concentration was measured by HPLC analysis in the same manner as above. The measurement results are shown in Figure 16.

As shown in Figure 16, the MHK4 strain was found to degrade AV more rapidly than the SYK-6 strain.

14. Substrate range of AcpA and AcpB enzymes
Plasmid pE16 acpA was introduced into Escherichia coli strain Lemo21(DE3), and plasmid pE16 acpB and the chaperone plasmid pKJE7 were introduced into Escherichia coli strain BL21(DE3).

The obtained Lemo21 (DE3) [pE16 acpA ] strain and BL21 (DE3) [pE16 acpB + pKJE7] were inoculated into 10 mL of LB containing 100 mg / L of Amp, and cultured overnight with shaking at 37 ° C. 0.1 mL of the obtained culture was inoculated into 10 mL of fresh LB containing 100 mg / L of Amp, and cultured with shaking at 30 ° C. until OD ₆₀₀ reached 0.5. When OD ₆₀₀ reached 0.5, isopropyl β-D-thiogalactopyranoside was added to a final concentration of 0.5 mM, and FeSO ₄ was further added to a final concentration of 0.1 mM. After addition, each strain was subjected to shaking culture at 15 ° C. for 20 hours.

The cells derived from the culture of Lemo21 (DE3) [pE16 acpA ] strain were washed with 50 mM Tris-HCl buffer (pH 7.5) + 3 mM dithiothreitol solution, and then suspended again in 30 mL of the same solution. The cells derived from the culture of BL21 (DE3) [pE16 acpB + pKJE7] strain were washed with 50 mM Tris-HCl buffer (pH 7.5), and then suspended again in 210 mL of the same buffer. The cells in each cell suspension were disrupted using an ultrasonic homogenizer, and the cell suspension after disruption was subjected to centrifugation to obtain a supernatant.

The resulting supernatant after cell lysis of the Lemo21 (DE3) [pE16 acpA ] strain was sent to 1 mL of a HisTrap HP column to bind Hisx10 fusion AcpA to the column carrier. 50 mM Tris-HCl buffer (pH 7.5) + 500 mM NaCl + 3 mM dithiothreitol + 100 mM imidazole solution was sent to the column to elute impurity proteins, and then 50 mM Tris-HCl buffer (pH 7.5) + 500 mM NaCl + 3 mM dithiothreitol + 500 mM imidazole solution was sent to obtain an elution fraction containing Hisx10 fusion AcpA. The obtained eluted fraction was desalted through a HiTrap Desalting column, and then a fraction containing Hisx10 fused AcpA was collected. The collected fraction was passed through an Amicon Ultra-0.5mL Centrifugal Filter 30kDa cutoff and concentrated until the solution volume was about 0.3mL, and the obtained concentrate was used as a purified AcpA solution.

Similarly, the supernatant obtained after cell lysis of the BL21 (DE3) [pE16 acpB + pKJE7] strain was sent to a HiTrap TALON crude column to bind Hisx10 fusion AcpB to the column carrier. After sending a 50 mM Tris-HCl buffer (pH 7.5) + 500 mM NaCl + 100 mM imidazole solution to elute impurity proteins, a 50 mM Tris-HCl buffer (pH 7.5) + 500 mM NaCl + 500 mM imidazole solution was sent to obtain an elution fraction containing Hisx10 fusion AcpB. The obtained eluted fraction was passed through a HiTrap Desalting column for desalting, and then a fraction containing Hisx10 fusion AcpB was collected. After adding dithiothreitol to the collected fraction to make it 3 mM, the fraction was passed through an Amicon Ultra-0.5mL Centrifugal Filter 30kDa cutoff and concentrated until the solution volume was about 0.3mL, and the resulting concentrate was used as a purified AcpB solution. The protein concentration of each of the obtained purified protein solutions was measured by the Bradford method.

The purified AcpA solution was mixed in 50 mM GTA buffer (pH 7.0) so that the final concentration of the purified AcpB solution was 50 μg/mL, the final concentration of the purified AcpB solution was 134.3 μg/mL, the final concentration of reduced nicotinamide adenine dinucleotide (NADH) was 0.4 mM, and the final concentration of each substrate was 0.2 mM, and the resulting mixture was reacted at 30°C for 1 hour. The substrates were AV, acetophenone, HAP, 3',5'-dimethoxy-4'-hydroxyacetophenone, 3',4'-dihydroxyacetophenone, 3',4'-dimethoxyacetophenone, 3'-hydroxy-4'-methoxyacetophenone, and 3'-methoxyacetophenone, respectively. The following compounds were used: 3'-hydroxyacetophenone, 3',4',5'-trimethoxyacetophenone, 4'-hydroxypropiophenone, 4'-hydroxybutyrophenone, 2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, VA, and HBA. After the reaction, a portion of the reaction solution was taken and mixed with an equal amount of acetonitrile to stop the reaction. The resulting reaction solution was centrifuged, and the resulting supernatant was subjected to HPLC analysis for each substrate and reaction product.

The results of subjecting each substrate to the enzymatic reaction with the AcpAB enzyme as described above are shown in Figure 17ABCDEFGH and Figure 17IJKLMNOP. Note that Figure 17ABCDEFGH includes Figures 17A-H, and Figure 17IJKLMNOP includes Figures 17I-P. Specifically, Figure 17A shows HPLC chromatograms immediately after the start of the reaction using AV as a substrate under conditions that did not contain an enzyme solution, one hour after the start of the reaction, and one hour after the start of the reaction under conditions that contained an enzyme solution. Similarly, acetophenone, HAP, 3',5'-dimethoxy-4'-hydroxyacetophenone, 3',4'-dihydroxyacetophenone, 3',4'-dimethoxyacetophenone, 3'-hydr oxy-4'-methoxyacetophenone, 3'-methoxyacetophenone, 3'-hydroxya HPLC chromatograms for cetophenone, 3',4',5'-trimethoxyacetophenone, 4'-hydroxypropiophenone, 4'-hydroxybutyrophenone, 2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, VA, and HBA as substrates are shown in Figures 17B to 17P, respectively.

As shown in Figures 17A, 17C, 17E, 17K, and 17L, when an enzyme solution consisting of a purified AcpA solution and a purified AcpB solution was used, peaks of the conversion products were detected at retention times of 10.6 minutes, 7.9 minutes, 5.5 minutes, 12.2 minutes, and 14.7 minutes, respectively, after 1 hour of reaction. This indicates that the AcpA enzyme and the AcpB enzyme have the ability to degrade (convert) AV, HAP, 3',4'-dihydroxyacetophenone, 4'-hydroxypropiophenone, and 4'-hydroxybutyrophenone.

On the other hand, as shown in Figures 17B, 17D, 17F, 17G, 17H, 17I, 17J, 17M, 17N, 17O and 17P, acetophenone, 3',5'-dimethoxy-4'-hydroxyacetophenone, 3',4'-dimethoxyacetophenone, 3'-hydroxy-4'-methoxyacetophenone, 3'-methoxyace When 3-tophenone, 3'-hydroxyacetophenone, 3',4',5'-trimethoxyacetophenone, 2-methoxy-4-methylphenol, 2-methoxy-4-ethylphenol, VA, and HBA were used as substrates, no peaks of conversion products were detected, indicating that the AcpA and AcpB enzymes do not have the ability to convert these substrates.

15. Substrate range of AcpC enzyme
The final concentration of the AcpC crude enzyme solution was 50 μg/mL, and the final concentration of the substrates α-hydroxyacetovanillone, 2,4'-dihydroxyacetophenone or α-hydroxyacetosyringone was 0.2 mM. The mixture was mixed in 50 mM Tris-HCl buffer (pH 7.5), and the resulting mixture was reacted at 30 ° C. for 1 hour. After the reaction, a portion of the reaction solution was taken and mixed with an equal amount of acetonitrile to stop the reaction. The resulting reaction solution was centrifuged, and the resulting supernatant was subjected to HPLC analysis of each substrate and reaction product in the same manner as above.

The results of the enzyme reaction of each substrate with the AcpC enzyme as described above are shown in Figures 18 to 20. Specifically, Figure 18A shows an HPLC chromatogram immediately after the start of the reaction using the AcpC crude enzyme solution and α-hydroxyacetovanillone, and Figure 18B shows an HPLC chromatogram one hour after the start of the reaction. Figure 19A shows an HPLC chromatogram immediately after the start of the reaction using the AcpC crude enzyme solution and 2,4'-dihydroxyacetophenone, and Figure 19B shows an HPLC chromatogram one hour after the start of the reaction. Figure 20A shows an HPLC chromatogram immediately after the start of the reaction using the AcpC crude enzyme solution and α-hydroxyacetosyringone, and Figure 20B shows an HPLC chromatogram one hour after the start of the reaction.

As shown in Figures 18 and 19, when α-hydroxyacetovanillone and 2,4'-dihydroxyacetophenone were used as substrates, VA and HBA peaks were detected at retention times of 8.2 and 7.1 minutes, respectively, after 1 hour of reaction. This indicates that the AcpC enzyme converts α-hydroxyacetovanillone to VA and 2,4'-dihydroxyacetophenone to HBA.

On the other hand, as shown in Figure 20, when α-hydroxyacetosyringone was used as a substrate, no peak of the conversion product was detected. This indicates that the AcpC enzyme does not have the ability to convert α-hydroxyacetosyringone.

[16. Evaluation of HBA accumulation from HAP using Pseudomonas sp. NGC7Δ pobA strain with acpCAB ^opt introduced]
It was confirmed that the genome of the NGC7 strain contains PSN_3542 ( pobA gene; SEQ ID NO: 58), a gene encoding an amino acid sequence that shows 94.4% sequence identity with the HBA monooxygenase gene (PP_3537) derived from the Pseudomonas putida KT2440 strain. Therefore, a mutant strain in which the pobA gene was disrupted was prepared to eliminate the ability to degrade HBA.

A DNA fragment of about 1.8 kbp including the pobA gene and about 0.5 kbp from the initiation codon to the 5'-end and about 0.5 kbp from the termination codon to the 3'-end was amplified by PCR using the genomic DNA of the NGC7 strain as a template and a primer set consisting of primers 30 and 31 of SEQ ID NOs: 59 and 60. The resulting DNA fragment was digested with EcoRI and HindIII, and ligated to pUC118 similarly digested with EcoRI and HindIII to prepare pUC118+ pobA .

Next, pUC118+ pobA was digested with SacI to delete about 0.3 kbp of the internal region of pobA , followed by self-ligation to prepare pUC118+Δ pobA . The DNA fragment of about 1.5 kbp obtained by digesting pUC118+Δ pobA with EcoRI and HindIII was ligated to pK19 mobsacB similarly digested with EcoRI and HindIII to prepare the plasmid pTS220 for preparing a strain lacking the pobA gene region.

The prepared plasmid pTS220 was introduced into the NGC7 strain by electroporation according to standard methods, and a strain capable of growing on LB agar medium containing 25 mg/L Km was selected.

The obtained Km-resistant strain was inoculated into 10 mL of LB liquid medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) containing 10% sucrose, and subjected to shaking culture at 30° C. for 16 hours. 0.1 mL of the obtained culture solution was further inoculated into the same medium, and subjected to shaking culture at 30° C. for 16 hours. This operation was repeated twice. The obtained culture solution was smeared on an LB agar medium, and subjected to stationary culture at 30° C. The transformant in which deletion of the pobA gene region on the genomic DNA was confirmed by colony direct PCR was designated as NGC7Δ pobA strain.

The resulting NGC7Δ pobA was transformed with the plasmid pSEVA acpCAB ^opt to obtain the strain NGC7Δ pobA [pSEVA acpCAB ^opt ].

The obtained NGC7Δ pobA [pSEVA acpCAB ^opt ] strain was inoculated into 10 mL of LB containing 25 mg / L Km and subjected to shaking culture at 30 ° C. overnight. 0.1 mL of the obtained culture was inoculated into 10 mL of LB containing new 25 mg / L Km and subjected to shaking culture at 30 ° C. for 16 hours. The obtained culture was subjected to centrifugation to recover the cells, and the obtained cells were suspended in MMx-3 buffer to prepare 1.2 mL of cell suspension so that the OD ₆₀₀ was 2.0. HAP was added to the obtained cell suspension so that the final concentration was 1.0 mM, and the resulting mixture was subjected to HAP conversion reaction by shaking at 30 ° C. and 1,500 rpm.

After the reaction started, samples were taken at regular intervals, and the reaction solution was centrifuged to obtain a reaction supernatant, and the concentrations of HAP and HBA were measured by HPLC analysis in the same manner as above.

The results of the conversion reaction from HAP to HBA using the NGC7Δ pobA [pSEVA acpCAB ^opt ] strain are shown in Figure 21. As shown in Figure 21, the NGC7Δ pobA [pSEVA acpCAB ^opt ] strain converted about 84% of HAP to HBA in a 24-hour reaction. From the above, it was found that HBA can be produced from HAP by introducing the acpC ^opt / acpA ^opt / acpB ^opt gene set into the NGC7Δ pobA strain, which is an HBA production host of the NGC7 strain.

[17. Evaluation of HBA productivity from a mixture of HAP and AV using Pseudomonas sp. NGC7Δ pobA strain with acpCAB ^opt introduced]
The NGC7Δ pobA [pSEVA acpCAB ^opt ] strain was inoculated into 10 mL of LB liquid medium containing 25 mg/L Km and subjected to shaking culture (180 rpm) at 30° C. overnight. 0.1 mL of the obtained culture was inoculated into 10 mL of fresh LB liquid medium containing 25 mg/L Km and subjected to shaking culture at 30° C. for 16 hours. The obtained culture was centrifuged to recover the cells, and the obtained cells were washed with MMx-3 medium and then suspended again in MMx-3 medium to prepare 1 mL of cell suspension so that the OD ₆₀₀ was 10.

The cell suspension was added to 10 mL of MMx-3 liquid medium [34.2 g/L Na ₂ HPO ₄ ·12H ₂ O, 6.0 g/L KH ₂ PO ₄ , 1.0 g/L NaCl, 2.5 g/L (NH ₄ ) ₂ SO ₄ , 49.3 mg/L MgSO ₄ ·7H ₂ O, 15 mg/L CaCl ₂ ·2H ₂ O, 5 mg/L FeSO ₄ ·7H ₂ O] (containing 25 mg/L Km and 15 mg/L Tc) containing 0.75 mL of 200 g/L glucose solution, 0.1 mL of 100 mM HAP solution, and 0.1 mL of 100 mM AV solution, so that the initial OD ₆₀₀ was 0.1. The medium containing the obtained cells was subjected to shaking culture at 30°C.

After the start of the culture, the culture was sampled at regular time intervals to measure the OD ₆₀₀ of the culture medium. The culture medium was then centrifuged to obtain a culture supernatant, and the concentrations of HAP, AV, HBA, VA, and glucose were measured. _{OD 600} was measured using a spectrophotometer (BioSpec-mini, Shimadzu Co., Ltd.). The concentrations of HAP, AV, HBA, and VA were measured using the above-mentioned HPLC analysis. The glucose concentration was measured using the above-mentioned biosensor BF-5.

The culture results of the HBA production test from a mixture of HAP and AV are shown in Figure 22. As shown in Figure 22, after 72 hours of culture, HAP and AV were completely consumed, and only HBA accumulated. This demonstrated that only HBA can be accumulated from a mixture of HAP and AV by introducing it into the NGC7Δ pobA strain, which is an HBA production host of the NGC7 strain.

The HBA yield after 72 hours of culture was 102%. The HBA yield was calculated by dividing the amount of HBA (mol) after 72 hours of culture by the amount of HAP (mol) at the start of culture x 100 (%).

18. Summary
The following points were confirmed through the above Examples 1 to 17.
A new strain of Pseudomonas sp. MHK4 capable of assimilating AV was isolated from soil samples collected from various parts of Japan.
The MHK4 strain did not have any genes having a sequence identity of 30% or more with the acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene , and vceA gene and vceB gene among the AV convertase gene group ( acvA gene, acvB gene, acvC gene, acvD gene, acvE gene, acvF gene, and vceA gene and vceB gene) possessed by the Sphingobium sp. SYK-6 strain.
The MHK4 strain possessed the acpA and acpB genes and thus converted AV into α-hydroxyacetovanillone.
The MHK4 strain possessed the acpA , acpB and acpC genes and thereby converted AV to VA.
AV was converted to α-hydroxyacetovanillone by AcpA and AcpB enzymes, which are the gene products of the acpA and acpB genes.
AV was converted to VA by AcpA enzyme, AcpB enzyme and AcpC enzyme, which are gene products of acpA gene, acpB gene and acpC gene.
The transformant in which the AV convertase genes of the SYK-6 strain were introduced into the Pseudomonas sp. NGC7 strain had a low efficiency of AV to VA conversion, whereas the transformant in which the acpA gene, acpB gene, and acpC gene of the MHK4 strain were introduced into the NGC7 strain had a very high efficiency of AV to VA conversion.
A transformant in which the AV convertase gene group of the SYK-6 strain was introduced into the NGC7 strain did not grow using AV as a carbon source, whereas a transformant in which the acpA gene, acpB gene, and acpC gene of the MHK4 strain were introduced into the NGC7 strain grew using AV as a carbon source.
By introducing the acpA , acpB , and acpC genes of the MHK4 strain into the NGC7 strain lacking the vanAB genes, conversion from AV to VA was possible regardless of whether the introduction was by plasmid introduction or genome introduction.
In the case of a model of coniferous sulfite lignin decomposition product containing 1.6 mM AV, the transformant in which the AV convertase genes of SYK-6 strain were introduced into NGC7 strain hardly consumed AV, whereas the transformant in which the acpA gene, acpB gene, and acpC gene of MHK4 strain were introduced into NGC7 strain completely consumed AV. In this case, the VA yield of the transformant in which the acpA gene, acpB gene, and acpC gene of MHK4 strain were introduced into NGC7 strain was higher.
The MHK4 strain was able to assimilate VA by expression of the vanA3 and vanB3 genes.
A transformant in which the vanA3 and vanB3 genes of the MHK4 strain were deleted produced VA from AV.
The MHK4 strain degraded AV more rapidly than the SKY-6 strain.
The AcpAB enzyme had decomposition activities against AV, HAP, 3',4'-dihydroxyacetophenone, 4'-hydroxypropiophenone, and 4'-hydroxybutyrophenone.
The AcpC enzyme converted α-hydroxyacetovanillone and 2,4'-dihydroxyacetophenone to VA and HBA, respectively.
A transformant in which the acpA , acpB and acpC genes of the MHK4 strain were introduced into the pobA gene-deleted NGC7 strain produced HBA from HAP alone or a mixture of HAP and AV.
It has been found that by using the AcpAB enzymes and AcpC enzymes and microorganisms expressing them, useful substances that can be used as monomers because they have carboxyl groups and hydroxyl groups in the molecule can be produced from H-nucleus-derived acetophenones (e.g., HAP, etc.) and G-nucleus-derived acetophenones (e.g., AV, etc.) obtained by alkaline oxidative decomposition of lignin.

[19. Sequence Listing]
The sequences described in the sequence listing are as shown in Tables 6A to 6D below.

By utilizing the gene, microorganism, transformed microorganism, and production method of one embodiment of the present invention, vanillic acid can be obtained from biomass containing acetovanillone, an aromatic compound derived from lignin. Vanillic acid can be converted into various industrially useful compounds, and can be used, for example, as a raw material for vanillic acid derivatives, which have applications such as heat-resistant and rigid plastics and coating agents. Similarly, the present invention can be used to obtain HBA from HAP, an aromatic compound derived from lignin, via DHAP.

Claims (17)

A transformed microorganism into which the acpA gene and the acpB gene have been introduced as foreign genes and which expresses the introduced genes. 2. The transformed microorganism according to claim 1, wherein the host microorganism is Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043). A method for producing α-hydroxyacetovanillone, comprising the step of: allowing acetovanillone to act on an expression product of acpA gene and acpB gene, or on the transformed microorganism according to claim 1 or 2, thereby obtaining α-hydroxyacetovanillone. A transformed microorganism into which the acpA gene, the acpB gene and the acpC gene have been introduced as foreign genes and which expresses the introduced genes. The transformed microorganism according to claim 4, wherein the host microorganism is a transformed microorganism of Pseudomonas sp. NGC7 strain (Accession Number: NITE BP-03043) in which the vanillate O -demethylase gene on the chromosome is deleted. 6. A method for producing vanillic acid, comprising the step of: allowing acetovanillone to act on an expression product of the acpA gene, the acpB gene and the acpC gene, or allowing it to act on the transformed microorganism according to claim 4 or 5, thereby obtaining vanillic acid. The transformed microorganism according to claim 4, wherein the transformed microorganism is a transformed microorganism of Pseudomonas sp. NGC7 strain (accession number: NITE BP-03043) in which the pobA gene on the chromosome of the host microorganism is deleted. A method for producing 4-hydroxybenzoic acid, comprising the step of: allowing 4'-hydroxyacetophenone to act on an expression product of the acpA gene, the acpB gene and the acpC gene, or allowing it to act on the transformed microorganism according to claim 7, thereby obtaining 4-hydroxybenzoic acid. An acpA gene comprising any one of the following nucleotide sequences (1) to (4), which encodes an amino acid sequence of an enzyme having an activity of catalyzing a reaction of converting acetovanillone to α-hydroxyacetovanillone and/or an activity of catalyzing a reaction of converting 4'-hydroxyacetophenone to 2,4'-dihydroxyacetophenone:
(1) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 1 or 4 in the sequence listing or a nucleotide sequence complementary to said nucleotide sequence, and in said nucleotide sequence set forth in SEQ ID NO: 1 or 4, has deletion, substitution and/or addition of 1 to 10 bases per unit consisting of 100 bases. (2) A nucleotide sequence which has 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or 4. (3) A nucleotide sequence which encodes an amino acid sequence which has 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 50. (4) A nucleotide sequence which encodes an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and/or added per unit consisting of 100 amino acids in the amino acid sequence set forth in SEQ ID NO: 50. An acpB gene comprising any one of the following nucleotide sequences (1) to (4), which encodes an amino acid sequence of an enzyme having an activity of catalyzing a reaction of converting acetovanillone to α-hydroxyacetovanillone and/or an activity of catalyzing a reaction of converting 4'-hydroxyacetophenone to 2,4'-dihydroxyacetophenone:
(1) A nucleotide sequence which hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 2 or 5 in the sequence listing or a nucleotide sequence complementary to said nucleotide sequence, and in said nucleotide sequence set forth in SEQ ID NO: 2 or 5, has deletion, substitution and/or addition of 1 to 10 bases per unit consisting of 100 bases. (2) A nucleotide sequence which has 80% or more sequence identity with a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2 or 5. (3) A nucleotide sequence which encodes an amino acid sequence which has 50% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 51. (4) A nucleotide sequence which encodes an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and/or added per unit consisting of 100 amino acids in the amino acid sequence set forth in SEQ ID NO: 51. Pseudomonas sp. MHK4 strain (accession number: NITE P-03794). the host microorganism is Pseudomonas sp. MHK4 strain (accession number: NITE P-03794); and the vanillate O -demethylase gene located on the chromosome is deleted;
Transformed microorganisms. 13. The transformed microorganism according to claim 12, wherein the vanillate O 2 -demethylase gene is at least one vanillate O 2 -demethylase gene selected from the group consisting of a vanA3 gene (SEQ ID NO: 23) and a vanB3 gene (SEQ ID NO: 24). A method for producing vanillic acid, comprising the step of obtaining vanillic acid by allowing acetovanillone to act on the transformed microorganism according to claim 12 or 13. The host microorganism is Pseudomonas sp. MHK4 strain (Accession Number: NITE P-03794), and the 4-hydroxybenzoate monooxygenase gene on the chromosome is deleted.
Transformed microorganisms. The transformed microorganism according to claim 15, wherein the 4-hydroxybenzoate monooxygenase gene is at least one 4-hydroxybenzoate monooxygenase gene selected from the group consisting of a PobA1 gene (SEQ ID NO: 61) and a pobA2 gene (SEQ ID NO: 62). A method for producing 4-hydroxybenzoic acid, comprising the step of obtaining 4-hydroxybenzoic acid by allowing 4'-hydroxyacetophenone to act on the transformed microorganism according to claim 15 or 16.