Process for producing a carbonyl compound

Abstract

Provided is a method for producing a carbonyl compound, and the like. The present invention is a method for producing a carbonyl compound, which includes bringing a protein having a motif represented by Formula I: GX1X2P[VL][AG]PS and a motif represented by Formula II: RVGX3H, and having ene reductase activity with respect to an α,β-unsaturated carbonyl compound and / or a cell capable of expressing the protein into contact with an α,β-unsaturated carbonyl compound, to produce a carbonyl compound derived from the compound.

Description

Technical Field

[0001] Carbonyl compounds have long been widely used as flavoring agents. For example, δ-decanolide and δ-dodecanolide are representative aroma components that contribute to the milky flavor and are widely used as blending materials in various flavoring compositions. Besides chemical synthesis methods, non-synthetic methods using fermentation are also known for manufacturing δ-decanolide and δ-dodecanolide. One known method involves reducing the corresponding α,β-unsaturated carbonyl compounds using strains belonging to the genera *Saccharomyces*, *Pseudomonas*, *Proteus*, *Bacillus*, *Cellulomonas*, *Micrococcus*, *Xanthomonas*, or *Acetobacter* (see Patent Documents 1-3). However, in these manufacturing methods, due to the low substrate concentration in the reaction solution, there are problems such as low carbonyl compound production (g / L) and insufficient production efficiency.

[0002] Furthermore, there is a method that converts unsaturated lactones into saturated lactones within host cells through the conjugated reaction of enoate reductase from Bacillus and glucose dehydrogenase (see Patent Document 4). However, this method requires the addition of expensive coenzymes and co-culturing with strains expressing glucose dehydrogenase. Due to the high cost and complex manufacturing process, there are challenges in industrialization.

[0003] Furthermore, while it is known that enoic acid reductases from the genus *Pseudomonas* reduce α,β-unsaturated carbonyl compounds (see Non-Patent Literature 1), there are no examples of their application to unsaturated lactones. Additionally, due to the low substrate concentration in the reaction solution, the amount of carbonyl compound produced (g / L) is low, resulting in insufficient production efficiency.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 3-155792

[0007] Patent Document 2: Japanese Patent Application Publication No. 6-225781

[0008] Patent Document 3: Japanese Patent Application Publication No. 10-042889

[0009] Patent Document 4: International Publication No. 2020 / 128644

[0010] Non-patent literature

[0011] Non-patent literature 1: Christin Peters, Regina Kolzsch, Maria Kadow, Lilly Skalden, Florian Rudroff, Marko D. Mihovilovic, and Uwe T. Bornscheuer, ChemCatChem, 6 (2014) 1021-1027. Summary of the Invention

[0012] The problem the invention aims to solve

[0013] In this context, it is desirable to develop techniques that can efficiently and / or easily generate carbonyl compounds derived from α,β-unsaturated carbonyl compounds.

[0014] Solution for solving the problem

[0015] The present invention was made in consideration of the above circumstances, and provides a method for manufacturing carbonyl compounds as shown below.

[0016] (1) A method for manufacturing a carbonyl compound, comprising: contacting a protein having the motif shown in Formula I: GX1X2P[VL][AG]PS and the motif shown in Formula II: RVGX3H, and having alkene reductase activity for α,β-unsaturated carbonyl compounds, and / or cells capable of expressing the protein, with the α,β-unsaturated carbonyl compound to generate a carbonyl compound derived from the compound.

[0017] (In Formula I, G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, X1 and X2 each independently represent any amino acid, [VL] represents V or L, and [AG] represents A or G.)

[0018] (In Formula II, R represents arginine, V represents valine, G represents glycine, H represents histidine, and X3 represents any amino acid.)

[0019] (2) According to the method described in (1) above, it satisfies at least one of the following:

[0020] X1 represents glutamic acid (E), alanine (A), or glutamine (Q);

[0021] X2 represents leucine (L); and

[0022] X3 represents either valine (V) or methionine (M).

[0023] (3) According to the method described in (1) above, it satisfies at least one of the following:

[0024] The base order shown in Equation I is:

[0025] GELPVAPS;

[0026] GALPVAPS; or

[0027] GQLPVAPS,

[0028] as well as

[0029] The base order shown in Equation II is:

[0030] RVGVH; or

[0031] RVGMH.

[0032] (4) According to the method described in (1) above, it satisfies any of the following:

[0033] The base sequence shown in Equation I is GELPVAPS, and the base sequence shown in Equation II is RVGVH;

[0034] The base sequence shown in Equation I is GALPVAPS, and the base sequence shown in Equation II is RVGVH;

[0035] The base sequence shown in Equation I is GQLPVAPS, and the base sequence shown in Equation II is RVGVH;

[0036] The motif shown in Equation I is GALPVAPS, and the motif shown in Equation II is RVGMH; and

[0037] The basis sequence shown in Equation I is GELPVAPS, and the basis sequence shown in Equation II is RVGMH.

[0038] (5) The method according to (1) above, wherein the protein is at least one selected from the group consisting of:

[0039] (i) Proteins composed of the amino acid sequence shown in sequence number 8, or

[0040] A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 8 and having the aforementioned olefin reductase activity;

[0041] (ii) Proteins composed of the amino acid sequence shown in sequence number 9, or

[0042] A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in sequence number 9 and having the aforementioned olefin reductase activity;

[0043] (iii) Proteins composed of the amino acid sequence shown in sequence number 11, or

[0044] A protein containing an amino acid sequence having more than 80% identity with the amino acid sequence shown in sequence number 11 and possessing the aforementioned olefin reductase activity; and

[0045] (iv) Proteins composed of the amino acid sequence shown in sequence number 13, or

[0046] A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in sequence number 13 and having the aforementioned olefin reductase activity.

[0047] (6) A method for manufacturing a carbonyl compound, comprising: contacting at least one protein selected from the group consisting of proteins (i) to (iv) below and / or a cell capable of expressing the protein with an α,β-unsaturated carbonyl compound to generate a carbonyl compound derived from the compound.

[0048] (i) Proteins composed of the amino acid sequence shown in sequence number 8, or

[0049] Proteins containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 8 and exhibiting alkene reductase activity for α,β-unsaturated carbonyl compounds;

[0050] (ii) Proteins composed of the amino acid sequence shown in sequence number 9, or

[0051] Proteins containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 9 and exhibiting alkene reductase activity for α,β-unsaturated carbonyl compounds;

[0052] (iii) Proteins composed of the amino acid sequence shown in sequence number 11, or

[0053] Proteins containing an amino acid sequence having more than 80% identity with the amino acid sequence shown in Serial No. 11 and exhibiting alkene reductase activity towards α,β-unsaturated carbonyl compounds; and

[0054] (iv) Proteins composed of the amino acid sequence shown in sequence number 13, or

[0055] Proteins containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 13 and exhibiting alkene reductase activity for α,β-unsaturated carbonyl compounds.

[0056] (7) According to the method described in (6) above, wherein the aforementioned protein has the motif shown in Formula I: GX1X2P[VL][AG]PS and the motif shown in Formula II: RVGX3H.

[0057] (In Formula I, G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, X1 and X2 each independently represent any amino acid, [VL] represents V or L, and [AG] represents A or G.)

[0058] (In Formula II, R represents arginine, V represents valine, G represents glycine, H represents histidine, and X3 represents any amino acid.)

[0059] (8) The method described in (7) above satisfies at least one of the following:

[0060] X1 represents glutamic acid (E), alanine (A), or glutamine (Q);

[0061] X2 represents leucine (L); and

[0062] X3 represents either valine (V) or methionine (M).

[0063] (9) The method described in (7) above satisfies at least one of the following:

[0064] The base order shown in Equation I is:

[0065] GELPVAPS;

[0066] GALPVAPS; or

[0067] GQLPVAPS,

[0068] as well as

[0069] The base order shown in Equation II is:

[0070] RVGVH; or

[0071] RVGMH.

[0072] (10) According to the method described in (7) above, wherein,

[0073] The base sequence shown in Equation I is GELPVAPS, and the base sequence shown in Equation II is RVGVH;

[0074] The base sequence shown in Equation I is GALPVAPS, and the base sequence shown in Equation II is RVGVH;

[0075] The base sequence shown in Equation I is GQLPVAPS, and the base sequence shown in Equation II is RVGVH;

[0076] The motif shown in Equation I is GALPVAPS, and the motif shown in Equation II is RVGMH; and

[0077] The basis sequence shown in Equation I is GELPVAPS, and the basis sequence shown in Equation II is RVGMH.

[0078] (11) The method according to any one of (1) to (10) above, wherein the aforementioned cell contains a heterologous expression unit, the heterologous expression unit containing a polynucleotide encoding the aforementioned protein and a promoter operatively linked thereto.

[0079] (12) The method according to any one of (1) to (10) above, wherein the aforementioned cells are bacteria or fungi.

[0080] (13) The method according to any one of (1) to (10) above, wherein the aforementioned α,β-unsaturated carbonyl compound is selected from at least one of the following:

[0081] 6-Heptyl-5,6-dihydropyran-2-one;

[0082] 6-Pentyl-5,6-dihydropyran-2-one;

[0083] Citral;

[0084] 2,4-Decadienal;

[0085] 2-Hexenal;

[0086] Cinnamaldehyde;

[0087] Coumarin;

[0088] Piperone;

[0089] Perillaldehyde;

[0090] α-Ionone;

[0091] Nocaketone;

[0092] Methyl dehydrojasmonate; and

[0093] Dehydromusketone (α,β).

[0094] The effects of the invention

[0095] According to the present invention, a method can be provided that can efficiently and / or easily generate carbonyl compounds derived from α,β-unsaturated carbonyl compounds. More specifically, the aforementioned method can be provided based on the use of a specific protein having alkene reductase activity for α,β-unsaturated carbonyl compounds and / or cells capable of expressing that protein. Attached Figure Description

[0096] Figure 1This is a diagram showing the comparative analysis and motif of enoate reductase (ER). Detailed Implementation

[0097] The method for manufacturing the carbonyl compound of the present invention (hereinafter also referred to as the manufacturing method of the present invention) will be specifically described below. The scope of the present invention is not limited by this description, and appropriate modifications and implementations may be made without prejudice to the spirit of the present invention, except for the examples below. It should be noted that this specification includes the entirety of Japanese Patent Application No. 2024-022764 (filed February 19, 2024), which forms the basis of the priority claim of this application. All publications cited in this specification, such as prior art documents and published documents, patent gazettes, and other patent documents, are incorporated herein by reference.

[0098] As described above, the manufacturing method of the present invention is a method for manufacturing carbonyl compounds derived from α,β-unsaturated carbonyl compounds.

[0099] Specifically, in the manufacturing method of the present invention, a protein is used that has a motif having an amino acid sequence shown in Formula I and an amino acid sequence shown in Formula II, and possesses alkene reductase activity for α,β-unsaturated carbonyl compounds. This protein is hereinafter also referred to as "the protein used in the manufacturing method of the present invention".

[0100] Formula I: GX1X2P[VL][AG]PS

[0101] Formula II: RVGX3H

[0102] Here, in Formula I above, 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.

[0103] In addition, X1 and X2 can each independently represent any amino acid without limitation. For example, X1 is preferably selected from at least one of the group consisting of glutamic acid (E), alanine (A) and glutamine (Q), and X2 is preferably leucine (L).

[0104] More specifically, as the basis sequence shown in Equation I above, the following can be preferably listed, for example:

[0105] GELPVAPS;

[0106] GALPVAPS; or

[0107] GQLPVAPS.

[0108] In Formula II above, R represents arginine, V represents valine, G represents glycine, and H represents histidine.

[0109] In addition, X3 can represent any amino acid without limitation, but is preferably valine (V) and / or methionine (M).

[0110] More specifically, as the basis sequence shown in Equation II above, the following can be preferably listed, for example:

[0111] RVGVH; or

[0112] RVGMH.

[0113] Various specific arrangements of the basis sequence of Formula I and Formula II described above can be used in any combination without limitation. They can be selected and used from all combinations that can be considered in the manufacturing method of the present invention. For example, the following combinations are preferred.

[0114] • The basis sequence shown in Equation I is a combination of GELPVAPS and the basis sequence shown in Equation II is a combination of RVGVH.

[0115] • The basis sequence shown in Equation I is GALPVAPS and the basis sequence shown in Equation II is RVGVH.

[0116] • The basis sequence shown in Equation I is a combination of GQLPVAPS and the basis sequence shown in Equation II is RVGVH.

[0117] • The basis sequence shown in Equation I is a combination of GALPVAPS and the basis sequence shown in Equation II is RVGMH.

[0118] • The basis sequence shown in Equation I is a combination of GELPVAPS and the basis sequence shown in Equation II is a combination of RVGMH.

[0119] The full-length amino acid sequence of the protein used in the manufacturing method of the present invention is not limited, but is preferably 200 to 500 amino acid residues or 300 to 400 amino acid residues.

[0120] The protein used in the manufacturing method of the present invention has the above-mentioned motifs. More specifically, it is preferable to include at least one protein from the group consisting of various protein compositions composed of the amino acid sequences shown in Serial Numbers 8, 9, 11 and 13.

[0121] In addition to the various proteins described above, proteins used in the manufacturing method of the present invention, as so-called mutants, are preferably listed as follows: proteins containing an amino acid sequence (or composed of) having at least 80% identity with any of the amino acid sequences shown in Serial Numbers 8, 9, 11, and 13, and possessing olefin reductase activity against α,β-unsaturated carbonyl compounds. More preferably, proteins containing an amino acid sequence (or composed of) having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with any of the above-described amino acid sequences, and possessing olefin reductase activity against α,β-unsaturated carbonyl compounds. Generally, the higher the value of the aforementioned identity, the more preferred. It should be noted that there is no limitation on the amino acid sequence containing more than 80% of the above-mentioned identity, but it is preferred to retain the amino acid sequence of each of the aforementioned motifs.

[0122] Here, regarding the alkene reductase activity of the aforementioned mutant protein for α,β-unsaturated carbonyl compounds, it can be evaluated and determined by methods such as measuring the specific activity per 1 mg of enzyme, or by detecting the product using qualitative / quantitative analysis methods such as gas chromatography or liquid chromatography (the same applies below).

[0123] Furthermore, the protein used in the manufacturing method of the present invention as the aforementioned mutant may include, for example, an amino acid sequence containing one or more amino acids deleted, substituted, or added in the amino acid sequences other than the amino acid sequences of the aforementioned motifs, and possessing alkene reductase activity against α,β-unsaturated carbonyl compounds. Here, the term "amino acid sequence with one or more amino acids deleted, substituted, or added" is not particularly limited; for example, amino acid sequences with 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 may be included. Generally, the smaller the number of deleted, substituted, or added amino acids, the more preferred. The introduction of mutations such as deletions, substitutions, or additions can be achieved using mutation introduction kits that utilize site-specific mutagenesis, such as GeneTailor. TMSite-directed mutagenesis systems (Invitrogen) and TaKaRa site-directed mutagenesis systems (Prime STAR Mutagenesis Basal kit, Mutan-Super Express Km, etc., manufactured by Takara Bio Inc.) are used. Furthermore, whether the introduced amino acid sequences contain deletions, substitutions, or additions can be confirmed using various amino acid sequencing methods and structural analysis methods such as X-ray diffraction and NMR.

[0124] Here, when the above-mentioned substitutions occur in the amino acid sequence, the substitution of amino acid residues can be a conservative substitution. "Conservative substitution" refers to replacing a specified amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are well known in the art. For example, such a family includes amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-electrolyte polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl-containing side chains (e.g., alcohols, phenoxy-containing side chains) (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Preferably, the above-mentioned conservative substitutions can be substitutions between aspartic acid and glutamic acid, substitutions between arginine and lysine and histidine, substitutions between tryptophan and phenylalanine, substitutions between phenylalanine and valine, substitutions between leucine and isoleucine and alanine, and substitutions between glycine and alanine.

[0125] The protein or its mutant used in the manufacturing method of the present invention can be a protein (peptide) derived from a natural source or a non-natural protein (peptide) obtained through artificial synthesis, and there is no limitation thereto. In the case of a non-natural protein, production is easy, and costs can be kept low for large-scale production, therefore it is preferred. In the case of a protein derived from a natural source, adverse effects such as cytotoxicity are generally absent, therefore it is preferred.

[0126] Chemically synthesized peptides can be obtained using well-known peptide synthesis methods. There are no particular limitations on the synthesis methods; examples include the azide method, acyl chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, carbonyl imidazole method, and redox method. Furthermore, this synthesis can employ either solid-phase synthesis or liquid-phase synthesis. Commercially available peptide synthesis equipment can also be used. After the synthesis reaction, the peptide can be purified using well-known purification methods such as chromatography.

[0127] In addition, peptides derived from natural sources can include naturally occurring oligopeptides, polypeptides, and proteins, or peptides in fragmented form. Peptides derived from natural products can be obtained directly from the natural products using known recovery and purification methods. Alternatively, they can be obtained using known gene recombination technologies, where the genome encoding the desired protein is inserted into various expression vectors and introduced into cells for expression, followed by known recovery and purification methods. Alternatively, they can be obtained using commercially available kits, such as the PROTEIOS kit. TM (Toyobo), TNT TM System (Promega), Synthesis Device PG-Mate TM The polypeptide is produced by cell-free protein synthesis systems such as Toyobo and RTS (Roche Diagnostics), and is obtained through known recovery and purification methods, without limitation.

[0128] As used in the manufacturing method of the present invention, the protein or its mutant may be used or may contain derivatives thereof. These derivatives refer to all substances that can be prepared from polypeptides derived from the protein or its mutant used in the manufacturing method of the present invention, such as substances in which a portion of the constituent amino acids is replaced with non-natural amino acids, substances in which a portion of the constituent amino acids (mainly their side chains) has been chemically modified, etc.

[0129] Furthermore, in this invention, the protein or its mutant, or its derivative, used in the manufacturing method of this invention may use or contain salts thereof. Preferably, these salts are 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; or 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; or salts with organic bases such as caffeine, piperidine, trimethylamine, and pyridine. The salts can be prepared using suitable acids such as hydrochloric acid or suitable bases such as sodium hydroxide. For example, they can be prepared by treatment in water or in a liquid containing an inactive aqueous mixed organic solvent such as methanol, ethanol, or dioxane using standard methods.

[0130] In this invention, the protein or its mutant used in the aforementioned manufacturing method of this invention may be used instead, or a cell capable of expressing the protein or mutant may be used in conjunction with the protein or mutant (hereinafter also referred to as "the cell used in the manufacturing method of this invention").

[0131] The cells used in the manufacturing method of the present invention are not limited, but are preferably cells containing a heterologous expression unit, which comprises a polynucleotide encoding the protein used in the manufacturing method of the present invention and a promoter operatively linked thereto. The base sequence of this polynucleotide is preferably, for example, a sequence of codons corresponding to each amino acid that is suitable for the type of cell used. The various base sequences contained in the heterologous expression unit, such as the promoter, can be appropriately designed and selected according to the type of cell used, and are not particularly limited. The type of cells used in the manufacturing method of the present invention is not limited; examples include bacterial and fungal cells. Specifically, examples of bacteria include *Escherichia coli*, *Bacillus subtilis*, *Actinomyces*, *Corynebacterium*, *Pseudomonas*, and *Bacillus brevis*, and examples of fungal cells include various yeasts and filamentous fungi.

[0132] The cells used in the manufacturing method of the present invention also include substances obtained by breaking down the cells, and various cell extracts prepared from cultured cells (including proteins used in the aforementioned manufacturing method of the present invention).

[0133] In the manufacturing method of the present invention, in order to enable cells to act on α,β-unsaturated carbonyl compounds as substrates, a suspension of cultured cells can usually be used directly in the reaction. If components generated during culture adversely affect the carbonyl compound formation reaction, a suspension can be prepared again using cells recovered once from the culture medium through centrifugation or similar operations, and then used in the reaction. Furthermore, as described above, cells obtained by disrupting cultured cells, enzyme proteins prepared from cultured cells, and various other cell extracts can also be used in the carbonyl compound formation reaction.

[0134] To ensure efficient reaction, the cell density in the suspension needs to be increased. However, if the density is too high, the reaction may be hindered by the production of autolysins and the accumulation of final metabolites. Therefore, a density of 10 is usually used. 6 ~10 12 cfu / mL (“cfu” refers to the number of colonies formed on agar medium, colony forming units), preferably 10 7 ~10 11 cfu / mL, more preferably 10 8 ~10 10 The concentration was increased to cfu / mL.

[0135] The amount of substrate added to the cell suspension can be any concentration that allows for the formation of the carbonyl compound, and there is no limit to the amount. Preferably, it is 0.05–50% (w / v) relative to the cell suspension, more preferably 0.1–40% (w / v). Furthermore, the method of adding the substrate to the cell suspension is not limited, but it is preferable to add it gradually while monitoring the formation of the carbonyl compound and maintaining the preferred range.

[0136] If the cell suspension is left to stand, the cells will settle and the reaction efficiency will decrease. Therefore, it is preferable to carry out the reaction while stirring the suspension. The reaction time can be determined based on the formation of the carbonyl compound of the target analyte, and is usually preferred to be 1 to 72 hours.

[0137] In the manufacturing method of the present invention, the reaction temperature for generating 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 its mutant used in the manufacturing method of the present invention, or the cell used in the manufacturing method of the present invention, typically between 5 and 50°C, preferably between 10 and 40°C, and more preferably between 20 and 30°C. Similarly, the pH during the reaction is typically between 4.0 and 10.0, preferably between 6.0 and 9.0.

[0138] The α,β-unsaturated carbonyl compound used as a substrate in the manufacturing method of the present invention is not limited, but preferably includes at least one selected from the group consisting of:

[0139] 6-Heptyl-5,6-dihydropyran-2-one;

[0140] 6-Pentyl-5,6-dihydropyran-2-one;

[0141] Citral;

[0142] 2,4-Decadienal;

[0143] 2-Hexenal;

[0144] Cinnamaldehyde;

[0145] Coumarin;

[0146] Piperone;

[0147] Perillaldehyde;

[0148] α-Ionone;

[0149] Nootkatone;

[0150] Methyl dehydrojasmonate; and

[0151] Dehydromusketone (α,β).

[0152] The carbonyl compounds derived from the substrates listed above are not limited, but preferably include at least one selected from the group consisting of:

[0153] δ-dodecanolide;

[0154] δ-decanolide;

[0155] Citronellol

[0156] Decenal;

[0157] Hexanal;

[0158] 3-Phenylacetal;

[0159] Dihydrocoumarin;

[0160] Menthone and isomenthone;

[0161] Dihydroperylaldehyde;

[0162] Dihydro-α-ionone;

[0163] dihydronorcaden;

[0164] Methyl dihydrojasmonic acid (hedione); and

[0165] Muskone.

[0166] In the manufacturing method of the present invention, the method for recovering the carbonyl compound, which is the target product, from the reaction termination solution is not limited, and for example, conventional separation methods in organic synthesis can be used. After the reaction is completed, conventional post-treatment operations such as extraction with an organic solvent are performed to obtain a crude product. The obtained crude product is then purified as needed by dehydration, activated carbon, distillation, recrystallization, column chromatography, etc., to obtain the carbonyl compound, which is the target product. It should be noted that operations to further improve the optical purity of the obtained carbonyl compound (e.g., chiral column chromatography, optical resolving agents, recrystallization) can also be performed.

[0167] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to these embodiments in any way.

[0168] Example 1

[0169] 1. Selection of ER candidate genes

[0170] Oleic acid reductases (ERs), NAD(P)H-dependent oxidoreductases with FMN binding sites, are known to catalyze stereoselective and enantioselective reductions of α,β-unsaturated carbonyl compounds (Peters et al., ChemCatChem 2014, 6, 1021-1027). Therefore, candidate ER genes were selected from enzymes sharing a common ER domain (Pfam database, PF00724: Oxidored_FMN) (Table 1 below). The base and amino acid sequences of each candidate ER gene are shown in SEQ ID NOs 1–7 and SEQ ID NOs 8–14. Furthermore, to efficiently express the ER gene, the codons were optimized to the base sequences used in *E. coli* and are shown in SEQ ID NOs 15–21.

[0171] [Table 1]

[0172]

[0173] 2. Construction of ER expression plasmids and preparation of Escherichia coli strains for ER expression

[0174] A 15-16 base sequence (Sequence Nos. 22-28) homologous to the ends of the dsDNA fragment digested with NdeI and XhoI enzymes at the multiple cloning site of pET23a was artificially synthesized and cloned into the pET23a plasmid as the ER expression plasmid. This plasmid was then used to transform *E. coli* BL21(DE3) to prepare *E. coli* strains for ER expression (Table 2, Nos. 2-8 below). Additionally, strains transformed with pET23a plasmids without ER gene insertion were used as control strains (Table 2, No. 1 below).

[0175] [Table 2]

[0176]

[0177] 3. Purification of histidine-tagged ER enzyme

[0178] Thaw the glycerol cryopreservation solution of each ER expression strain of *E. coli*, and inoculate 50 μl into 5 mL of LB medium containing 50 μg / mL carbenicillin. Incubate for 16 hours using a reciprocating shaking incubator (Tokyo Rika K.K. Co., Ltd.) at 37°C and 160 rpm. Inoculate 1 mL of the resulting pre-culture solution into 50 mL of TB medium containing 50 μg / mL carbenicillin and 1% glucose, and incubate using a rotary shaking incubator (Tokyo Rika K.K. Co., Ltd.) at 37°C and 125 rpm. At OD... 600 Once the concentration reached 0.6–1.0, IPTG was added to a final concentration of 0.5 mM, and the culture was incubated using a rotary shaker at 25°C and 150 rpm. The bacterial cells were recovered from the culture medium 24 hours after the start of incubation by centrifugation and analyzed using Capture™. TM Enzyme purification was performed using the His-Tagged Purification Maxiprep Kit (manufactured by Takara Bio Co., Ltd.) to obtain the eluent fraction. The eluent fraction was then replaced with 0.1M HEPES (pH 7.5) buffer using a PD-10 Desalting Column (cytiva) to prepare the purified enzyme.

[0179] 4. Determination of the specific activity of ER-purified enzyme against 6-heptyl-5,6-dihydropyran-2-one

[0180] The enzymes were mixed according to the compositions shown in Table 3 below, and the rate of decrease in absorbance of NADPH (340 nm) at 37°C was measured using a spectrophotometer (Scrum Inc.). The amount of enzyme required to change 1 μmol of substrate per minute was defined as 1 U. Specific activity (the amount of substrate change per unit time per 1 mg of enzyme) was calculated from the absorbance measurement using Equation 1 below. The specific activities of each purified enzyme against 6-heptyl-5,6-dihydropyran-2-one are shown in Table 4 below.

[0181] [Table 3]

[0182]

[0183] [Mathematical Expression 1]

[0184]

[0185] [Table 4]

[0186]

[0187] 5. Comparative analysis of ER enzymes and exploration of locally conserved amino acid sequence motifs in highly active ER enzymes.

[0188] The amino acid sequences of the ER enzymes discovered in this invention were compared and analyzed using Clustal Omega. As a common motif for ER enzymes, NX1RX2DX3X4GG (where each X independently represents any amino acid) is known (Peters et al., ChemCatChem 2014, 6, 1021-1027). All the ER enzymes discovered in this invention contain the NX1RX2DX3X4GG motif. Furthermore, as amino acid sequence motifs that are locally conserved only in highly active ER enzymes with specific activities above 1.160 U / mg, motifs a: GX1X2P[VL][AG]PS (where the two characters within [ ] represent any amino acid residue) and motif b: RVGX1H ( Figure 1 ).

[0189] 6. Determination of the specific activity of ER-purified enzymes for α,β-unsaturated carbonyl compounds

[0190] The enzymes were mixed according to the compositions shown in Table 5 below, and the rate of decrease in absorbance of NADPH (340 nm) at 37°C was measured using a spectrophotometer. The amount of enzyme required to change 1 μmol of substrate per minute was defined as 1 U. Specific activity (the amount of substrate change per unit time per 1 mg of enzyme) was calculated from the absorbance measurements using Equation 1 above. The specific activities of each purified enzyme against each α,β-unsaturated carbonyl compound are shown in Table 7 below.

[0191] [Table 5]

[0192]

[0193] [Table 6]

[0194]

[0195] [Table 7]

[0196]

[0197] 7. Production of δ-dodecanolide from Escherichia coli strains by culturing ER expression.

[0198] The glycerol cryopreservation solution of ER-expressing *Escherichia coli* strain (Table 2, No. 3, 7) was thawed, and 50 μl was inoculated into 5 mL of LB medium containing 50 μg / mL carbenicillin. The medium was then cultured for 16 hours at 37°C and 160 rpm using a reciprocating shaking incubator. 1 mL of the resulting pre-culture solution was inoculated into 50 mL of TB medium containing 50 μg / mL carbenicillin and 1% glucose, with 10 mL of isopropyl myristate added. The medium was then cultured using a rotary shaking incubator at 37°C and 150 rpm. At OD... 600 After reaching a concentration of 0.6–1.0, a final concentration of 0.5 mM IPTG and 2.5 g of 6-heptayl-5,6-dihydropyran-2-one (Table 6, No. a) were added, and the cultures were incubated using a rotary shaker at 25°C and 150 rpm. The control strain (Table 2, No. 1) was cultured in the same manner. The culture medium was centrifuged 48 hours after the start of incubation, and the concentration of δ-dodecanolide in isopropyl myristate was determined by gas chromatography (Shimadzu Corporation). The δ-dodecanolide concentrations are shown in Table 8 below in terms of converted culture medium volume. Increased δ-dodecanolide production was confirmed in the ER expression strains inoculated with the ER gene compared to the control strain.

[0199] [Table 8]

[0200]

[0201] Industrial availability

[0202] According to the present invention, a method can be provided that can efficiently and / or easily generate carbonyl compounds derived from α,β-unsaturated carbonyl compounds. More specifically, the aforementioned method can be provided based on the use of a specific protein having alkene reductase activity for α,β-unsaturated carbonyl compounds and / or cells capable of expressing that protein.

[0203] Sequence list text

[0204] Sequence numbers 15–28: Synthetic DNA.

Claims

1. A method for manufacturing a carbonyl compound, comprising: Proteins having the motifs shown in Formula I: GX1X2P[VL][AG]PS and Formula II: RVGX3H, and possessing alkene reductase activity towards α,β-unsaturated carbonyl compounds, and / or cells capable of expressing such proteins, are contacted with α,β-unsaturated carbonyl compounds to generate carbonyl compounds derived from those compounds. In Formula I, G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, X1 and X2 each independently represent any amino acid, [VL] represents V or L, and [AG] represents A or G. In Formula II, R represents arginine, V represents valine, G represents glycine, H represents histidine, and X3 represents any amino acid.

2. The method according to claim 1, wherein at least one of the following is satisfied: X1 represents glutamic acid (E), alanine (A), or glutamine (Q); X2 represents leucine (L); and X3 represents either valine (V) or methionine (M).

3. The method according to claim 1, wherein at least one of the following is satisfied: The base order shown in Equation I is: GELPVAPS; GALPVAPS; or GQLPVAPS, as well as The base order shown in Equation II is: RVGVH; or RVGMH.

4. The method according to claim 1, wherein it satisfies any of the following: The base sequence shown in Equation I is GELPVAPS, and the base sequence shown in Equation II is RVGVH; The base sequence shown in Equation I is GALPVAPS, and the base sequence shown in Equation II is RVGVH; The base sequence shown in Equation I is GQLPVAPS, and the base sequence shown in Equation II is RVGVH; The motif shown in Equation I is GALPVAPS, and the motif shown in Equation II is RVGMH; and The basis sequence shown in Equation I is GELPVAPS, and the basis sequence shown in Equation II is RVGMH.

5. The method according to claim 1, wherein, The protein is at least one selected from the group consisting of: (i) Proteins composed of the amino acid sequence shown in sequence number 8, or A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 8 and having the olefin reductase activity described above; (ii) Proteins composed of the amino acid sequence shown in sequence number 9, or A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 9 and having the olefin reductase activity described above; (iii) Proteins composed of the amino acid sequence shown in sequence number 11, or A protein comprising an amino acid sequence having more than 80% identity with the amino acid sequence shown in sequence number 11 and possessing the said olefin reductase activity; and (iv) Proteins composed of the amino acid sequence shown in sequence number 13, or A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in sequence number 13 and having the olefin reductase activity.

6. A method for manufacturing a carbonyl compound, comprising: By contacting at least one protein selected from the group consisting of proteins (i) to (vi) below and / or a cell capable of expressing that protein with an α,β-unsaturated carbonyl compound, a carbonyl compound derived from that compound is generated. (i) Proteins composed of the amino acid sequence shown in sequence number 8, or Proteins containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 8 and exhibiting alkene reductase activity for α,β-unsaturated carbonyl compounds; (ii) Proteins composed of the amino acid sequence shown in sequence number 9, or Proteins containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 9 and exhibiting alkene reductase activity for α,β-unsaturated carbonyl compounds; (iii) Proteins composed of the amino acid sequence shown in sequence number 11, or Proteins containing an amino acid sequence having more than 80% identity with the amino acid sequence shown in Serial No. 11 and exhibiting alkene reductase activity towards α,β-unsaturated carbonyl compounds; and (iv) Proteins composed of the amino acid sequence shown in sequence number 13, or A protein containing an amino acid sequence that has more than 80% identity with the amino acid sequence shown in Serial No. 13 and exhibiting alkene reductase activity for α,β-unsaturated carbonyl compounds.

7. The method according to claim 6, wherein, The protein has the motif shown in Formula I: GX1X2P[VL][AG]PS and the motif shown in Formula II: RVGX3H. In Formula I, G represents glycine, P represents proline, V represents valine, L represents leucine, A represents alanine, S represents serine, X1 and X2 each independently represent any amino acid, [VL] represents V or L, and [AG] represents A or G. In Formula II, R represents arginine, V represents valine, G represents glycine, H represents histidine, and X3 represents any amino acid.

8. The method of claim 7, wherein at least one of the following is satisfied: X1 represents glutamic acid (E), alanine (A), or glutamine (Q); X2 represents leucine (L); and X3 represents either valine (V) or methionine (M).

9. The method of claim 7, wherein at least one of the following is satisfied: The base order shown in Equation I is: GELPVAPS; GALPVAPS; or GQLPVAPS, as well as The base order shown in Equation II is: RVGVH; or RVGMH.

10. The method of claim 7, wherein it satisfies any of the following: The base sequence shown in Equation I is GELPVAPS, and the base sequence shown in Equation II is RVGVH; The base sequence shown in Equation I is GALPVAPS, and the base sequence shown in Equation II is RVGVH; The base sequence shown in Equation I is GQLPVAPS, and the base sequence shown in Equation II is RVGVH; The motif shown in Equation I is GALPVAPS, and the motif shown in Equation II is RVGMH; and The basis sequence shown in Equation I is GELPVAPS, and the basis sequence shown in Equation II is RVGMH.

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

12. The method according to any one of claims 1 to 10, wherein, The cells are bacteria or fungi.

13. The method according to any one of claims 1 to 10, wherein, The α,β-unsaturated carbonyl compound is selected from at least one of the following: 6-Heptyl-5,6-dihydropyran-2-one; 6-Pentyl-5,6-dihydropyran-2-one; Citral; 2,4-Decadienal; 2-Hexenal; Cinnamaldehyde; Coumarin; Piperone; Perillaldehyde; α-Ionone; Nocaketone; Methyl dehydrojasmonate; and Dehydromusketone (α,β).

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