JP7531311B2 - Gallic acid synthase - Google Patents

Description

The present invention relates to gallic acid synthase.

Gallic acid (3,4,5-trihydroxybenzoic acid) has strong reducing power and is used as a reducing agent and photographic developer. It is also used in the manufacture of inks because it has the property of coloring by forming iron salts. Furthermore, many derivatives such as gallic acid esters can be obtained from gallic acid, and these are widely used in various fields such as the food industry and electronic materials industry.

A method for producing gallic acid from inexpensive raw materials such as aromatic carboxylic acids and glucose using microorganisms has been reported. Patent Documents 1 and 2 disclose an enzyme derived from Corynebacterium glutamicum ATCC13032 strain that has the enzymatic activity of oxidizing the 5-position of protocatechuic acid as an enzyme that converts protocatechuic acid to gallic acid.

JP 2009-065839 A JP 2009-213392 A

The present invention provides a novel gallic acid synthase.

That is, the present invention provides a polypeptide having the function of converting protocatechuic acid to gallic acid, which consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity to said sequence.
The present invention also provides a polynucleotide encoding the polypeptide.
The present invention also provides a vector containing the polynucleotide.
The present invention also provides a transformant containing the polynucleotide or vector.
The present invention also provides a method for producing gallic acid, which comprises converting protocatechuic acid to gallic acid using the above-mentioned polypeptide.

The polypeptide of the present invention is a highly active gallic acid synthase that has the function of converting protocatechuic acid to gallic acid. The present invention is useful for the biochemical production of gallic acid.

All patents, non-patent literature, and other publications cited herein are hereby incorporated by reference in their entirety.

In this specification, the identity of amino acid sequences or nucleotide sequences is calculated by the Lipman-Pearson method (Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis using the homology analysis program of the genetic information processing software GENETY Ver. 12 with the unit size to compare (ktup) set to 2.

As used herein, "at least 90% identity" with respect to an amino acid sequence or a nucleotide sequence means identity of 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, even more preferably 99% or more, even more preferably 99.5% or more, even more preferably 99.7% or more.

As used herein, a "corresponding position" or "corresponding region" on an amino acid sequence or a nucleotide sequence can be determined by aligning a target sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 2) to maximize homology. Alignment of amino acid sequences or nucleotide sequences can be performed using known algorithms, the procedures of which are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Thompson, J.D. et al., 1994, Nucleic Acids Res. 22: 4673-4680) with default settings. Clustal W can be used, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk/index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp/searches-j.html]) operated by the National Institute of Genetics. The position of the target sequence aligned to any position of the reference sequence by the above-mentioned alignment is considered to be a "position corresponding to" that any position. In addition, a region sandwiched between corresponding positions or a region consisting of a corresponding motif is considered to be a corresponding region.

Those skilled in the art can further fine-tune the alignment of the amino acid sequences obtained above to optimize it. Such an optimal alignment is preferably determined taking into consideration the similarity of the amino acid sequences, the frequency of gaps to be inserted, and the like. Here, the similarity of the amino acid sequences refers to the percentage (%) of the number of positions at which identical or similar amino acid residues exist in both sequences when the two amino acid sequences are aligned, relative to the total number of amino acid residues. Similar amino acid residues refer to amino acid residues that have similar properties in terms of polarity and charge among the 20 types of amino acids that make up proteins, and that cause so-called conservative substitutions. Such groups of similar amino acid residues are well known to those skilled in the art, and examples thereof include, but are not limited to, arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; leucine and isoleucine.

In this specification, "amino acid residue" refers to the 20 types of amino acid residues that make up proteins: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

As used herein, "operably linked" between a control region such as a promoter and a gene means that the gene and the control region are linked in such a way that the gene can be expressed under the control of the control region. The procedure for "operably linked" between a gene and a control region is well known to those skilled in the art.

As used herein, "upstream" and "downstream" in relation to a gene refer to the upstream and downstream of the transcription direction of the gene. For example, "a gene located downstream of a promoter" means that the gene is present on the 3' side of the promoter on the DNA sense strand, and "upstream" of a gene means the region on the 5' side of the gene on the DNA sense strand.

In this specification, "gallic acid synthase" refers to an enzyme that catalyzes the synthesis reaction of gallic acid (3,4,5-trihydroxybenzoic acid). Examples of gallic acid synthase include enzymes that have the function of converting protocatechuic acid to gallic acid, and more specific examples include enzymes that catalyze the reaction of oxidizing the 5-position of protocatechuic acid to convert it to gallic acid.

The present invention provides a polypeptide that functions as a gallic acid synthase. In one embodiment, the polypeptide of the present invention consists of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity thereto. The polypeptide of the present invention has the function of converting protocatechuic acid to gallic acid. More specifically, the polypeptide of the present invention has the function of converting protocatechuic acid to gallic acid by oxidizing the 5-position of protocatechuic acid.

The polypeptide of the present invention can be obtained by a conventional chemical synthesis method or a genetic engineering method. For example, the polypeptide of the present invention can be chemically synthesized based on the amino acid sequence of SEQ ID NO: 2. For chemical synthesis of peptides, a commercially available peptide synthesis service provided by, for example, GenScript, Inc. can be used. Alternatively, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 can be obtained by introducing a mutation into the amino acid sequence of 4-hydroxybenzoate-3-monooxygenase (NCBI: WP_040354425.1) derived from Corynebacterium ammoniagenes. For example, a gene encoding 4-hydroxybenzoate-3-monooxygenase (WP_040354425.1) derived from Corynebacterium ammoniagenes is mutated to prepare a gene encoding the polypeptide of the present invention (for example, a polynucleotide of SEQ ID NO: 6), and a mutant polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 can be expressed from the obtained gene.

A polypeptide having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2 can be produced by introducing a mutation into the polypeptide having the amino acid sequence of SEQ ID NO:2. Various mutation introduction techniques known in the art can be used as a means for mutating amino acid residues of a polypeptide. For example, in a gene encoding the amino acid sequence of SEQ ID NO:2 (e.g., a polynucleotide of SEQ ID NO:6), a nucleotide sequence encoding an amino acid residue to be mutated is mutated into a nucleotide sequence encoding an amino acid residue after mutation, and the mutated gene is expressed to obtain a mutant polypeptide of interest. Alternatively, a nucleotide sequence corresponding to the amino acid sequence of the mutant polypeptide of interest can be chemically synthesized and expressed to obtain a mutant polypeptide of interest. Preferably, the polypeptide of the present invention having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO:2 has Val at a position corresponding to position 208 of SEQ ID NO:2 and Phe at a position corresponding to position 397 of SEQ ID NO:2.

Introduction of the desired mutation into a gene can basically be carried out using various site-specific mutagenesis methods well known to those skilled in the art. Site-specific mutagenesis can be carried out by any method, such as inverse PCR or annealing. Commercially available site-specific mutagenesis kits (e.g., QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit, etc., from Stratagene) can also be used.

Site-specific mutagenesis can be performed most commonly using a mutagenesis primer containing the nucleotide mutation to be introduced. The mutagenesis primer can be designed to anneal to a region containing a nucleotide sequence encoding the amino acid residue to be mutated in the target gene, and to contain a nucleotide sequence having a nucleotide sequence (codon) encoding the mutated amino acid residue instead of the nucleotide sequence (codon) encoding the amino acid residue to be mutated. A person skilled in the art can appropriately recognize and select the nucleotide sequences (codons) encoding the amino acid residues before and after the mutation based on a typical textbook or the like. Alternatively, site-specific mutagenesis can be performed by a method in which DNA fragments obtained by amplifying the upstream and downstream sides of the mutation site using two complementary primers containing the nucleotide mutation to be introduced separately are linked together by SOE (splicing by overlap extension)-PCR (Gene, 1989, 77(1): pp. 61-68).

Template DNA containing the target gene can be prepared from Corynebacterium ammoniagenes by extracting genomic DNA using standard methods, or by extracting RNA and synthesizing cDNA by reverse transcription. Alternatively, a nucleotide sequence corresponding to the amino acid sequence of the desired polypeptide can be chemically synthesized and used as the template DNA.

The mutation primer can be prepared by a well-known oligonucleotide synthesis method such as the phosphoramidite method (Nucleic Acids R4 research, 1989, 17: 7059-7071). Such primer synthesis can also be performed using, for example, a commercially available oligonucleotide synthesizer (such as that manufactured by ABI). A primer set including the mutation primer can be used to perform site-specific mutagenesis as described above using the target gene as template DNA to obtain a polynucleotide encoding the desired mutant polypeptide.

Therefore, the present invention also provides a polynucleotide encoding the polypeptide of the present invention. The polynucleotide of the present invention may include single-stranded or double-stranded DNA, cDNA, RNA, or other artificial nucleic acid. The DNA, cDNA, and RNA may be chemically synthesized. The polynucleotide of the present invention may also include a nucleotide sequence of an untranslated region (UTR) in addition to an open reading frame (ORF). The polynucleotide of the present invention may also be codon-optimized according to the species of the transformant used to produce the polypeptide of the present invention. Information on codons used by various organisms is available from the Codon Usage Database ([www.kazusa.or.jp/codon/]). An example of a polynucleotide encoding the polypeptide of the present invention is a polynucleotide comprising the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 90% identity to said sequence.

The present invention also provides a DNA fragment containing a polynucleotide encoding a polypeptide of the present invention. Preferably, the DNA fragment contains a polynucleotide encoding the polypeptide of the present invention and a control region operably linked upstream thereof. The control region contained in the DNA fragment preferably contains a promoter, and may further contain a cis-element, a terminator, or the like that improves the transcription activity of the promoter. Furthermore, the DNA fragment may contain a selection marker gene such as a drug resistance gene or an auxotrophic marker gene. Preferably, the DNA fragment is a DNA expression cassette for expressing a polynucleotide encoding a polypeptide of the present invention.

The DNA fragment may have a restriction enzyme recognition sequence at both ends. The restriction enzyme recognition sequence may be used to introduce the DNA fragment into a vector. For example, the vector may be cleaved with a restriction enzyme, and a DNA fragment having the restriction enzyme cleavage sequence may be added thereto, thereby introducing the DNA fragment into the vector (restriction enzyme method).

The present invention also provides a vector containing a polynucleotide encoding the polypeptide of the present invention. The vector can be prepared by inserting the polynucleotide of the present invention into any vector by a conventional method. The type of vector is not particularly limited, and may be any vector such as a plasmid, cosmid, phasmid, phage, transposon, or BAC vector. The vector may be a vector for introduction into the chromosome of a host cell, or a vector that is retained extrachromosomally. A vector that can be amplified in a host cell is preferred.

The vector is preferably, but not limited to, a bacterial vector, more preferably a vector for bacteria belonging to the genus Escherichia or Corynebacterium. Examples of preferred vectors include, but are not limited to, pET21-a(+), pUC18/19, pUC118/119, pBR322, pMW218/219, pZ1 (Applied and Environmental Microbiology, 1989, 55:684-688), pEKEx1 (Gene, 1991, 102:93-98), pHS2-1 (Gene, 1991, 107:69-74), pCLiK5MCS (JP-T-2005-522218), pCG2 (JP-A-58-35197), pNG2 (FEMS Microbiology Letters, 1990, 66: 119-124), pAG1 (JP Patent Publication 61-52290), pHKPsacB1 (WO2014/007273), etc.

When recombinantly producing the polypeptide of the present invention, the vector is preferably an expression vector. The expression vector may contain useful sequences as necessary, such as various elements essential for gene expression in a host organism, such as a transcription promoter and a terminator; cis elements, such as a polylinker and an enhancer; a polyA addition signal; a ribosome binding sequence (SD sequence); and a selection marker gene, such as a drug resistance gene or an auxotrophic marker gene.

The present invention also provides a transformant comprising a polynucleotide encoding the above-mentioned polypeptide of the present invention or a vector containing the same. The transformant can be produced by introducing a polynucleotide encoding the polypeptide of the present invention, or a DNA fragment or vector containing the same, into a host.

The host of the transformant is not particularly limited, but is preferably a bacterium, more preferably a bacterium belonging to the genus Escherichia or Corynebacterium. An example of Escherichia is Escherichia coli. Examples of Corynebacterium are preferably Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes, Corynebacterium halotolerans, Corynebacterium alkanolyticum, Corynebacterium callunae, etc., more preferably Corynebacterium glutamicum and Corynebacterium ammoniagenes, and even more preferably Corynebacterium glutamicum. In addition, according to molecular biological classification, coryneform bacteria such as Brevibacterium flavum, Brevibacterium lactofermentum, Brevibacterium divaricatum, and Corynebacterium lilium have been unified under the name Corynebacterium glutamicum (Liebl W et al., Int J Syst Bacteriol, 1991, 41: 255-260; Komagata Kazuo et al., Fermentation and Industry, 1987, 45: 944-963).

To introduce a polynucleotide or vector into a host cell, a general transformation method, such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium, can be used.

The polypeptide of the present invention can be produced by culturing the transformant of the present invention. Thus, the present invention also provides a method for producing the polypeptide of the present invention using the transformant of the present invention. More specifically, the method for producing the polypeptide of the present invention includes culturing the transformant of the present invention. For example, when the transformant is Escherichia coli, preferred media include LB medium, M9 medium, etc., and the culture conditions include a culture temperature of 15°C to 45°C and a culture time of 1 to 7 days. When the transformant is Corynebacterium, preferred media include LB medium, CGXII medium (Journal of Bacteriology, 1993, 175:5595-5603), etc., and the culture conditions include a culture temperature of 15°C to 45°C and a culture time of 1 to 7 days.

Alternatively, the polypeptide of the present invention may be expressed from a polynucleotide encoding the polypeptide of the present invention or a transcription product thereof using a cell-free translation system. A "cell-free translation system" is an in vitro transcription/translation system or an in vitro translation system that is constructed by adding reagents such as amino acids necessary for protein translation to a suspension obtained by mechanically disrupting host cells.

The polypeptide of the present invention produced by the transformant or cell-free translation system can be recovered from the culture medium, cell lysate, reaction solution of the cell-free translation system, etc., by using general methods used in protein purification, such as cell disruption, centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., either alone or in appropriate combinations.

The polypeptide of the present invention is a gallic acid synthase, and has a higher gallic acid synthesis activity (activity to convert protocatechuic acid to gallic acid) than a conventionally known gallic acid synthase derived from Corynebacterium (for example, SEQ ID NO: 1: a mutant enzyme in which Leu at position 200 of parahydroxybenzoate hydroxylase (HFM145) of Corynebacterium glutamicum ATCC13032 strain is replaced with Val and Tyr at position 385 is replaced with Phe, as disclosed in Patent Document 2). Preferably, the gallic acid synthesis activity (activity to convert protocatechuic acid to gallic acid) of the polypeptide of the present invention can be 110% or more compared to the polypeptide of SEQ ID NO: 1. The gallic acid synthesis activity of the polypeptide can be measured, for example, by quantifying the amount of gallic acid produced by the reaction of the polypeptide with the substrate protocatechuic acid. Chromatography can be used, for example, to quantify the amount of gallic acid.

Therefore, the present invention also provides a method for producing gallic acid using the polypeptide of the present invention. The method for producing gallic acid according to the present invention includes converting protocatechuic acid to gallic acid by the polypeptide of the present invention. In a more detailed example, the method includes adding the polypeptide of the present invention to a liquid containing protocatechuic acid as a substrate, and converting protocatechuic acid to gallic acid. In the method, the polypeptide of the present invention may be used in the form of a purified polypeptide or a solution containing the same, or may be used in the form of a cell disruption liquid, a cell extract, or a supernatant thereof, or a solution or suspension thereof, of the transformant of the present invention described above. Alternatively, the method for producing gallic acid according to the present invention includes culturing the transformant of the present invention described above in a medium containing protocatechuic acid as a substrate, whereby protocatechuic acid is converted to gallic acid by the polypeptide of the present invention expressed by the transformant. Alternatively, the method for producing gallic acid according to the present invention includes producing a transformant of the present invention capable of producing protocatechuic acid and culturing it. That is, a transformant that produces the polypeptide of the present invention and the substrate protocatechuic acid is produced by introducing a polynucleotide encoding the polypeptide of the present invention, or a DNA fragment or vector containing the polynucleotide, into a microorganism capable of producing protocatechuic acid as a host. Gallic acid is produced from the produced polypeptide of the present invention and protocatechuic acid by culturing the transformant. The initial concentration of protocatechuic acid in the reaction solution (or culture) is preferably about 0.1 to 500 mM, and the concentration of the polypeptide of the present invention is preferably about 1 mg/L to 10 g/L. The temperature of the reaction solution is preferably 10 to 60°C, more preferably 15 to 50°C, and the pH of the reaction solution is preferably pH 5 to 10, more preferably pH 6 to 9. The reaction solution may contain a buffer, a pH adjuster, a coenzyme, and the like. When the reaction solution is a culture, the temperature and pH conditions, and the composition of the medium are preferably in accordance with the culture conditions of the transformant.

The present invention also includes the following substances, manufacturing methods, uses, methods, etc. as exemplary embodiments. However, the present invention is not limited to these embodiments.

[1] A polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90% identity to said sequence, and having the function of converting protocatechuic acid into gallic acid.
[2] The polypeptide according to [1], preferably having Val at the position corresponding to position 208 of SEQ ID NO:2 and Phe at the position corresponding to position 397.
[3] A polynucleotide encoding the polypeptide according to [1] or [2].
[4] A vector containing the polynucleotide described in [3].
[5] A DNA fragment containing the polynucleotide according to [3].
[6] The DNA fragment according to [5], which is preferably a DNA expression cassette.
[7] A transformant comprising the polynucleotide according to [3], the vector according to [4], or the DNA fragment according to [5] or [6].
[8] The transformant according to [7], which is preferably a bacterium belonging to the genus Escherichia or Corynebacterium.
[9] The transformant according to [7] or [8], preferably having the ability to produce protocatechuic acid.
[10] A method for producing gallic acid, comprising converting protocatechuic acid to gallic acid using the polypeptide according to [1] or [2].
[11] The method according to [10], preferably comprising culturing the transformant according to [7] or [8] in a medium containing the protocatechuic acid.
[12] The method according to [10], which preferably comprises culturing the transformant according to [7] or [8], wherein the transformant has the ability to produce protocatechuic acid.

The present invention will be explained in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

Example 1 Preparation of gallic acid synthase 1) Preparation of gene encoding gallic acid synthase Cg145 Patent Document 2 discloses that a mutant enzyme (disclosed as SEQ ID NO: 22 in Patent Document 2) in which Leu at position 200 is replaced with Val and Tyr at position 385 is replaced with Phe in the amino acid sequence of parahydroxybenzoate hydroxylase (Genbank Accession No.: NP_600305.1) of Corynebacterium glutamicum ATCC13032 strain has a high activity of converting protocatechuic acid to gallic acid. In the following examples, the mutant enzyme is referred to as Cg145, and its amino acid sequence is disclosed as SEQ ID NO: 1. DNA encoding parahydroxybenzoate hydroxylase (Genbank Accession No.: NP_600305.1) was amplified by PCR from the genomic DNA of Corynebacterium glutamicum ATCC13032 strain and subcloned. The above amino acid substitutions were introduced into the resulting DNA by PCR to prepare DNA of SEQ ID NO:5 encoding the polypeptide of SEQ ID NO:1.

2) Preparation of genes encoding gallic acid synthases Ca145, Cc145, and Ce145 The amino acid sequences of 4-hydroxybenzoate-3-monooxygenase derived from Corynebacterium ammoniagenes, Corynebacterium callunae, and Corynebacterium efficiens (WP_040354425.1, WP_015650893.1, and WP_006769907.1, respectively) were obtained from the Genbank database. For each amino acid sequence, an amino acid sequence was designed in which Leu at the position corresponding to position 200 of SEQ ID NO: 1 was replaced with Val and Tyr at the position corresponding to position 385 was replaced with Phe, similar to Cg145. That is, an amino acid sequence (SEQ ID NO: 2) was designed in which Leu at position 208 of the amino acid sequence of WP_040354425.1 was replaced with Val and Tyr at position 397 was replaced with Phe. Similarly, SEQ ID NO: 3 was designed from WP_015650893.1, and SEQ ID NO: 4 was designed from WP_006769907.1. In the following examples, the polypeptides shown in SEQ ID NOs: 2, 3, and 4 are referred to as Ca145, Cc145, and Ce145, respectively. DNAs encoding the polypeptides of SEQ ID NOs: 2, 3, and 4 (SEQ ID NOs: 6, 7, and 8, respectively) were artificially synthesized by requesting GenScript.

3) Preparation of expression vector The DNAs prepared in 1) and 2) above were introduced into pET21-a(+) plasmid (MERCK). A pET21-a(+) vector fragment was amplified by PCR using primers His-pET21a-F and pET21a-R (Table 1) with pET21-a(+) as a template. A Cg145 gene fragment was amplified by PCR using primers pET-Cg145-F and pET-Cg145-R (Table 1) with a plasmid into which DNA encoding Cg145 (SEQ ID NO: 5) was introduced as a template. PrimeSTAR Max DNA Polymerase (TaKaRa) was used as the PCR enzyme. The pET21-a(+) vector fragment and the Cg145 gene fragment were ligated using an In-Fusion HD cloning kit (Clontech) to obtain pET21-Cg145.

In a similar manner, a Ca145 gene fragment was amplified by PCR using a plasmid containing DNA (SEQ ID NO: 6) encoding Ca145 as a template and primers pET-Ca145-F and pET-Ca145-R (Table 1). The pET21-a(+) vector fragment and the Ca145 gene fragment were ligated using the In-Fusion HD cloning kit (Clontech) to obtain pET21-Ca145.

In a similar manner, a Cc145 gene fragment was amplified by PCR using a plasmid containing DNA encoding Cc145 (SEQ ID NO: 7) as a template and primers pET-Cc145-F and pET-Cc145-R (Table 1). The pET21-a(+) vector fragment and the Cc145 gene fragment were ligated using the In-Fusion HD cloning kit (Clontech) to obtain pET21-Cc145.

Using a similar procedure, a Ce145 gene fragment was amplified by PCR using primers pET-Ce145-F and pET-Ce145-R (Table 1) with a plasmid containing DNA encoding Ce145 (SEQ ID NO: 8) as a template. The pET21-a(+) vector fragment and the Ce145 gene fragment were ligated using the In-Fusion HD cloning kit (Clontech) to obtain pET21-Ce145.

The obtained plasmid solution was used to transform ECOS Competent E. coli DH5α strain (Nippon Gene Co., Ltd.), and the cell solution was spread on LB agar medium containing ampicillin and left to stand overnight at 37°C. Colony PCR was performed using the obtained colonies as templates and Sapphire Amp (TaKaRa Co., Ltd.) as the enzyme. The primers used were T7promoter-2 and T7terminator-2 (Table 1). The transformant carrying the plasmid in which the introduction of the target gene was confirmed was inoculated into 2 mL of LB liquid medium containing ampicillin and cultured overnight at 37°C. The plasmid was purified from this culture using NucleoSpin Plasmid EasyPure (TaKaRa Co., Ltd.).

4) Expression of each gallic acid synthase The plasmid obtained in 3) above was transformed into ECOS Competent E. coli BL21 (DE3). Each transformant was inoculated into 2 mL of LB liquid culture containing ampicillin and cultured overnight at 37 ° C. Then, 1 mL of this culture was inoculated into 10 mL of Overnight Express Instant LB Medium (Merck) liquid culture containing ampicillin and cultured at 250 rpm and 37 ° C. for 24 hours. After the culture was completed, the cells were centrifuged at 4 ° C. and 3000 rpm for 10 minutes to collect the cells. The cells were disrupted using xTractor buffer kit (TaKaRa), followed by centrifugation at 4 ° C. and 14500 rpm for 5 minutes to collect the supernatant. The resulting supernatant was used as an enzyme solution containing gallic acid synthase. The protein concentration of the enzyme solution was quantified using a TaKaRa Bradford Protein Assay Kit (TaKaRa).

Example 2 Enzyme Activity Measurement 100 μL of the enzyme solution obtained in Example 1, appropriately diluted, was added to 100 μL of substrate solution (a mixture of 60 μL of 100 mM Tris-HCl (pH 7.5) solution, 20 μL of 20 mM NADH solution, and 20 μL of 20 mM protocatechuic acid solution), and the mixture was allowed to stand at room temperature for 1 hour. 10 μL of the reaction solution was sampled and mixed with 190 μL of 37 mM sulfuric acid to stop the reaction. The amount of gallic acid produced in this solution was quantified according to the method of Reference Example 1. The activity of each gallic acid synthase was measured from the amount of gallic acid produced per protein concentration. The relative activity of each gallic acid synthase when the activity of Cg145 was taken as 100 is shown in Table 2. Compared with the known enzyme Cg145, Ca145 had a gallic acid conversion activity 18% higher.

Reference Example 1: Quantification of Gallic Acid Gallic acid was quantified by HPLC. Chromaster (Hitachi High-Tech Science) was used as the HPLC device. L-column ODS (4.6 mm ID x 150 mm, x Chemicals Evaluation and Research Institute, Japan) was used as the analytical column. Gallic acid was separated by gradient elution under the conditions of a flow rate of 1.0 mL/min and a column temperature of 40°C using a 0.1% phosphoric acid solution containing 0.1 M potassium dihydrogen phosphate as eluent A and 70% methanol as eluent B. A UV detector (detection wavelength 210 nm) was used to detect gallic acid. A concentration calibration curve was created using a standard sample (gallic acid, Kanto Chemical, product number 40205), and gallic acid was quantified based on the concentration calibration curve. The reaction solution of Example 2 and the standard sample subjected to HPLC analysis were appropriately diluted with 37 mM sulfuric acid, and insoluble matters were removed using AcroPrep 96-well Filter Plates (0.2 μm GHP membrane, Pall Corporation).

Claims (7)

A method for producing gallic acid, comprising converting protocatechuic acid to gallic acid using a polypeptide having the amino acid sequence of SEQ ID NO:2. A method for producing gallic acid, comprising converting protocatechuic acid to gallic acid by a polypeptide having an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 2 and having a function of converting protocatechuic acid to gallic acid ,
The polypeptide has Val at a position corresponding to position 208 of SEQ ID NO:2 and Phe at a position corresponding to position 397 of SEQ ID NO:2, and has a higher activity of converting protocatechuic acid to gallic acid than an enzyme consisting of the amino acid sequence of SEQ ID NO:1.
method. The method of claim 2, wherein the polypeptide consists of an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:2. The method according to any one of claims 1 to 3, comprising culturing a transformant containing a polynucleotide encoding the polypeptide in a medium containing protocatechuic acid. The method according to any one of claims 1 to 4, comprising culturing a transformant containing a polynucleotide encoding the polypeptide, the transformant having the ability to produce protocatechuic acid. The method according to claim 4 or 5, wherein the transformant contains a vector containing the polynucleotide or a DNA expression cassette containing the polynucleotide. The method according to any one of claims 4 to 6, wherein the transformant is a bacterium belonging to the genus Escherichia or Corynebacterium.