Method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid

Abstract

To provide production methods capable of efficiently producing 3-(4-Hydroxy-3-methoxyphenyl) propionic acid (HMPA).SOLUTION: Provided is a method for producing HMPA, which comprises acting on HMCA a protein that has an amino acid sequence of a protein having 90% or more sequence identity to a particular amino acid sequence and having an enzymatic activity of reducing 4-hydroxy-3-methoxycinnamic acid (HMCA) to HMPA, in the presence of a protein having 90% or more sequence identity to a particular amino acid sequence and having an amino acid sequence of a protein belonging to NADH-dependent FMN reductase family.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid, and more specifically to a method for efficiently producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid using 4-hydroxy-3-methoxycinnamic acid as a raw material through an enzymatic reduction reaction. [Background technology]

[0002] In recent years, lifestyle-related diseases such as obesity, type 2 diabetes, hypertension, and insulin resistance have become a major problem due to lifestyle habits such as overeating and lack of exercise. In order to improve these lifestyle-related diseases, attention is being paid not only to treatment with pharmaceuticals but also to prevention and treatment using ingredients derived from natural products or foods.

[0003] 3-(4-hydroxy-3-methoxyphenyl)propionic acid is recognized to have an effect related to such lifestyle-related diseases, and its functionality has attracted attention. For example, it has been reported to have inhibitory effects on cAMP phosphodiesterase activity and dipeptidyl peptidase IV activity (see Patent Document 1).

[0004] Lactobacillus plantarum, a lactic acid bacterium isolated from pickles and other foods, has been identified as having two pathways for degrading phenylpropenoic acids, such as 4-hydroxy-3-methoxycinnamic acid, contained in plant cell walls. For example, when 4-hydroxy-3-methoxycinnamic acid, a type of phenylpropenoic acid, is added to a medium and cultured, 3-(4-hydroxy-3-methoxyphenyl)propionic acid, which is the reduction of the propenoic acid moiety of 4-hydroxy-3-methoxycinnamic acid, and 4-vinylguaiacol, which is the decarboxylation of 4-hydroxy-3-methoxycinnamic acid, which is considered an off-flavor in foods and alcoholic beverages, are detected. The present inventors have shown that when 4-hydroxy-3-methoxycinnamic acid is added to a medium in which dissolved oxygen has been removed and cultured, the reduction reaction takes precedence over the decarboxylation reaction, resulting in the preferential production of 3-(4-hydroxy-3-methoxyphenyl)propionic acid (see Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Publication No. 2016-079186 [Patent Document 2] Japanese Patent Application Publication No. 2019-017381 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a method for efficiently producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the present inventors identified 4-hydroxy-3-methoxycinnamate reductase as a protein whose expression is induced in the presence of 4-hydroxy-3-methoxycinnamic acid, and discovered that the reductase activity of 4-hydroxy-3-methoxycinnamate reductase alone is insufficient, but that the reductase activity is significantly increased in the presence of a specific protein belonging to the NADH-dependent flavin mononucleotide reductase family.

[0008] The present invention, which was completed based on this discovery, is as follows. [1] A method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid by reducing 4-hydroxy-3-methoxycinnamic acid, The following amino acid sequence (a1) or (a2): (a1) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; or (a2) an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; In the presence of a protein (A) comprising The following amino acid sequence (b1) or (b2): (b1) the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or (b2) an amino acid sequence of a protein having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and having an enzymatic activity of reducing 4-hydroxy-3-methoxycinnamic acid to 3-(4-hydroxy-3-methoxyphenyl)propionic acid; A method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid, comprising a step of reacting the protein (B) comprising the formula: [2] The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to [1], wherein at least one of the protein (A) and the protein (B) is a recombinant protein. [3] The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to [1] or [2], wherein in the step, the molar ratio of the amount of protein (A) used to the amount of protein (B) used is 1 or more. [4] The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to any one of [1] to [3], wherein the step is carried out in an environment of pH 2 to 7. [5] The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to any one of [1] to [4], wherein the step is carried out in the presence of NADH. [6] The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to any one of [1] to [5], wherein the step is carried out in the presence of flavin mononucleotide (FMN) and / or flavin adenine dinucleotide (FAD). [Effects of the Invention]

[0009] According to the production method of the present invention, 3-(4-hydroxy-3-methoxyphenyl)propionic acid can be produced efficiently. [Brief explanation of the drawings]

[0010] [Figure 1a] FIG. 1 shows the amino acid sequence (SEQ ID NO: 3) of the hcrB gene product obtained from Lactobacillus plantarum 22A-3 (FERM P-21411). [Figure 1b] FIG. 1 shows the amino acid sequence (SEQ ID NO: 4) of the hcrB gene product obtained from Lactobacillus plantarum WCFS1. [Figure 2a] FIG. 1 shows the amino acid sequence (SEQ ID NO: 1) of the hcrA gene product obtained from Lactobacillus plantarum 22A-3 (FERM P-21411). [Figure 2b] FIG. 1 shows the amino acid sequence (SEQ ID NO: 2) of the hcrA gene product obtained from Lactobacillus plantarum WCFS1. [Figure 3]FIG. 1 shows the construction of the expression plasmids constructed in the Examples. [Figure 4] 1 is a graph showing the results of testing 4-hydroxy-3-methoxycinnamate reductase activity for HcrB and HcrA. Error bars indicate standard deviation (n=3). [Figure 5] Graphs showing that HcrB-mediated 4-hydroxy-3-methoxycinnamate reductase activity is enhanced in a concentration-dependent manner by HcrA. (A) FAD was used as the flavin nucleotide, and (B) FMN was used. Error bars indicate standard deviation (n=3). [Figure 6] FIG. 1 shows a putative redox balance control mechanism in Lactobacillus plantarum 22A-3. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment of the present invention will be described. The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to this embodiment is a method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid by reducing 4-hydroxy-3-methoxycinnamic acid, and includes a step of allowing a protein (B) having a predetermined amino acid sequence to act on 4-hydroxy-3-methoxycinnamic acid in the presence of a protein (A) having a predetermined amino acid sequence.

[0012] 1. Protein (A) and Protein (B) The protein (A) used in this embodiment has the following amino acid sequence (a1) or (a2): (a1) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; or (a2) An amino acid sequence of a protein having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and belonging to the NADH-dependent flavin mononucleotide reductase family.

[0013] Furthermore, the protein (B) used in this embodiment has the following amino acid sequence (b1) or (b2): (b1) the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or (b2) An amino acid sequence of a protein having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and having the enzymatic activity of reducing 4-hydroxy-3-methoxycinnamic acid to 3-(4-hydroxy-3-methoxyphenyl)propionic acid.

[0014] Protein (B) has the enzyme activity of reducing 4-hydroxy-3-methoxycinnamic acid (hereinafter sometimes abbreviated as "HMCA") to 3-(4-hydroxy-3-methoxyphenyl)propionic acid (hereinafter sometimes abbreviated as "HMPA"). A protein having the amino acid sequence (b1) has been confirmed to have this reductase activity. Here, the phrase "protein (B) has the enzymatic activity to reduce HMCA to HMPA" specifically means that HMPA can be detected when protein (B) is allowed to react with 1 mM HMCA in the presence of 100 mM acetate buffer (pH 5.0), 10 mM NADH, and 5 mM flavin adenine dinucleotide (FAD) for 30 minutes at 25° C. Details of the methods for measuring the reductase activity and detecting HMPA are provided in the Examples below. The enzymatic activity of protein (B) to reduce HMCA to HMPA is preferably 0.02 U / mg or more, and particularly preferably 0.1 U / mg or more, calculated as enzymatic activity (U) per mg of protein (B), where 1 U represents the amount of enzyme that reduces 1 μmol of HMCA per minute at 25° C.

[0015] Among the amino acid sequences (b1), SEQ ID NO: 3 is the amino acid sequence of the hcrB gene product obtained from Lactobacillus plantarum 22A-3 (FERM P-21411), and is shown in FIG. 1a. SEQ ID NO: 4 is the amino acid sequence of the hcrB gene product obtained from Lactobacillus plantarum WCFS1 and is shown in Figure 1b. The amino acid sequence of SEQ ID NO: 4 can be referenced in databases such as GenPept under the accession number YP_004889276.1.

[0016] As long as protein (B) has the enzymatic activity of reducing HMCA to HMPA, the above amino acid sequence (b1) may be replaced with a protein having an amino acid sequence (b2) that has 90% or more, 92% or more, 95% or more, 97% or more, or 98% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or 4.

[0017] Furthermore, as long as protein (B) has the enzymatic activity of reducing HMCA to HMPA, it may contain, instead of the amino acid sequence (b1), one or several amino acid residue mutations (e.g., substitution, deletion, insertion, etc.) in the amino acid sequence of SEQ ID NO: 3 or 4. The number of mutations can be, for example, 1 to 30, 1 to 20, or 1 to 10 (e.g., 1, 2, 3, 4, or 5).

[0018] Protein (A) is a protein having amino acid sequence (a1) or a similar amino acid sequence (a2). Protein (A) can be said to belong to the NADH-dependent flavin mononucleotide (FMN) reductase family, and amino acid sequence (a1) is annotated as a protein belonging to the NADH-dependent FMN reductase family. Among the amino acid sequences (a1), SEQ ID NO: 1 is the amino acid sequence of the hcrA gene product obtained from Lactobacillus plantarum 22A-3 (FERM P-21411), and is shown in FIG. 2a. SEQ ID NO: 2 is the amino acid sequence of the hcrA gene product obtained from Lactobacillus plantarum WCFS1, and is shown in Figure 2b. The amino acid sequence of SEQ ID NO: 2 can be referenced in databases such as GenPept under the accession number YP_004889275.1.

[0019] Protein (A) may be a protein having an amino acid sequence (a2) that has 90% or more, 92% or more, 95% or more, 97% or more, or 98% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, instead of the amino acid sequence (a1).

[0020] Furthermore, instead of the amino acid sequence (a1), protein (A) may contain one or several amino acid residue mutations (e.g., substitutions, deletions, insertions, etc.) in the amino acid sequence of SEQ ID NO: 1 or 2. The number of mutations can be, for example, 1 to 30, 1 to 20, or 1 to 10 (e.g., 1, 2, 3, 4, or 5).

[0021] The genes encoding the above proteins (A) and (B) can be easily obtained from, for example, lactic acid bacteria. Specific examples of lactic acid bacteria include the following lactic acid bacteria belonging to the families Lactobacillaceae, Streptococcaceae, Leuconostocaceae, Enterococcaceae, and Bacillaceae.

[0022] Examples of lactic acid bacteria belonging to the Lactobacillaceae family include lactic acid bacteria belonging to the genus Lactobacillus and Pediococcus. Lactic acid bacteria belonging to the genus Lactobacillus include Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus gallinarum, Lactobacillus fermentum, and Lactobacillus delbrueckii. delbrueckii, Lactobacillus buchneri, Lactobacillus parabuchneri, Lactobacillus kefiranofaciens, Lactobacillus curvatus, Lactobacillus rossiae, Lactobacillus salivarius, Lactobacillus casei, Lactobacillus kitasatonis, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillus coeliformis coryniformis, Lactobacillus farciminus, Lactobacillus rhamnosusExamples include Lactobacillus rhamnosus and Lactobacillus bulgaricus. When Lactobacillus plantarum is used, Lactobacillus plantarum 22A-1 (FERM P-21409), Lactobacillus plantarum 22A-3 (FERM P-21411), or Lactobacillus plantarum 22B-2 (FERM P-21410) may be used. These three strains were isolated and identified by the applicant from pickles and have been deposited at the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology. Examples of lactic acid bacteria belonging to the genus Pediococcus include Pediococcus damnosus, Pediococcus halophilus, Pediococcus parvulus, and Pediococcus pentosaceus.

[0023] Examples of lactic acid bacteria belonging to the family Streptococcus include lactic acid bacteria belonging to the genera Streptococcus and Lactococcus. Examples of lactic acid bacteria belonging to the genus Streptococcus include Streptococcus thermophilus and Streptococcus salivarius. Examples of lactic acid bacteria belonging to the genus Lactococcus include Lactococcus lactis.

[0024] Examples of lactic acid bacteria belonging to the family Leuconostoc include lactic acid bacteria belonging to the genera Leuconostoc, Weissella, and Oenococcus. Examples of lactic acid bacteria belonging to the genus Leuconostoc include Leuconostoc lactis and Leuconostoc mesenteroides. Examples of lactic acid bacteria belonging to the genus Weissella include Weissella cibaria and Weissella confusa. Examples of lactic acid bacteria belonging to the genus Oenococcus include Oenococcus oeni.

[0025] Lactic acid bacteria belonging to the Enterococcus family include those belonging to the genus Enterococcus, and examples of lactic acid bacteria belonging to the genus Enterococcus include Enterococcus faecalis.

[0026] Examples of lactic acid bacteria belonging to the Bacillaceae family include lactic acid bacteria belonging to the genus Bacillus, and examples of lactic acid bacteria belonging to the genus Bacillus include Bacillus coagulans. When Bacillus coagulans is used, Bacillus coagulans NBRC12714 may be used. This strain is preserved at the National Institute of Technology and Evaluation (NITE) Biotechnology Center and is listed in the NBRC catalog as a distributable strain.

[0027] The above proteins (A) and / or (B) may have other peptide components (e.g., tag moieties, other proteins) at the C-terminus or N-terminus. Examples of other peptide components that can be added to the above proteins (A) and / or (B) include peptide components that facilitate the purification of the target protein (e.g., tag moieties such as histidine tag and Strep-tag II; proteins commonly used in the purification of target proteins such as glutathione S-transferase and maltose-binding protein), peptide components that improve the solubility of the target protein (e.g., Nus-tag), peptide components that act as chaperones (e.g., trigger factor), proteins or protein domains with other functions, and peptide components that serve as linkers connecting these other peptide components.

[0028] In this embodiment, at least one of the proteins (A) and (B) is preferably a recombinant protein. By using at least one of the proteins (A) and (B) as a recombinant protein, it becomes easier to adjust the amount of protein (A) used relative to the amount of protein (B) used, and the reduction reaction of HMCA can proceed more efficiently.

[0029] The reduction reaction step described below may be carried out using a microorganism transformed with an expression vector containing a polynucleotide encoding protein (A) or (B), or may be carried out using a microbial extract obtained by disrupting or lysing the cells of the microorganism, or may be carried out using protein (A) or (B) purified from the microbial extract. Protein (A) or (B) may also be expressed using a cell-free system or the like.

[0030] When transforming a microorganism, various prokaryotic cells, including Escherichia bacteria such as Escherichia coli and Bacillus bacteria such as Bacillus subtilis, and various eukaryotic cells, including Saccharomyces bacteria such as Saccharomyces cerevisiae and Aspergillus bacteria such as Aspergillus oryzae, can be used as hosts. A strain lacking a specific gene may also be used as a host. The transformant may, for example, possess an expression vector in its cytoplasm, or may have a target gene introduced into its genome.

[0031] The transformant cells can be disrupted or lysed by conventional methods. Examples of methods for disrupting cells include sonication and homogenization, and examples of methods for lysing cells include lysozyme treatment. The resulting disrupted or lysed cells may be further purified. The purification method is not particularly limited, and techniques commonly used by those skilled in the art, such as extraction, precipitation, filtration, and column chromatography, can be used. When other peptide components that facilitate purification are added to protein (A) and / or (B), the properties of such peptide components can be utilized to facilitate purification.

[0032] 2. Reduction reaction process In this embodiment, the method includes a step of reacting the protein (B) with HMCA in the presence of the protein (A). Hereinafter, this step may be referred to as a "reduction reaction step."

[0033] As described above, protein (B) has the enzymatic activity of reducing HMCA to HMPA. However, as a result of studies by the present inventors, it was found that the reductase activity of protein (B) alone is insufficient, and that the reductase activity of protein (B) is significantly increased in the presence of protein (A).

[0034] In the reduction reaction step, the molar ratio of the amount of protein (A) used to the amount of protein (B) used is preferably at least 1, more preferably at least 2, even more preferably at least 5, and particularly preferably at least 10. When a large amount of protein (A) is used, the reductase activity of protein (B) increases more significantly, making it easier to produce HMPA efficiently. On the other hand, there is no particular upper limit to the amount of protein (A) used relative to the amount of protein (B) used. However, from the viewpoint of ensuring a sufficient amount of protein (B) used, for example, the molar ratio of the above amounts used may be 100 or less, or may be 50 or less.

[0035] In the reduction reaction step, the type of reaction solution is not particularly limited, and in addition to buffer solutions that can be used in enzymatic reactions, when the transformant is used as is, the medium used for culturing the transformant can also be used. The reduction reaction step is preferably carried out in an environment of pH 2 to 7, more preferably pH 3 to 6, and particularly preferably pH 4 to 5.5. The reduction reaction step is preferably carried out in an environment of 15 to 40°C, more preferably 18 to 33°C, and particularly preferably 20 to 28°C.

[0036] The reduction reaction step is preferably carried out in the presence of NADH. The concentration of NADH can be, for example, 1 mM or more, 2 mM or more, or 5 mM or more, and can be 100 mM or less, 50 mM or less, or 20 mM or less.

[0037] The reduction reaction step is preferably carried out in the presence of flavin mononucleotide (FMN) and / or flavin adenine dinucleotide (FAD). As shown in the Examples below, the reduction reaction proceeds favorably regardless of whether FMN or FAD is used. The concentration of FMN or FAD can be, for example, 0.5 mM or more, 1 mM or more, or 2 mM or more, or 50 mM or less, 20 mM or less, or 10 mM or less.

[0038] In the reduction reaction step, the amount of HMCA used as the substrate may be, for example, 0.1 mM or more, 0.5 mM or more, 1 mM or more, or even 10 mM or more, or 25 mM or more. On the other hand, although there is no particular upper limit, when phenolic acid decarboxylase (Pdc) is present in the reduction reaction step, using a high concentration of HMCA may facilitate the decarboxylation reaction, resulting in a reduced yield of HMPA. In such cases, the amount of HMCA used may be 10 mM or less, 5 mM or less, or 2 mM or less.

[0039] According to the above-described method for producing HMPA, protein (B) is allowed to act on HMCA in the presence of protein (A), which significantly increases the reduction reaction of HMCA, particularly compared to when protein (B) is used alone, and allows HMPA to be obtained in high yield.

[0040] The above-described embodiments have been described to facilitate understanding of the present invention, and are not intended to limit the present invention. Therefore, each element disclosed in the above embodiments is intended to include all design modifications and equivalent methods that fall within the technical scope of the present invention. [Example]

[0041] The present invention will be specifically explained below by showing test examples, but the present invention is not limited to the following examples.

[0042] 1. Identification of genes induced by HMCA Genes whose expression was induced by the addition of HMCA under low dissolved oxygen conditions were searched for by RNA-seq analysis as follows.

[0043] Lactobacillus plantarum 22A-3 (FERM P-21411) was inoculated into 5 mL of MRS liquid medium and cultured at 28°C for 1 day (preculture). 1.2 mL of the resulting preculture was added to MRS liquid medium containing 1 mM HMCA and cultured with shaking at 28°C and 120 rpm for 16 hours (main culture). For the main culture, 117.6 mL of MRS liquid medium was dispensed into a 100 mL medium bottle (screw-capped), filled to the brim with medium, autoclaved (121°C for 20 minutes), and then immediately sealed to remove dissolved oxygen. HMCA was added using a 100 mM HMCA solution prepared with 500 mM potassium phosphate buffer (pH 7.0).

[0044] After the main culture, the culture medium was centrifuged (6,000 × g, 5 minutes) four times, and bacteria were collected from a total of 6 mL of culture medium. The obtained bacteria were lysed using TRIzol® Reagent (Ambion) according to the attached protocol, and extracted with chloroform. 400 to 600 μL of the colorless upper fraction from the chloroform extraction was collected, to which an equal volume of ethanol was added and mixed. RNA was extracted from the mixture using the Monarch Total RNA Miniprep Kit (New England Biolabs) according to the attached protocol. The obtained RNA was analyzed by Qubit TM RNA IQ Assay Kit (Invitrogen) was used, and a fluorometer (Qubit TM The degree of decomposition of total RNA was confirmed using a Fluorometer (Invitrogen).

[0045] Using the obtained RNA, we performed a comprehensive search for genes induced by HMCA by RNA-seq analysis (GeneWiz Japan) based on the genome information of Lactobacillus plantarum WCFS1 (GenBank accession number: NC_004567.2). The results are shown in Table 1.

[0046] [Table 1]

[0047] A comprehensive search for genes induced by HMCA using RNA-seq analysis revealed four genes, as shown in Table 1: hcrB, annotated as a fumarate reductase and reported to have HMCA reduction activity; hcrA, annotated as an NAD(P)H-dependent FMN reductase and thought to be involved in electron transfer during reduction; lp_1426, which appears to form the same operon as hcrA and hcrB; and pdc, annotated as a phenolic acid decarboxylase and reported to have HMCA decarboxylation activity. However, these four genes were the only ones induced by HMCA. These results suggest that the phenomenon of HMCA reduction occurring exclusively under low dissolved oxygen conditions is due to the reaction of these enzymes alone.

[0048] 2. Construction of an HMCA-induced gene expression system Of the four genes identified in 1 above, a gene expression system in Escherichia coli for hcrA, hcrB, and pdc was constructed as follows.

[0049] Lactobacillus plantarum 22A-3 (FERM P-21411) was inoculated into 5 mL of MRS liquid medium and cultured at 28°C and 260 rpm for 24 hours with shaking. The culture was centrifuged twice (15,000 × g, 2 minutes) to collect bacteria from a total of 1 mL of culture. Genomic DNA was extracted from the resulting bacteria using the Wizard® Genomic DNA Purification Kit (Promega) according to the attached protocol. The concentration of the obtained genomic DNA was measured using an absorbance meter.

[0050] Using the resulting genomic DNA as a template and the oligo DNAs shown in Table 2 as primers, genomic DNA fragments were amplified by PCR. The PCR reaction reagents used were KOD-Plus-Neo (Toyobo Co., Ltd.), and the thermal cycler was a SimpliAmp Thermal Cycler (Thermo Fisher Scientific). The reaction conditions were 94°C (2 minutes), followed by 30 cycles of 98°C (10 seconds), 60°C (30 seconds), and 68°C (3 minutes). The resulting PCR reaction mixture was used to purify the PCR-amplified fragments using the Monarch® PCR & DNA Cleanup Kit (New England Biolabs).

[0051] [Table 2]

[0052] The pET21(+) plasmid (Novagen) was used as a cloning vector for the genomic DNA fragment. pET21(+) was digested with the restriction enzyme BamHI, and the vector fragment was purified using the Monarch® PCR & DNA Cleanup Kit (New England Biolabs). The PCR amplified fragment obtained as described above and the vector fragment were reacted in NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs). The resulting assembly product was transformed into Escherichia coli DH5α, and transformants were selected on LB solid medium containing 100 mg / mL ampicillin. A single colony of the selected transformant was cultured on LB solid medium containing 100 mg / mL ampicillin at 37°C for 16 hours, and the plasmid was recovered using a High Pure Plasmid Isolation Kit (Roche Diagnostics). The recovered plasmid was subjected to restriction enzyme treatment and DNA sequence analysis to confirm that the target DNA fragment had been inserted into the desired position on the vector.

[0053] Using the above procedures, we constructed the following plasmids: pRM601, which contains a sequence in which a 6xHis-tag is fused to the C-terminus of Pdc derived from Lactobacillus plantarum 22A-3, pRM602, which contains a sequence in which a 6xHis-tag is fused to the C-terminus of HcrA, and pRM603, which contains a sequence in which a 6xHis-tag is fused to the C-terminus of HcrB. The construction of the resulting plasmid is shown in FIG.

[0054] 3. Expression and purification of HMCA-induced genes in E. coli Using the plasmids constructed in 2 above, Pdc, HcrA, and HcrB were expressed in E. coli and purified as follows.

[0055] Plasmid pRM601 carrying recombinant Pdc, plasmid pRM602 carrying recombinant HcrA, and plasmid pRM603 carrying recombinant HcrB were used to transform E. coli BL21(DE3) or Rosetta 2(DE3) strains, respectively, to obtain strains BL21(DE3)[pRM601], Rosetta 2(DE3)[pRM602], and Rosetta 2(DE3)[pRM603], respectively.

[0056] The BL21(DE3)[pRM601] strain was pre-cultured in 5 mL of LB liquid medium containing 100 mg / mL ampicillin, and then 4 mL of the pre-culture medium and IPTG (final concentration 0.5 mM) were added to 400 mL of the same liquid medium, and the mixture was shaken at 28°C and 105 rpm for 24 hours to induce expression. On the other hand, the Rosetta 2(DE3)[pRM602] and Rosetta 2(DE3[pRM603] strains were pre-cultured in 5 mL of LB liquid medium containing 100 mg / mL ampicillin, and then 4 mL of the pre-culture solution was added to 400 mL of the same liquid medium. The bacterial cell turbidity (OD 610 The strain was cultured at 28°C with shaking at 105 rpm for approximately 4 hours until the β-aspartate ratio (β) reached 0.4-0.6. IPTG (final concentration 0.5 mM) was then added, and the strain was cultured at 28°C with shaking at 105 rpm for 20 hours to induce expression.

[0057] The induced cells were harvested by centrifugation (8,000 × g, 10 minutes, 4°C). The wet weight of the harvested cells was measured, and the cells were suspended in 50 mM potassium phosphate buffer (pH 7.0) to a cell concentration of 10% (w / v). The cells were then disrupted by ultrasonication (5 mL, 180 W, 5 minutes, 4°C), and the supernatant was collected by centrifugation (15,000 × g, 20 minutes, 4°C) to obtain a crude enzyme extract.

[0058] The crude enzyme extract was purified by affinity chromatography using a Ni-Sepharose column (GE Healthcare) at room temperature under the following chromatography conditions.

[0059] =Columns and equipment used= Chromatography system: AKTA start (GE Healthcare) Fraction collector: Frac 30 (GE Healthcare) Column: Ni-Sepharose column (GE Healthcare) Column volume: 5 mL Flow rate: 5mL / min

[0060] =Buffer Used= A buffer: 20mM sodium phosphate buffer (pH 7.4) 500mM NaCl 20mM imidazole B buffer: 20mM sodium phosphate buffer (pH 7.4) 500mM NaCl 500mM imidazole

[0061] =Separation condition= 0 mL → 50 mL: 100% A buffer 50mL → 100mL: 100% A buffer (apply) 100mL → 250mL: 100% A buffer 250mL → 450mL: 100% A buffer → 100% B buffer 450mL → 500mL: 100% B buffer

[0062] The purified HcrA, HcrB, and Pdc proteins obtained as described above were used in the enzymatic reactions described below. The purified proteins were quantified by the Bradford method (reagents used: Bio-Rad Protein assay kit, manufactured by BIO-RAD).

[0063] For HcrA and Pdc, the purified products obtained by Ni-affinity chromatography were subjected to SDS-PAGE, revealing homogeneous bands at the estimated molecular weights (HcrA: 25 kDa, Pdc: 25 kDa), confirming that the target proteins had been purified. Furthermore, the native molecular weights of HcrA and Pdc were determined by gel filtration chromatography, revealing that they were monomers, with a molecular weight of approximately 17 kDa for HcrA and approximately 25 kDa for Pdc.

[0064] On the other hand, when HcrB was purified by Ni-affinity chromatography, SDS-PAGE revealed a band around the estimated molecular weight of 93 kDa, as well as several bands at lower molecular weights that were presumed to be degradation products. A second purification step by gel filtration chromatography resulted in a single band on SDS-PAGE. The amino acid sequence of the resulting single band was analyzed by Edman degradation, and it matched the N-terminal sequence of HcrB perfectly. Furthermore, the native molecular weight of HcrB was determined by gel filtration chromatography to be approximately 145 kDa, suggesting that HcrB is a homodimer.

[0065] 4. Detection of HcrB and HcrA Enzyme Activities The HMCA reductase activity was measured using the purified HcrB and HcrA obtained in step 3. Furthermore, since HcrA is annotated as a flavin mononucleotide (FMN) reductase, its FMN reductase activity was also measured.

[0066] [HMCA reductase activity] A total of 1000 μL of reaction mixture was prepared in a 1.5 mL tube using 100 μL of 500 mM acetate buffer (pH 5.0), 100 μL of 100 mM NADH, 100 μL of 50 mM FAD (or FMN), 10 μL of 100 mM HMCA, 100 μL of enzyme solution, and the remainder was purified water. The amount of enzyme added per 1000 μL of reaction mixture was 0.34 μg of HcrA and 1.22 μg of HcrB. The reaction was allowed to proceed in a 25°C water bath for 30 minutes, and then terminated with 6N HCl. An equal volume of ethyl acetate was then added, and the mixture was suspended using a vortex mixer to extract HMCA and HMPA. After centrifugation (13,000 × g, 5 minutes), 250 μL of the organic layer was collected, dried in a centrifugal evaporator, and dissolved in an eluent of 1% acetic acid:methanol = 13:7. This was passed through a 0.22 μm filter and analyzed by HPLC under the following conditions.

[0067] =HPLC conditions= Product name: TOSOH HPLC Stationary phase:TSK-gel ODS-80T M (Manufactured by TOSOH BIOSCIECE) Column length: φ4.6mm x 250mm Mobile phase: methanol:water:acetic acid = 35:64:1 Mobile phase flow rate: 0.8mL / min Detection: Fluorescence detector FP-2020 (excitation wavelength: 279 nm, detection wavelength: 314 nm, response: STD)

[0068] The HMCA reductase activity was calculated by measuring the amount of HMPA produced by reduction of HMCA using a fluorescence detector (Ex: 279 nm, Em: 314 nm) and calculating it using the following formula:

[0069] Activity value (U / mL) = {HMPA production amount (μmol / mL) / min × Enzyme dilution factor × Reaction volume (mL)} / Enzyme volume (μL) 1 U represents the amount of enzyme that reduces 1 μmol of HMCA in 1 minute at 25°C.

[0070] [FMN reductase activity] A total of 1000 μL of reaction mixture was prepared using 600 μL of 500 mM potassium phosphate buffer (pH 7.0), 100 μL of 2.5 mM NADH, 100 μL of 1.25 mM FMN, 100 μL of enzyme solution (HcrA), and 600 μL of purified water. The decrease in NADH associated with the reduction of FMN was measured at 25°C, 300 s, and 340 nm using a UV-Vis spectrophotometer (U-2001, Hitachi High-Tech Science Corporation). The substrate was changed from FMN to flavin adenine dinucleotide (FAD), and similar measurements were performed. From the obtained results, FMN reductase activity was calculated based on the following formula.

[0071] Activity (U / mL) = {-ΔABS / min × enzyme dilution factor × reaction solution volume (mL)} / 6.22 × enzyme solution volume (mL) The molar absorption coefficient of NADH is β = 6.22 × 10 3 L / mol / cm, and 1 U represents the amount of enzyme that reduces 1 μmol of FMN (or FAD) in 1 minute at 25°C.

[0072] The results for HMCA reductase activity are shown in Figure 4. When HcrB was incubated with HMCA in the presence of NADH (final concentration 10 mM) as a coenzyme, no reduction activity was detected. However, when FAD (final concentration 5 mM) was added in addition to NADH, approximately 0.4 U / mg of HMCA reductase activity was detected. Furthermore, when FMN was used instead of FAD, HMCA reductase activity was also detected.

[0073] On the other hand, HcrA alone did not exhibit HMCA reductase activity, even in the presence of both NADH and FAD.Furthermore, HcrA did not exhibit reductase activity toward FMN or FAD in the above test system. However, when HcrA was added in an equimolar amount to HcrB in the HcrB reaction system described above (HcrA:HcrB = 0.34 μg:1.22 μg in 1 mL of reaction solution), the HMCA reductase activity relative to the mass of HcrB increased to approximately 1.1 U / mg.

[0074] Furthermore, HMCA reductase activity was measured using a fixed concentration of HcrB (0.0081 mg / mL) and varying concentrations of HcrA, with NADH (final concentration 10 mM) and either FAD or FMN (final concentration 5 mM) as coenzymes. Figure 5A shows the results for FAD, and Figure 5B shows the results for FMN. As shown in Figure 5, increasing the amount of HcrA increased HMCA reductase activity in both FAD and FMN.

[0075] These results demonstrate that the HMCA reductase activity of HcrB is significantly enhanced by the coexistence of HcrA.

[0076] Based on these results, the predicted redox balance control mechanism in Lactobacillus plantarum 22A-3 is shown in Figure 6. The protons required for HMCA reduction by HcrB are supplied as FADH2, which is produced by reducing FAD using NADH accumulated within the cell. Because HcrB contains an FAD reduction domain in the N-terminal region of the molecule, it is possible for it to simultaneously generate FADH2 from NADH and reduce HMCA. Furthermore, it is thought that the coexistence of HcrA increases the supply of FADH2, thereby enabling higher HMCA reduction activity. Thus, HMCA reduction is a novel redox regulation mechanism that counteracts the redox regulation mediated by the reduction of pyruvate to lactate when excessive NADH accumulates under fermentation conditions. Furthermore, coexpression of HcrA and HcrB may provide more FADH2 to HcrB, enabling it to efficiently reduce HMCA and regulate the redox balance. The HcrB and HcrA genes and their operon-like structures are widely conserved among lactic acid bacteria, suggesting that similar mechanisms for HMCA reduction and redox balance regulation are widely conserved among lactic acid bacteria.

[0077] 5. Measurement of the Km value of Pdc It has been reported that Pdc has the enzyme activity to decarboxylate HMCA to produce 4-vinylguaiacol. Using the purified Pdc obtained in 3 above, the Km value of HMCA decarboxylase activity was calculated as follows.

[0078] A total of 1000 μL of reaction solution was prepared in a 1.5 mL tube using 100 μL of 500 mM acetate buffer (pH 5.0), 100 μL of 100 mM HMCA, 100 μL of enzyme solution, and the remainder purified water. Also, the amount of 100 mM HMCA was varied to 200, 300, 400, and 500 μL, and the total volume was mixed with purified water. The reaction was allowed to proceed in a 25°C water bath for 30 minutes, and then terminated with 6N HCl. An equal volume of ethyl acetate was then added, and the mixture was suspended using a vortex mixer to extract HMCA and 4-vinylguaiacol. After centrifugation (13,000 × g, 5 minutes), 250 μL of the organic layer was collected, dried in a centrifugal evaporator, and dissolved in an eluent of 1% acetic acid:methanol = 13:7. This was passed through a 0.22 μm filter and analyzed by HPLC under the following conditions.

[0079] =HPLC conditions= Product name: TOSOH HPLC Stationary phase:TSK-gel ODS-80T M(Manufactured by TOSOH BIOSCIECE) Column length: φ4.6mm x 250mm Mobile phase: methanol:water:acetic acid = 35:64:1 Mobile phase flow rate: 0.8mL / min Detection: Fluorescence detector FP-2020 (excitation wavelength: 279 nm, detection wavelength: 314 nm, response: STD)

[0080] The HMCA decarboxylase activity was calculated by measuring the amount of 4-vinylguaiacol produced by decarboxylation of HMCA using a fluorescence detector (Ex: 279 nm, Em: 314 nm) and calculating it using the following formula:

[0081] Activity value (U / mL) = {4-vinylguaiacol production (μmol / mL) / min × enzyme dilution ratio × reaction solution volume (mL)} / enzyme solution volume (mL) 1 U represents the amount of enzyme that decarboxylates 1 μmol of HMCA in 1 minute at 25°C.

[0082] Based on the above experiments, the HMCA decarboxylase activity of Pdc was measured, and the Km value calculated using a Lineweaver-Burk plot was 30.4 mM. This result may explain the phenomenon whereby the product 4-vinylguaiacol was hardly produced in the cells cultured under low dissolved oxygen conditions. Specifically, Pdc is a gene induced by HMCA. Under low dissolved oxygen conditions and in the presence of HMCA, the enzyme is expressed in cultured cells, and HMCA is also present as a substrate. However, under these conditions, the product 4-vinylguaiacol is hardly produced. This suggests that Pdc has a low affinity for HMCA. In fact, the Km value of Pdc for HMCA is extremely high (30.4 mM), suggesting that the reaction with low concentrations of HMCA, such as 1 mM, is extremely slow. Therefore, under normal culture conditions with 1 mM HMCA, only a small amount of 4-vinylguaiacol is produced in the culture medium. Furthermore, under anaerobic conditions, the reduction activity of HcrA and HcrB is high, presumably resulting in HMPA production. [Industrial Applicability]

[0083] The production method of the present invention is suitable as a method for efficiently producing HMPA, and is expected to promote the use of HMPA, which has been reported to have various physiological activities, and will make a significant contribution to the food, pharmaceutical, cosmetics, and other fields.

Claims

1. A method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid by reducing 4-hydroxy-3-methoxycinnamic acid, comprising the steps of: The following amino acid sequence (a1) or (a2): (a1) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; or (a2) an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; In the presence of a protein (A) comprising The following amino acid sequence (b1) or (b2): (b1) the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or (b2) an amino acid sequence having 90% or more sequence identity to the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4; The protein (B) is reacted with the 4-hydroxy-3-methoxycinnamic acid, the protein (B) has an enzyme activity of reducing 4-hydroxy-3-methoxycinnamic acid to 3-(4-hydroxy-3-methoxyphenyl)propionic acid; the protein (A) has an activity of increasing the enzymatic activity of the protein (B) when coexisting with the protein (B); At least one of the protein (A) and the protein (B) is a recombinant protein; The step is carried out in the presence of NADH and flavin mononucleotide (FMN) and / or flavin adenine dinucleotide (FAD). A method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid.

2. 2. The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to claim 1, wherein in the step, the molar ratio of the amount of protein (A) used to the amount of protein (B) used is 1 or more.

3. The method for producing 3-(4-hydroxy-3-methoxyphenyl)propionic acid according to claim 1 or 2, wherein the step is carried out in an environment of pH 2 to 7.

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