Method for producing carbonyl compound

Abstract

The present invention provides, for example, a method for producing a carbonyl compound. The present invention is a method for producing a carbonyl compound, said method comprising generating a carbonyl compound derived from an α,β-unsaturated carbonyl compound by bringing into contact with the α,β-unsaturated carbonyl compound a protein that includes a motif represented by formula I: GX1X2P[VL][AG]PS and a motif represented by formula II: RVGX3H and that has ene-reductase activity with respect to the α,β-unsaturated carbonyl compound, and / or a cell that is capable of expressing said protein.

Description

Method for producing carbonyl compounds

[0001] Carbonyl compounds have traditionally been widely used as flavoring substances. For example, δ-decalactone and δ-dodecalactone are typical aroma components that contribute to the flavor of milk and are widely used as ingredients in various flavoring compositions. Methods for producing δ-decalactone and δ-dodecalactone include chemical synthesis and non-synthetic fermentation methods. For example, a method for producing them involves reducing the corresponding α,β-unsaturated carbonyl compound using a strain of bacteria belonging to the genera Saccharomyces, Pseudomonas, Proteus, Bacillus, Cellulomonas, Micrococcus, Xanthomonas, or Acetobacter (see Patent Documents 1 to 3). However, these production methods have the problem that the substrate concentration in the reaction solution is low, resulting in a low amount (g / L) of carbonyl compounds produced and insufficient production efficiency.

[0002] Furthermore, there is a method for converting unsaturated lactones to saturated lactones within host cells by the coupled reaction of enoate reductase and glucose dehydrogenase from the genus Bacillus (see Patent Document 4). However, this method requires the addition of expensive coenzymes and co-cultivation with a strain that expresses glucose dehydrogenase, and the cost and complexity of the production method pose challenges for industrialization.

[0003] Furthermore, although enoate reductase from the genus Pseudomonas is known to reduce α,β-unsaturated carbonyl compounds (see Non-Patent Document 1), there are no examples of its application to unsaturated lactones. Furthermore, because the substrate concentration in the reaction solution is low, the amount of carbonyl compound produced (g / L) is low, resulting in insufficient production efficiency.

[0004] JP-A-3-155792 JP-A-6-225781 JP-A-10-042889 International Publication No. 2020 / 128644

[0005] Christin Peters, Regina Kolzsch, Maria Kadow, Lilly Skalden, Florian Rudroff, Marko D. Mihovilovic, and Uwe T. Bornscheuer, ChemCatChem, 6 (2014) 1021-1027.

[0006] Under these circumstances, there has been a demand for the development of a technique that can efficiently and / or simply produce carbonyl compounds derived from α,β-unsaturated carbonyl compounds.

[0007] The present invention has been made in consideration of the above circumstances, and provides the following method for producing a carbonyl compound.

[0008] (1) Formula I: GX １ X ２ P[VL][AG]PS (wherein G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, and X represents a ... １ and X ２ each independently represents any amino acid, [VL] represents V or L, and [AG] represents A or G.) A motif represented by Formula II: RVGX ３ H (wherein R represents arginine, V represents valine, G represents glycine, H represents histidine, and X represents a ３ represents any amino acid.) and a protein having ene-reductase activity for an α,β-unsaturated carbonyl compound, and / or a cell capable of expressing said protein, is contacted with an α,β-unsaturated carbonyl compound to produce a carbonyl compound derived from the compound.

[0009] (2) X １ represents glutamic acid (E), alanine (A) or glutamine (Q); X ２ represents leucine (L), and X ３ represents valine (V) or methionine (M).

[0010] (3) The method according to (1) above, wherein the motif represented by formula I is GELPVAPS; GALPVAPS; or GQLPVAPS, and the motif represented by formula II is RVGVH; or RVGMH.

[0011] (4) The method according to (1), wherein the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GQLPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGMH; and the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGMH.

[0012] (5) The method according to (1), wherein the protein is at least one selected from the group consisting of: (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 8, and having the ene-reductase activity; (ii) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 9, and having the ene-reductase activity; (iii) a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 11, and having the ene-reductase activity; and (iv) a protein consisting of the amino acid sequence shown in SEQ ID NO: 13, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 13, and having the ene-reductase activity.

[0013] (6) Proteins of the following (i) to (iv): (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 8, and having ene-reductase activity for α,β-unsaturated carbonyl compounds; (ii) a protein consisting of the amino acid sequence shown in SEQ ID NO: 9, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 9, and having ene-reductase activity for α,β-unsaturated carbonyl compounds; (iii) a protein consisting of the amino acid sequence shown in SEQ ID NO: 11, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 11, and having ene-reductase activity for α,β-unsaturated carbonyl compounds; and (iv) a protein consisting of the amino acid sequence shown in SEQ ID NO: 13, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 13, and having ene-reductase activity for α,β-unsaturated carbonyl compounds. and / or a cell capable of expressing said protein, with an α,β-unsaturated carbonyl compound to produce a carbonyl compound derived from said compound.

[0014] (7) The protein is represented by Formula I: GX １ X ２ P[VL][AG]PS (wherein G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, and X represents a ... １ and X ２ each independently represents any amino acid, [VL] represents V or L, and [AG] represents A or G.) A motif represented by Formula II: RVGX ３ H (wherein R represents arginine, V represents valine, G represents glycine, H represents histidine, and X represents a ３ (5) The method according to (4), wherein the motif is represented by the following formula:

[0015] (8) X １ represents glutamic acid (E), alanine (A) or glutamine (Q); X ２ represents leucine (L), and X ３ represents valine (V) or methionine (M).

[0016] (9) The method according to (7) above, wherein the motif represented by formula I is GELPVAPS; GALPVAPS; or GQLPVAPS, and the motif represented by formula II is RVGVH; or RVGMH.

[0017] (10) The method according to (7), wherein the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GQLPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGMH; and the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGMH.

[0018] (11) The method according to any one of (1) to (10), wherein the cell contains a heterologous expression unit comprising a polynucleotide encoding the protein and a promoter operably linked thereto.

[0019] (12) The method according to any one of (1) to (10), wherein the cell is a bacterium or a fungus.

[0020] (13) The method according to any one of (1) to (10), wherein the α,β-unsaturated carbonyl compound is at least one selected from the group consisting of 6-heptyl-5,6-dihydropyran-2-one; 6-pentyl-5,6-dihydropyran-2-one; citral; 2,4-decadienal; 2-hexenal; cinnamaldehyde; coumarin; piperitone; perillaldehyde; α-ionone; nootkatone; dehydrohedione; and dehydromuscone(α,β).

[0021] The present invention provides a method for efficiently and / or simply producing a carbonyl compound derived from an α,β-unsaturated carbonyl compound, specifically, the method using a specific protein having ene-reductase activity for an α,β-unsaturated carbonyl compound and / or a cell capable of expressing the protein.

[0022] FIG. 1 shows alignment analysis and motifs of enoate reductase (ER) enzymes.

[0023] The method for producing a carbonyl compound according to the present invention (hereinafter also referred to as the production method of the present invention) will be specifically described below. The scope of the present invention is not limited to the description, and other than the examples below, appropriate modifications can be made and implemented within the scope that does not impair the spirit of the present invention. This specification encompasses the entire specification of Japanese Patent Application No. 2024-022764 (filed February 19, 2024), which is the basis for claiming priority to this application. All publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference.

[0024] As described above, the production method of the present invention is a method for producing a carbonyl compound derived from an α,β-unsaturated carbonyl compound. Specifically, the production method of the present invention uses a protein having an amino acid sequence motif represented by the following formula I and an amino acid sequence motif represented by the following formula II, and having ene-reductase activity toward an α,β-unsaturated carbonyl compound. Hereinafter, this protein will also be referred to as the "protein used in the production method of the present invention."

[0025] Formula I: GX １ X ２ P[VL][AG]PS Formula II: RVGX ３ H

[0026] In the above formula I, G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, [VL] represents V or L, and [AG] represents A or G. １ and X ２ may each independently represent any amino acid, and are not limited to, for example, X １ is preferably at least one selected from the group consisting of glutamic acid (E), alanine (A) and glutamine (Q), and X ２ is preferably leucine (L). More specifically, preferred examples of the motif represented by the above formula I include: GELPVAPS; GALPVAPS; or GQLPVAPS.

[0027] In the above formula II, R represents arginine, V represents valine, G represents glycine, and H represents histidine. ３ may represent any amino acid, and is not limited thereto, but is preferably, for example, valine (V) and / or methionine (M). More specifically, preferred examples of the motif represented by formula II above include RVGVH; or RVGMH.

[0028] The various specific embodiments of the motif of formula I and the various specific embodiments of the motif of formula II described above may be employed in any combination, and are not limited thereto. In the production method of the present invention, any combination can be selected and employed from all conceivable combinations. For example, the combinations exemplified below are preferred.

[0029] - A combination in which the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGVH. - A combination in which the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGVH. - A combination in which the motif represented by formula I is GQLPVAPS and the motif represented by formula II is RVGVH. - A combination in which the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGMH. - A combination in which the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGMH.

[0030] The full-length amino acid sequence constituting the protein used in the production method of the present invention is not limited, but is preferably, for example, 200 to 500 amino acid residues, or 300 to 400 amino acid residues.

[0031] The protein used in the production method of the present invention has each of the motifs described above, and more specifically, at least one protein selected from the group consisting of various proteins having the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13 is preferably used.

[0032] In addition to the various proteins described above, preferred proteins used in the production method of the present invention include so-called mutant proteins, such as proteins that contain (or consist of) an amino acid sequence that is 80% or more identical to any of the amino acid sequences set forth in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, and SEQ ID NO:13 and that have ene-reductase activity for α,β-unsaturated carbonyl compounds. More preferred proteins include (or consist of) an amino acid sequence that is 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 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 identical to any of the above amino acid sequences and that have ene-reductase activity for α,β-unsaturated carbonyl compounds. Generally, the greater the identity value, the more preferable. The protein containing the amino acid sequence having 80% or more identity is not limited, but preferably contains the amino acid sequence of each of the motifs described above.

[0033] Here, whether the mutant protein has ene-reductase activity against α,β-unsaturated carbonyl compounds can be evaluated and measured by methods such as measuring the specific activity, which is the activity per 1 mg of enzyme, or detecting the product by qualitative or quantitative analysis such as gas chromatography or liquid chromatography (the same applies hereinafter).

[0034] Furthermore, examples of the proteins used in the production method of the present invention as the above-mentioned so-called mutants include proteins that contain an amino acid sequence in which one or several amino acids are deleted, substituted, or added in an amino acid sequence other than the amino acid sequences of each of the above-mentioned motifs in the amino acid sequences shown in SEQ ID NO: 8, SEQ ID NO: 19, SEQ ID NO: 11, and SEQ ID NO: 13, and that have ene-reductase activity against α,β-unsaturated carbonyl compounds. Here, the "amino acid sequence in which one or several amino acids have been deleted, substituted, or added" is not particularly limited, but includes, for example, amino acid sequences in which 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6 (1 to several), 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid has been deleted, substituted, or added, and the number of deletions, substitutions, or additions is generally preferably as small as possible. Introduction of mutations such as deletions, substitutions, or additions can be performed using a mutation introduction kit utilizing site-directed mutagenesis, for example, GeneTailor TM This can be done using the Site-Directed Mutagenesis System (Invitrogen), the TakaRa Site-Directed Mutagenesis System (Prime STAR® Mutagenesis Basal kit, Mutan®-Super Express Km, etc.: TakaRa Bio Inc.), etc. Furthermore, whether or not the amino acid sequence has the above-mentioned deletion, substitution, or addition mutation introduced can be confirmed using various amino acid sequencing methods, structural analysis methods such as X-ray and NMR, etc.

[0035] Here, when the amino acid sequence is substituted as described above, the substitution of the amino acid residue may be a conservative substitution. "Conservative substitution" refers to replacing a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), hydroxyl-containing side chains (e.g., alcohols, phenoxy-containing side chains) (e.g., serine, threonine, tyrosine), and sulfur-containing side chains (e.g., cysteine, methionine). Preferably, the conservative substitution may be a substitution between aspartic acid and glutamic acid, a substitution between arginine, lysine and histidine, a substitution between tryptophan and phenylalanine, a substitution between phenylalanine and valine, a substitution between leucine, isoleucine and alanine, and a substitution between glycine and alanine.

[0036] The protein or its variant used in the production method of the present invention may be a naturally occurring protein (polypeptide) or a non-naturally occurring protein (polypeptide) obtained by artificial chemical synthesis, and is not limited thereto. Non-naturally occurring proteins are preferred because they are easy to produce and can be mass-produced at low cost. Naturally occurring proteins are preferred because they often do not have adverse effects such as cytotoxicity.

[0037] Chemically synthesized polypeptides can be obtained using known peptide synthesis methods. The synthesis method is not particularly limited, but examples include the azide method, acid chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, carboimidazole method, and oxidation-reduction method. Both solid-phase synthesis and liquid-phase synthesis can be used for the synthesis. A commercially available peptide synthesizer may also be used. After the synthesis reaction, the polypeptide can be purified by a combination of known purification methods such as chromatography.

[0038] Furthermore, examples of polypeptides derived from natural products include naturally occurring oligopeptides, polypeptides, and proteins, as well as fragments thereof. Naturally occurring polypeptides may be obtained directly from natural products by known recovery and purification methods, or may be obtained by incorporating a gene encoding the desired protein into various expression vectors or the like using known genetic recombination techniques, introducing the vector into cells, allowing the protein to be expressed, and then by known recovery and purification methods. Alternatively, commercially available kits, such as the PROTEIOS Reagent Kit, may be used. TM (Toyobo), TNT TM System (Promega), synthesis equipment PG-Mate TM The polypeptide may be produced in a cell-free protein synthesis system using, for example, Toyobo (Toyobo) and RTS (Roche Diagnostics), and then recovered and purified by known methods, without any particular limitation.

[0039] The protein or mutant thereof used in the production method of the present invention may be or may include a derivative thereof. The derivative means all of those that can be prepared from the polypeptide of the protein or mutant thereof used in the production method of the present invention, and examples thereof include those in which some of the constituent amino acids have been substituted with unnatural amino acids, and those in which some of the constituent amino acids (mainly their side chains) have been chemically modified.

[0040] Furthermore, in the present invention, the protein or its variant, or its derivative used in the production method of the present invention can use or contain these salts. Such salts are preferably physiologically acceptable acid addition salts or basic salts. Examples of acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid, and salts with organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and trifluoroacetic acid. Examples of basic salts include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, and magnesium hydroxide, and salts with organic bases such as caffeine, piperidine, trimethylamine, and pyridine. Salts can be prepared using an appropriate acid such as hydrochloric acid or an appropriate base such as sodium hydroxide. For example, they can be prepared by treating in water or in a liquid containing an inert, water-miscible organic solvent such as methanol, ethanol, or dioxane using standard protocols.

[0041] In the present invention, cells capable of expressing the protein or mutant thereof (hereinafter also referred to as "cells used in the production method of the present invention") may be used in place of or together with the protein or mutant used in the production method of the present invention described above.

[0042] The cells used in the production method of the present invention are preferably, but not limited to, cells containing a heterologous expression unit including a polynucleotide encoding the protein used in the production method of the present invention and a promoter operably linked thereto. The base sequence of the polynucleotide, for example, the base sequence of the codons corresponding to each amino acid, is preferably an arrangement suitable for the type of cell used. The various base sequences contained in the heterologous expression unit, such as the promoter, can also be appropriately designed and selected depending on the type of cell used and are not particularly limited. The types of cells used in the production method of the present invention are not limited to, but include, for example, bacterial and fungal cells. Specific examples of bacteria include Escherichia coli, Bacillus subtilis, actinomycetes, coryneform bacteria, Pseudomonas bacteria, and Brevibacillus bacteria, and examples of fungal cells include various yeasts and filamentous fungi.

[0043] The cells used in the production method of the present invention include those obtained by disrupting the cells, etc., and various cell extracts prepared from cultured cells (which also include the proteins used in the production method of the present invention described above).

[0044] In the production method of the present invention, when cells are allowed to act on the substrate α,β-unsaturated carbonyl compound, a suspension of cultured cells can usually be used in the reaction as is. If components produced during the culture adversely affect the carbonyl compound production reaction, the cells may be recovered from the culture medium by centrifugation or other procedures, and a suspension prepared using the cells may be used again for the reaction. Furthermore, as described above, various cell extracts, such as those obtained by disrupting cultured cells or enzyme proteins prepared from cultured cells, can also be used in the carbonyl compound production reaction.

[0045] To efficiently promote this reaction, it is necessary to increase the cell density in the suspension. However, if the density is too high, the reaction may be inhibited by the production of autolytic enzymes and the accumulation of end metabolites. Therefore, the cell density is usually set at 10 6 ~10 12cfu / mL ("cfu" means the number of colonies formed on an agar medium, colony forming units), preferably 10 7 ~10 11 cfu / mL, more preferably 10 8 ~10 10 Performed in cfu / mL.

[0046] The amount of substrate added to the cell suspension is not limited as long as it is a concentration that promotes the carbonyl compound production reaction, but is preferably 0.05 to 50% (w / v) of the cell suspension, and more preferably 0.1 to 40% (w / v). The method of adding the substrate to the cell suspension is not limited, but it is preferable to add it gradually while monitoring the carbonyl compound production reaction so as to maintain the concentration within a preferred range.

[0047] If the cell suspension is left standing, the cells will settle and the reaction efficiency will decrease, so it is preferable to carry out the reaction while stirring the suspension. The reaction time can be determined depending on the degree of production of the target carbonyl compound, and is usually preferably 1 to 72 hours.

[0048] In the production method of the present invention, the reaction temperature when producing a carbonyl compound derived from an α,β-unsaturated carbonyl compound as a substrate is preferably maintained within a range suitable for the enzymatic reaction of the protein or mutant thereof used in the production method of the present invention, and the cell used in the production method of the present invention, and is typically 5 to 50° C., preferably 10 to 40° C., and more preferably 20 to 30° C. Similarly, the pH during the reaction is preferably typically 4.0 to 10.0, and preferably 6.0 to 9.0.

[0049] The α,β-unsaturated carbonyl compound used as a substrate in the production method of the present invention is not limited, and preferred examples thereof include at least one selected from the group consisting of 6-heptyl-5,6-dihydropyran-2-one; 6-pentyl-5,6-dihydropyran-2-one; citral; 2,4-decadienal; 2-hexenal; cinnamaldehyde; coumarin; piperitone; perillaldehyde; α-ionone; nootkatone; dehydrohedione; and dehydromuscone(α,β).

[0050] Preferred examples of carbonyl compounds derived from the substrates listed above include, but are not limited to, at least one selected from the group consisting of δ-dodecalactone; δ-decalactone; citronellal; decenal; hexanal; 3-phenylpropionaldehyde; dihydrocoumarin; menthone and isomenthone; dihydroperillaldehyde; dihydro-α-ionone; dihydronootkatone; hedione; and muscone.

[0051] In the production method of the present invention, the method for recovering the target product, the carbonyl compound, from the reaction-terminated solution is not limited, and for example, a general isolation method used in organic synthesis can be adopted. After completion of the reaction, a crude product can be obtained by carrying out a typical post-treatment operation such as extraction with an organic solvent. The obtained crude product can be purified, as necessary, using dehydration, activated carbon, distillation, recrystallization, column chromatography, etc., to obtain the target product, the carbonyl compound. In addition, operations (e.g., chiral column chromatography, optical resolution agent, recrystallization) can also be carried out to further increase the optical purity of the obtained carbonyl compound.

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0053] 1. Selection of ER Candidate Genes. Enoate reductase (ER), an NAD(P)H-dependent oxidoreductase with an FMN-binding site, is known to catalyze the stereoselective and enantioselective reduction of α,β-unsaturated carbonyl compounds (Peters et al., ChemCatChem 2014, 6, 1021-1027). Therefore, we selected ER candidate genes from enzymes that share a common domain with ER (Pfam database, PF00724: Oxidored_FMN) (Table 1 below). The nucleotide and amino acid sequences of each ER candidate gene are shown in SEQ ID NOS: 1-7 and 8-14, respectively. Furthermore, the nucleotide sequences of the ER candidate genes, codon-optimized for efficient expression in E. coli, are shown in SEQ ID NOS: 15-21.

[0054]

[0055] 2. Construction of ER Expression Plasmids and Creation of E. coli Strains for ER Expression. The nucleotide sequences of each ER gene were optimized for E. coli (SEQ ID NOS: 15-21). To each end of the resulting dsDNA fragment, pET23a was digested with NdeI and XhoI in the multicloning site. 15-16 bases homologous to the respective ends of the fragments were added to create artificially synthesized sequences (SEQ ID NOS: 22-28). These sequences were then cloned into the pET23a plasmid to create ER expression plasmids. Furthermore, E. coli BL21(DE3) was transformed with each ER expression plasmid to create ER expression strains (Table 2, Nos. 2-8). A strain transformed with the pET23a plasmid without the ER gene inserted served as a control (Table 2, No. 1).

[0056]

[0057] 3. Purification of ER Enzyme Using Histidine Tags Glycerol stocks of each ER-expressing E. coli strain were thawed, and 50 μl of each aliquot was inoculated into 5 mL of LB medium containing 50 μg / mL carbenicillin and cultured for 16 hours at 37°C and 160 rpm in a reciprocal shaker (Tokyo Rikakikai Co., Ltd.). 1 mL of the resulting preculture was inoculated into 50 mL of TB medium containing 50 μg / mL carbenicillin and 1% glucose and cultured in a rotary shaker (Tokyo Rikakikai Co., Ltd.) at 37°C and 125 rpm. OD 600 When the pH reached 0.6 to 1.0, IPTG was added to a final concentration of 0.5 mM, and the mixture was cultured in a rotary shaker at 25°C and 150 rpm. After 24 hours of culture, the culture medium was centrifuged to recover the bacterial cells, and the cells were then subjected to Capture™. TM The enzyme was purified using a His-Tagged Purification Maxiprep Kit (Takara Bio Inc.) to obtain an eluted fraction, which was then buffer-exchanged with 0.1 M HEPES (pH 7.5) using a PD-10 Desalting Column (Cytiva) to obtain the purified enzyme.

[0058] 4. Measurement of specific activity of ER-purified enzymes toward 6-heptyl-5,6-dihydropyran-2-one The compositions shown in Table 3 below were mixed, and the rate of decrease in absorbance of NADPH (340 nm) was measured at 37°C using a spectrophotometer (Scrum Co., Ltd.). The amount of enzyme required to convert 1 μmol of substrate per minute was defined as 1 U, and specific activity (amount of substrate converted per unit time per 1 mg of enzyme) was calculated from the absorbance measurement results using Equation 1 below. Table 4 below shows the specific activity of each purified enzyme toward 6-heptyl-5,6-dihydropyran-2-one.

[0059]

[0060]

[0061]

[0062] 5. ER Enzyme Alignment Analysis and Search for Locally Conserved Amino Acid Sequence Motifs in Highly Active ER Enzymes The amino acid sequences of the ER enzymes discovered in this study were aligned using Clustal Omega. A common motif among ER enzymes is NX1RX2DX3X4GG (where each X independently represents any amino acid) (Peters et al., ChemCatChem 2014, 6, 1021-1027). All of the ER enzymes discovered in this study contained the NX1RX2DX3X4GG motif. Furthermore, we identified two amino acid sequence motifs locally conserved only in highly active ERs with a specific activity of 1.160 U / mg: motif a: GX1X2P[VL][AG]PS (the two letters in [ ] indicate either amino acid residue) and motif b: RVGX1H (Figure 1).

[0063] 6. Measurement of specific activity of ER-purified enzymes toward α,β-unsaturated carbonyl compounds The compositions shown in Table 5 below were mixed, and the rate of decrease in absorbance of NADPH (340 nm) was measured at 37°C using a spectrophotometer. The amount of enzyme converting 1 μmol of substrate per minute was defined as 1 U, and specific activity (amount of substrate converted per unit time per 1 mg of enzyme) was calculated from the absorbance measurement results using Equation 1 above. Table 7 below shows the specific activity of each purified enzyme toward each α,β-unsaturated carbonyl compound.

[0064]

[0065]

[0066]

[0067] 7. Production of δ-dodecalactone by culturing E. coli strains for ER expression. Glycerol stocks of E. coli strains for ER expression (Table 2, Nos. 3 and 7) were thawed, and 50 μl of the thawed aliquot was inoculated into 5 mL of LB medium containing 50 μg / mL carbenicillin and cultured in a reciprocal shaker at 37°C and 160 rpm for 16 hours. 1 mL of the resulting preculture was inoculated into 50 mL of TB medium containing 50 μg / mL carbenicillin and 1% glucose, and 10 mL of isopropyl myristate was added. The culture was then cultured in a rotary shaker at 37°C and 150 rpm. OD 600 When the pH reached 0.6-1.0, a final concentration of 0.5 mM IPTG and 2.5 g of 6-heptyl-5,6-dihydropyran-2-one (Table 6, No. a) were added, and the strains were cultured in a rotary shaker at 25°C and 150 rpm. The control strain (Table 2, No. 1) was also cultured in the same manner. After 48 hours of culture, the culture medium was centrifuged, and the δ-dodecalactone concentration in the isopropyl myristate was measured using a gas chromatograph (Shimadzu Corporation). The δ-dodecalactone concentration was converted to the medium volume and is shown in Table 8 below. The ER-expressing E. coli strains containing the ER gene exhibited increased δ-dodecalactone production compared to the control strains.

[0068]

[0069] The present invention provides a method for efficiently and / or simply producing a carbonyl compound derived from an α,β-unsaturated carbonyl compound, specifically, the method using a specific protein having ene-reductase activity for an α,β-unsaturated carbonyl compound and / or a cell capable of expressing the protein.

[0070] SEQ ID NOs: 15 to 28: synthetic DNA

Claims

1. Formula I: GX １ X ２ P[VL][AG]PS (wherein G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, and X represents a ... １ and X ２ each independently represents any amino acid, [VL] represents V or L, and [AG] represents A or G.) A motif represented by Formula II: RVGX ３ H (wherein R represents arginine, V represents valine, G represents glycine, H represents histidine, and X represents a ３ represents any amino acid.) and a protein having ene-reductase activity for an α,β-unsaturated carbonyl compound, and / or a cell capable of expressing said protein, is contacted with an α,β-unsaturated carbonyl compound to produce a carbonyl compound derived from the compound.

2. X １ represents glutamic acid (E), alanine (A) or glutamine (Q); X ２ represents leucine (L), and X ３ The method of claim 1 , wherein at least one of the following conditions is satisfied: represents valine (V) or methionine (M).

3. The method according to claim 1, wherein the motif represented by formula I satisfies at least one of: GELPVAPS; GALPVAPS; or GQLPVAPS; and the motif represented by formula II is RVGVH; or RVGMH.

4. The method according to claim 1, wherein any of the following is satisfied: the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GQLPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGMH; and the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGMH.

5. The method of claim 1, wherein the protein is at least one selected from the group consisting of: (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 8, and having the ene-reductase activity; (ii) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 9, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 9, and having the ene-reductase activity; (iii) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 11, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 11, and having the ene-reductase activity; and (iv) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 13, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 13, and having the ene-reductase activity.

6. Proteins selected from the following (i) to (vi): (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 8, and having ene-reductase activity for α,β-unsaturated carbonyl compounds; (ii) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 9, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 9, and having ene-reductase activity for α,β-unsaturated carbonyl compounds; (iii) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 11, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 11, and having ene-reductase activity for α,β-unsaturated carbonyl compounds; and (iv) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 13, or a protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 13, and having ene-reductase activity for α,β-unsaturated carbonyl compounds. and / or a cell capable of expressing said protein, with an α,β-unsaturated carbonyl compound to produce a carbonyl compound derived from said compound.

7. The protein is represented by Formula I: GX １ X ２ P[VL][AG]PS (wherein G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, and X represents a ... １ and X ２ each independently represents any amino acid, [VL] represents V or L, and [AG] represents A or G.) A motif represented by Formula II: RVGX ３ H (wherein R represents arginine, V represents valine, G represents glycine, H represents histidine, and X represents a ３ The method of claim 6, wherein the motif is represented by the following formula: and 8. X １ represents glutamic acid (E), alanine (A) or glutamine (Q); X ２ represents leucine (L), and X ３ The method according to claim 7, wherein at least one of the following conditions is satisfied: represents valine (V) or methionine (M).

9. The method according to claim 7, wherein the motif represented by formula I satisfies at least one of: GELPVAPS; GALPVAPS; or GQLPVAPS; and the motif represented by formula II is RVGVH; or RVGMH.

10. The method according to claim 7, wherein any one of the following is satisfied: the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GQLPVAPS and the motif represented by formula II is RVGVH; the motif represented by formula I is GALPVAPS and the motif represented by formula II is RVGMH; and the motif represented by formula I is GELPVAPS and the motif represented by formula II is RVGMH.

11. The method of any one of claims 1 to 10, wherein the cell contains a heterologous expression unit comprising a polynucleotide encoding the protein and a promoter operably linked thereto.

12. The method of any one of claims 1 to 10, wherein the cell is a bacterium or a fungus.

13. The method according to any one of claims 1 to 10, wherein the α,β-unsaturated carbonyl compound is at least one selected from the group consisting of 6-heptyl-5,6-dihydropyran-2-one; 6-pentyl-5,6-dihydropyran-2-one; citral; 2,4-decadienal; 2-hexenal; cinnamaldehyde; coumarin; piperitone; perillaldehyde; α-ionone; nootkatone; dehydrohedione; and dehydromuscone(α,β).

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