Carbonyl reductase mutants and their application in the reduction of cyclopentanedione compounds

Abstract

The present invention provides a carbonyl reductase mutant and its application in the reduction of cyclopentanedione compounds. Specifically, the present invention provides a carbonyl reductase mutant, wherein the mutant protein is a non-natural protein, and the mutant protein has a catalytic activity of catalyzing cyclopentanedione compounds to generate cyclopentanone alcohol, and the mutant protein is mutated in 4 or more core amino acids related to the catalytic activity of the enzyme in the wild-type carbonyl reductase. The carbonyl reductase mutant of the present invention can significantly improve the catalytic activity of the carbonyl reductase, increase the ratio of 2R and 3S chiral products of cyclopentanone alcohol, and improve the yield, time-space yield, ee and de value of cyclopentanone alcohol compounds.

Description

[0001] This application is a divisional application of an invention patent application with an application date of May 31, 2018, application number 201810555674.6, and invention name “Carbonyl reductase mutants and their application in the reduction of cyclopentanedione compounds”. Technical Field

[0002] The present invention relates to the field of enzyme and enzyme engineering, and in particular to a carbonyl reductase mutant and application thereof in the reduction of cyclopentanedione compounds. Background Art

[0003] Asymmetric reduction of the carbonyl group of 2,2-disubstituted cyclopentanedione is an effective way to synthesize many key intermediates of drugs, such as levonorgestrel and its hydroxylate in hormone drugs. Levonorgestrel (Formula I) is the most common emergency contraceptive drug in the Chinese market. It is the active ingredient of the single progestogen drug, levonorgestrel tablets (0.75 mg per tablet or 1.5 mg per tablet), which has been included in my country's "National Over-the-Counter Drug Catalogue".

[0004]

[0005] The synthetic route of levonorgestrel involves the synthesis of chiral intermediates by asymmetric reduction of dicarbonyl intermediates. The current production route is to prepare chiral intermediates by transformation using Saccharomyces cerevisiae (Route 1). The transformation of this route requires the first cultivation of Saccharomyces cerevisiae for two to three days, followed by the addition of the substrate ethyl condensate, and the concentration of the ethyl condensate is 4g / L, and the shortest reported transformation time is 3 days. In recent years, reports are still about wild bacteria for ethyl condensates, such as using Pichia minuta CBS 1708 to achieve the transformation of ethyl condensates, but the ee value of the reaction product is only 90%, which cannot meet the requirements for stereoselectivity.

[0006]

[0007] The use of wild bacteria for transformation not only takes a long time to cultivate the bacteria, but also has low catalytic efficiency because the wild bacteria do not over-express the required carbonyl reductase for transformation.

[0008] Therefore, there is an urgent need in the art to develop efficient carbonyl reductases to achieve high stereoselective conversion of cyclopentanedione compounds, especially ethyl condensates. Summary of the invention

[0009] The invention provides an efficient carbonyl reductase, thereby realizing the high stereoselective synthesis of cyclopentanone alcohol.

[0010] The first aspect of the present invention provides a carbonyl reductase mutant protein, wherein the mutant protein is a non-natural protein, and the mutant protein has catalytic activity of catalyzing cyclopentanedione compounds to generate cyclopentanone alcohol compounds, and the mutant protein has mutations in four or more core amino acids related to the enzyme catalytic activity corresponding to SEQ ID NO.: 1 in the wild-type carbonyl reductase selected from the following group:

[0011] Isoleucine (I) at position 91;

[0012] Glutamic acid (E) at position 92;

[0013] Glutamine (Q) at position 93;

[0014] Leucine (L) at position 142;

[0015] Leucine (L) at position 144;

[0016] Histidine (H) at position 147;

[0017] Tyrosine (Y) at position 150;

[0018] Isoleucine (I) at position 187;

[0019] Isoleucine (I) at position 188;

[0020] Glutamine (Q) at position 191;

[0021] Leucine (L) at position 201;

[0022] Lysine (K) at position 204;

[0023] Phenylalanine (F) at position 205; and

[0024] Leucine (L) at position 246.

[0025] In another preferred embodiment, the mutant protein is mutated in a core amino acid corresponding to SEQ ID NO.: 1 in the wild-type carbonyl reductase and is selected from the following group of core amino acids associated with enzyme catalytic activity:

[0026] Isoleucine (I) at position 91;

[0027] Glutamic acid (E) at position 92;

[0028] Glutamine (Q) at position 93;

[0029] Leucine (L) at position 142;

[0030] Leucine (L) at position 144;

[0031] Histidine (H) at position 147;

[0032] Position 150: Tyrosine (Y);

[0033] Isoleucine (I) at position 187;

[0034] Isoleucine (I) at position 188;

[0035] Glutamine (Q) at position 191;

[0036] Leucine (L) at position 201;

[0037] Lysine (K) at position 204;

[0038] Phenylalanine (F) at position 205; and

[0039] Leucine (L) at position 246.

[0040] In another preferred embodiment, the mutant protein is mutated in a core amino acid corresponding to SEQ ID NO.: 1 in the wild-type carbonyl reductase and is selected from the following group of core amino acids associated with enzyme catalytic activity:

[0041] Isoleucine (I) at position 91;

[0042] Glutamic acid (E) at position 92;

[0043] Glutamine (Q) at position 93;

[0044] Leucine (L) at position 142;

[0045] Leucine (L) at position 144;

[0046] Histidine (H) at position 147;

[0047] Tyrosine (Y) at position 150;

[0048] Isoleucine (I) at position 187;

[0049] Isoleucine (I) at position 188;

[0050] Glutamine (Q) at position 191;

[0051] Leucine (L) at position 201;

[0052] Lysine (K) at position 204; and

[0053] Phenylalanine (F) at position 205.

[0054] In another preferred embodiment, the mutant protein is mutated in a core amino acid corresponding to SEQ ID NO.: 1 in the wild-type carbonyl reductase and is selected from the following group of core amino acids associated with enzyme catalytic activity:

[0055] Isoleucine (I) at position 91;

[0056] Isoleucine (I) at position 187;

[0057] Isoleucine (I) at position 188;

[0058] Lysine (K) at position 204; and

[0059] Phenylalanine (F) at position 205.

[0060] In another preferred embodiment, the mutant protein is mutated in a core amino acid corresponding to SEQ ID NO.: 1 in the wild-type carbonyl reductase and is selected from the following group of core amino acids associated with enzyme catalytic activity:

[0061] Isoleucine (I) at position 91;

[0062] Isoleucine (I) at position 187;

[0063] Isoleucine (I) at position 188; and

[0064] Phenylalanine (F) at position 205.

[0065] In another preferred embodiment, the isoleucine (I) at position 91 is mutated to alanine (A), valine (V), leucine (L), or a combination thereof, preferably alanine (A) and / or valine (V).

[0066] In another preferred embodiment, the glutamic acid (E) at position 92 is mutated to valine (V), leucine (L), or a combination thereof, preferably valine (V).

[0067] In another preferred embodiment, the 93rd glutamine (Q) is mutated to glutamic acid (E), aspartic acid (D), or a combination thereof, preferably glutamic acid (E).

[0068] In another preferred embodiment, the leucine (L) at position 142 is mutated to valine (V), alanine (A), glycine (G), or a combination thereof, preferably valine (V).

[0069] In another preferred embodiment, the leucine (L) at position 144 is mutated to alanine (A), valine (V), or a combination thereof, preferably alanine (A).

[0070] In another preferred embodiment, the histidine (H) at position 147 is mutated to phenylalanine (F), leucine (L), or a combination thereof, preferably phenylalanine (F).

[0071] In another preferred embodiment, the 150th tyrosine (Y) is mutated to glycine (G), alanine (A), or a combination thereof, preferably glycine (G).

[0072] In another preferred embodiment, the isoleucine (I) at position 187 is mutated to threonine (T), serine (S), or a combination thereof, preferably serine (S).

[0073] In another preferred embodiment, the isoleucine (I) at position 188 is mutated to leucine (L), valine (V), or a combination thereof, preferably leucine (L).

[0074] In another preferred embodiment, the 191st glutamine (Q) is mutated to asparagine (N), alanine (A), glycine (G), or a combination thereof, preferably asparagine (N) and / or glycine (G), more preferably asparagine (N).

[0075] In another preferred embodiment, the leucine (L) at position 201 is mutated to proline (P), alanine (A), or a combination thereof, preferably proline (P).

[0076] In another preferred embodiment, the lysine (K) at position 204 is mutated to threonine (T), serine (S), aspartic acid (D), or a combination thereof, preferably threonine (T).

[0077] In another preferred embodiment, the phenylalanine (F) at position 205 is mutated to alanine (A), glycine (G), or a combination thereof, preferably alanine (A).

[0078] In another preferred embodiment, the leucine (L) at position 246 is mutated to cysteine ​​(C), serine (S), proline (P), or a combination thereof, preferably cysteine ​​(C).

[0079] In another preferred embodiment, the wild-type carbonyl reductase is RasADH.

[0080] In another preferred embodiment, except for the mutations (such as position 91, 92, 93, 142, 144, 147, 150, 187, 188, 191, 201, 204, 205, and / or 246), the remaining amino acid sequence of the mutant protein is the same or substantially the same as the sequence shown in SEQ ID NO.: 1.

[0081] In another preferred embodiment, the substantially identical amino acids are at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5) different amino acids, wherein the differences include substitution, deletion or addition of amino acids, and the mutant protein still has the catalytic activity of catalyzing the production of cyclopentanone alcohol compounds.

[0082] In another preferred embodiment, the homology with the sequence shown in SEQ ID NO.: 1 is at least 80%, preferably at least 85% or 90%, more preferably at least 95%, and most preferably at least 98% or 99%.

[0083] In another preferred embodiment, the amino acid sequence of the mutant protein is shown in any one of SEQ ID NOs: 6-9.

[0084] In another preferred embodiment, the carbonyl reductase is derived from Ralstonia sp.

[0085] In another preferred embodiment, the catalytic substrate of the carbonyl reductase includes cyclopentanedione compounds.

[0086] In another preferred embodiment, the cyclopentanedione compound is selected from the following group:

[0087] Wherein, R is vinyl, ethynyl, phenyl or

[0088] In another preferred embodiment, the cyclopentanedione compound includes cyclopentanedione and / or ethyl condensate.

[0089] In another preferred embodiment, the mutant protein of the carbonyl reductase catalyzes the reaction of cyclopentanedione compounds to generate cyclopentanone alcohol compounds.

[0090] In another preferred embodiment, the mutant protein of carbonyl reductase catalyzes the following reaction:

[0091] In another preferred embodiment, the reaction has one or more characteristics selected from the following group:

[0092] (i) the pH of the reaction system is 6.0-9.0, preferably 6.5-8, more preferably 6.5-7.5;

[0093] (ii) the reaction temperature is 20-45°C, preferably 25-35°C, more preferably 28-32°C;

[0094] (iii) The reaction time is 4-24 hours, preferably 6-12 hours, more preferably 6-8 hours.

[0095] In another preferred embodiment, the catalytic activity of the mutant protein of the carbonyl reductase in catalyzing the generation of cyclopentanone alcohol from dicarbonyl cyclopentanedione is 100-2000% of that of the wild-type carbonyl reductase (SEQ ID NO.: 1), preferably, 1000%-2000%.

[0096] In another preferred embodiment, the mutant protein of the carbonyl reductase has one or more characteristics selected from the following group:

[0097] (a) compared with the wild-type carbonyl reductase, the ratio of the 2R, 3S chiral product of the cyclopentanone alcohol compound catalyzed is ≥85%, preferably, ≥90%, and more preferably 95-99%;

[0098] (b) compared with the wild-type carbonyl reductase, the yield of the cyclopentanone alcohol compound obtained by catalysis is ≥ 95%, preferably, ≥ 98%;

[0099] (c) compared with the wild-type carbonyl reductase, the space-time yield of the cyclopentanone alcohol compound obtained by catalysis is 0.1-6 g / Lh, preferably, 2-4 g / Lh;

[0100] (d) compared with the wild-type carbonyl reductase, the ee value of the cyclopentanone alcohol compound obtained by catalysis is ≥95%, preferably, >98%;

[0101] (e) Compared with the wild-type carbonyl reductase, the content of cyclopentanone alcohol compound obtained by catalysis is greater than 99%, and no cyclopentanediol product is generated.

[0102] The second aspect of the present invention provides a polynucleotide, which encodes the mutant protein described in the first aspect of the present invention.

[0103] In another preferred embodiment, the polynucleotide is selected from the following group:

[0104] (a) encoding a polypeptide as shown in any one of SEQ ID NOs.: 6-9;

[0105] (b) a polynucleotide whose sequence is shown in any one of SEQ ID NOs.: 2-5;

[0106] (c) a polynucleotide having a nucleotide sequence homology of ≥95% (preferably ≥98%) to the sequence shown in any one of SEQ ID NOs.: 2-5, and encoding a polypeptide shown in any one of SEQ ID NOs.: 1, 6-9;

[0107] (d) A polynucleotide complementary to the polynucleotide described in any one of (a) to (c).

[0108] In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the following groups on the flank of the ORF of the mutant protein of carbonyl reductase: a signal peptide, a secretory peptide, a tag sequence (such as 6His), or a combination thereof.

[0109] In another preferred embodiment, the polynucleotide is selected from the following group: a DNA sequence, an RNA sequence, or a combination thereof.

[0110] The third aspect of the present invention provides a vector, wherein the vector contains the polynucleotide described in the second aspect of the present invention.

[0111] In another preferred embodiment, the vector includes an expression vector, a shuttle vector, and an integration vector.

[0112] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the third aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0113] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0114] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0115] The fifth aspect of the present invention provides a method for producing a mutant protein of the carbonyl reductase according to the first aspect of the present invention, comprising the steps of:

[0116] Cultivating the host cell according to the fourth aspect of the present invention under conditions suitable for expression, thereby expressing a mutant protein of carbonyl reductase; and / or

[0117] A mutant protein of the carbonyl reductase is isolated.

[0118] The sixth aspect of the present invention provides an enzyme preparation, which comprises a mutant protein of the carbonyl reductase described in the first aspect of the present invention.

[0119] In another preferred embodiment, the enzyme preparation includes an injection and / or a lyophilized preparation.

[0120] A seventh aspect of the present invention provides a method for preparing a cyclopentanone alcohol compound, comprising the steps of:

[0121] (i) contacting the mutant protein of the carbonyl reductase described in the first aspect of the present invention with a reaction substrate to carry out a catalytic reaction, thereby obtaining the cyclopentanone alcohol compound; and

[0122] (ii) optionally, isolating and purifying the cyclopentanone alcohol compound.

[0123] In another preferred embodiment, the cyclopentanone alcohol compound is a chiral cyclopentanone alcohol compound.

[0124] In another preferred embodiment, in step (i), in the presence of a cofactor, the mutant protein of the carbonyl reductase acts together with a coenzyme regeneration system.

[0125] In another preferred embodiment, the coenzyme regeneration system is selected from the following group: glucose dehydrogenase / glucose, formate dehydrogenase / sodium formate, or a combination thereof.

[0126] In another preferred embodiment, the cofactor includes: nicotinamide adenine dinucleotide phosphate in an oxidized state (such as NADP+), and / or nicotinamide adenine dinucleotide phosphate in a reduced state (such as NADPH).

[0127] In another preferred embodiment, the reaction substrate is a cyclopentanedione compound.

[0128] In another preferred embodiment, in step (i), the catalytic reaction time is 2-20 h, preferably 3-10 h, more preferably 6-8 h.

[0129] In another preferred embodiment, in step (i), the temperature of the catalytic reaction is 20-40°C, preferably 25-35°C, more preferably 28-32°C.

[0130] The eighth aspect of the present invention provides a use of the mutant protein described in the first aspect of the present invention, wherein the mutant protein is used to catalyze cyclopentanedione compounds to generate cyclopentanone alcohol compounds, or is used to prepare a catalytic preparation for catalyzing cyclopentanedione compounds to generate cyclopentanone alcohol compounds.

[0131] The ninth aspect of the present invention provides a use of the mutant protein described in the first aspect of the present invention or the host cell described in the fourth aspect of the present invention for preparing a cyclopentanone alcohol compound.

[0132] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0133] Figure 1 The results after purification of RasADH mutant protein are shown.

[0134] Among them, M represents Marker, and 1 represents the representative mutant protein. DETAILED DESCRIPTION

[0135] Through extensive and in-depth research, the inventors unexpectedly screened out the key amino acid sites that can significantly improve the catalytic activity of the mutant protein of carbonyl reductase through a large number of screenings. The present invention finds that after the key sites in the wild-type carbonyl reductase of the present invention are modified, the catalytic activity of carbonyl reductase, the ratio of 2R and 3S chiral products, and the yield, space-time yield, ee, and de values ​​of cyclic pentanol compounds can be significantly improved. On this basis, the inventors have completed the present invention.

[0136] the term

[0137] As used herein, the term "AxxB" means that the amino acid A at position xx is changed to amino acid B, for example, "L87I" means that the amino acid L at position 87 is mutated to I, and so on.

[0138] Mutant protein and its encoding nucleic acid of the present invention

[0139] As used herein, the terms "mutant protein", "mutant protein of the present invention", "carbonyl reductase mutant protein of the present invention", and "carbonyl reductase mutant of the present invention" are used interchangeably, and all refer to non-naturally occurring carbonyl reductase mutant proteins, and the mutant protein is an artificially modified protein based on the protein shown in SEQ ID NO.: 1, wherein the mutant protein contains core amino acids related to the catalytic activity of the enzyme, and at least one of the core amino acids is artificially modified; and the mutant protein of the present invention has the enzyme activity of catalyzing cyclopentanedione compounds to form cyclopentanone alcohol compounds.

[0140] The term "core amino acids" refers to a sequence based on SEQ ID NO.: 1, and having at least 80%, such as 84%, 85%, 90%, 92%, 95%, 98% homology to SEQ ID NO.: 1, wherein the corresponding positions are specific amino acids described herein, such as based on the sequence shown in SEQ ID NO.: 1, the core amino acids are:

[0141] Isoleucine (I) at position 91; and / or

[0142] Glutamic acid (E) at position 92; and / or

[0143] Glutamine (Q) at position 93; and / or

[0144] Leucine (L) at position 142; and / or

[0145] Leucine (L) at position 144; and / or

[0146] Histidine (H) at position 147; and / or

[0147] Tyrosine (Y) at position 150; and / or

[0148] Isoleucine (I) at position 187; and / or

[0149] Isoleucine (I) at position 188; and / or

[0150] Glutamine (Q) at position 191; and / or

[0151] Leucine (L) at position 201; and / or

[0152] Lysine (K) at position 204; and / or

[0153] Phenylalanine (F) at position 205; and / or

[0154] Leucine (L) at position 246.

[0155] The mutant protein obtained by mutating the core amino acids has the enzyme activity of catalyzing cyclopentanedione compounds to form cyclopentanone alcohol compounds.

[0156] Preferably, in the present invention, the core amino acids of the present invention are mutated as shown in Table 1.

[0157] Table 1

[0158] Location wild type The most preferred mutant Mutation site 1 (amino acid 91) I A or V Mutation site 2 (amino acid 92) E V Mutation site 3 (amino acid 93) Q E Mutation site 4 (amino acid 142) L V Mutation site 5 (amino acid 144) L A Mutation site 6 (amino acid 147) H F Mutation site 7 (amino acid 150) Y G Mutation site 8 (amino acid 187) I S Mutation site 9 (amino acid 188) I L Mutation site 10 (amino acid 191) Q N Mutation site 11 (amino acid 201) L P Mutation site 12 (amino acid 204) K T Mutation site 13 (amino acid 205) F A Mutation site 14 (amino acid 246) L C

[0159] It should be understood that the amino acid numbering in the mutant protein of the present invention is based on SEQ ID NO.: 1. When the homology between a specific mutant protein and the sequence shown in SEQ ID NO.: 1 reaches 80% or more, the amino acid numbering of the mutant protein may be misplaced relative to the amino acid numbering of SEQ ID NO.: 1), such as misplacement of 1-5 positions to the N-terminus or C-terminus of the amino acid. Using conventional sequence alignment techniques in the art, those skilled in the art can generally understand that such misplacement is within a reasonable range, and mutant proteins with a homology of 80% (such as 90%, 95%, 98%) and the same or similar catalytic activity for producing cyclopentanedione compounds should not be excluded from the scope of the mutant protein of the present invention due to the misplacement of amino acid numbering.

[0160] Mutants of the present invention are synthetic proteins or recombinant proteins, i.e., they can be the product of chemical synthesis, or they can be produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant technology. Depending on the host used in the recombinant production scheme, the mutagen of the present invention can be glycosylated, or it can be non-glycosylated. The mutagen of the present invention may also include or not include an initial methionine residue.

[0161] The present invention also includes fragments, derivatives and analogs of the mutant protein. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity of the mutant protein.

[0162] Mutant protein fragments, derivatives or analogs of the present invention can be (i) substituted mutant proteins with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) mutant proteins with substituent groups in one or more amino acid residues, or (iii) mutant proteins formed by fusion of mature mutant proteins with another compound (such as a compound that prolongs the half-life of the mutant protein, such as polyethylene glycol), or (iv) mutant proteins formed by fusion of additional amino acid sequences to the mutant protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the mutant protein, or a fusion protein formed with an antigen IgG fragment). According to the teachings of this article, these fragments, derivatives and analogs belong to the well-known scope of those skilled in the art. In the present invention, the conservatively substituted amino acids are preferably produced by amino acid substitution according to Table I.

[0163] Table I

[0164] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0165] The active mutant protein of the invention has the enzyme activity of catalyzing cyclopentanedione compounds to form cyclopentanone alcohol compounds.

[0166] Preferably, the mutant protein is as shown in SEQ ID NO.: 6, 7, 8, 9. It should be understood that the mutant protein of the present invention generally has a higher homology (identity) than the sequence shown in SEQ ID NO.: 6, 7, 8, 9. Preferably, the homology of the mutant protein to the sequence shown in SEQ ID NO.: 6, 7, 8, 9 is at least 80%, preferably at least 85%-90%, more preferably at least 95%, most preferably at least 98%, and most preferably ≥ 99%. In addition, the mutant protein of the present invention can also be modified. Modifications (usually without changing the primary structure) include: chemical derivatization forms of the mutant protein in vivo or in vitro, such as acetylation or carboxylation. Modifications also include glycosylation, such as those mutant proteins produced by glycosylation modification during the synthesis and processing of the mutant protein or in further processing steps. This modification can be achieved by exposing the mutant protein to a glycosylation enzyme (such as a mammalian glycosylase or deglycosylase). Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine), and also include mutant proteins that have been modified to improve their anti-proteolytic properties or optimize their solubility properties.

[0167] The term "polynucleotide encoding a mutant protein" may include a polynucleotide encoding a mutant protein of the present invention, or may include a polynucleotide further including additional coding and / or non-coding sequences.

[0168] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides or mutant proteins having the same amino acid sequence as the present invention. These nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the mutant protein encoded by it.

[0169] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions (or stringent conditions). In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably 95%.

[0170] The mutant proteins and polynucleotides of the present invention are preferably provided in an isolated form, and more preferably, purified to homogeneity.

[0171] The full-length sequence of the polynucleotide of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify and obtain the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0172] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0173] In addition, artificial synthesis methods can also be used to synthesize related sequences, especially when the fragment length is shorter. Usually, a long fragment of sequence can be obtained by synthesizing multiple small fragments first and then connecting them.

[0174] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0175] The method of amplifying DNA / RNA using PCR technology is preferably used to obtain the polynucleotides of the present invention. In particular, when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA terminal rapid amplification method) can be preferably used. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.

[0176] The carbonyl reductase of the invention has a broad substrate spectrum, has high reaction activity and stereoselectivity to a series of cyclopentanedione substrates, and has high stereoselectivity to ethyl condensates in particular.

[0177] In a preferred embodiment of the present invention, the preparation method of the recombinant carbonyl reductase of the present invention is: culturing the recombinant expression transformant as described above to obtain the recombinant expression carbonyl reductase. The culture medium used for culturing the recombinant expression transformant is any culture medium in the art that can grow the transformant and produce the recombinant carbonyl reductase of the present invention. There is no special limitation on the culture method and culture conditions, and they can be appropriately selected according to the conventional knowledge in the art according to factors such as the host cell type and the culture method, as long as the transformant can grow and produce the carbonyl reductase.

[0178] In a preferred embodiment of the present invention, the preparation method of the carbonyl reductase mutant (RasADH mutant) of the present invention is as follows: Escherichia coli is used as the expression host.

[0179] Specifically, the preparation method comprises the following steps: (1) constructing the gene of the corresponding mutation site of RasADH into the pET21a expression vector to obtain a recombinant plasmid carrying the target enzyme gene. (2) transferring the recombinant plasmid into a host bacterial cell (preferably Escherichia coli BL21 (DE3)) to obtain a corresponding engineered strain. (3) inoculating the engineered strain into LB medium and culturing at 25°C for 16 hours. (4) collecting the bacterial cells by centrifugation, breaking the bacteria and retaining the supernatant.

[0180] The present invention also provides a method for converting cyclopentanedione using RasADH and mutant recombinant bacteria as biocatalysts. Specifically, a reaction system is constructed by combining a cyclopentanedione substrate with recombinant bacteria or a broken bacterial solution and a pure enzyme, and cofactor NADPH is regenerated using glucose dehydrogenase, glucose or formate dehydrogenase, and sodium formate. The reaction system is a buffer solution with a pH of 6.0-9.0, and the reaction temperature is 20° C. to 45° C.

[0181] After the asymmetric reduction reaction is completed, the reaction solution is extracted with an equal amount of a conventional water-insoluble organic solvent in the art, such as ethyl acetate, butyl acetate, toluene, dichloromethane, chloroform, isopropyl ether, methyl tert-butyl ether, etc., and the extraction is repeated twice, and the extracts are combined, and anhydrous sodium sulfate is added to dry overnight. The solvent is removed by rotary evaporation to obtain an optically pure chiral product, which can be further purified by conventional methods, such as reduced pressure distillation, recrystallization, etc. to obtain a highly chemically pure and optically pure product.

[0182] Wild-type carbonyl reductase

[0183] As used herein, "wild-type carbonyl reductase" refers to a naturally occurring, unmodified carbonyl reductase, whose nucleotide sequence can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and whose amino acid sequence can be derived from the nucleotide sequence. The amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID NO.: 1.

[0184] The sequence information of the wild protein and mutant protein of the present invention mentioned above is shown in Table 2 (see Examples).

[0185] Expression vector

[0186] The present invention also relates to a vector comprising the polynucleotide of the present invention, a host cell produced by genetic engineering using the vector of the present invention or the mutant protein coding sequence of the present invention, and a method for producing the polypeptide of the present invention by recombinant technology.

[0187] The polynucleotide sequences of the present invention can be used to express or produce recombinant mutant proteins by conventional recombinant DNA techniques. Generally, the following steps are involved:

[0188] (1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a mutant protein of the present invention, or a recombinant expression vector containing the polynucleotide;

[0189] (2) Host cells cultured in a suitable culture medium;

[0190] (3) Isolate and purify proteins from culture medium or cells.

[0191] In the present invention, the polynucleotide sequence encoding the mutant protein can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. As long as they can replicate and be stable in the host, any plasmid and vector can be used. An important feature of an expression vector is that it usually contains a replication origin, a promoter, a marker gene and a translation control element.

[0192] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the mutant protein of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to guide mRNA synthesis. Representative examples of these promoters include: lac or trp promoters of Escherichia coli; λ phage PL promoter; eukaryotic promoters include CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, retroviral LTRs and other known promoters that can control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0193] In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0194] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0195] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast.

[0196] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include the SV40 enhancer of 100 to 270 base pairs on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0197] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0198] Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be carried out using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0199] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0200] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0201] The promoter of the carbonyl reductase gene of the present invention can be changed by replacement, insertion or deletion of one or more nucleotides, but these changes have no negative impact on the function of the promoter. Moreover, the expression level of the carbonyl reductase can be increased by changing the sequence of the promoter or using a more effective promoter from a different source to replace it completely.

[0202] The recombinant expression vector of the present invention can be constructed by cloning the carbonyl reductase gene into various vectors by conventional methods in the art. The expression vector preferably includes various conventional vectors in the art, such as commercially available plasmids, cosmids, phages or virus vectors, etc. The vector is preferably a pET28a plasmid for Escherichia coli.

[0203] The main advantages of the present invention include:

[0204] (i) After a large number of screening and transformation, the present invention discovered the catalytic active site of carbonyl reductase for the first time. After the relevant site was transformed, the catalytic activity of carbonyl reductase, the ratio of 2R and 3S chiral products, the yield (equivalent to the output), the space-time yield, and the ee value of cyclic pentanol compounds can be significantly improved.

[0205] (ii) The present invention is the first to discover a carbonyl reductase (RasADH mutant protein) that can improve the activity and stereoselectivity towards ethyl condensates, especially cyclopentanedione compounds.

[0206] (iii) The carbonyl reductase of the present invention has a broad substrate spectrum and has high reactivity and stereoselectivity for a series of cyclopentanedione compound substrates, especially high stereoselectivity for ethyl condensates.

[0207] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0208] Unless otherwise specified, the reagents and materials in the examples of the present invention are all commercially available products.

[0209] Example 1 Preparation of RasADH carbonyl reductase recombinant expression plasmid and recombinant expression transformant

[0210] The sequence of SEQ ID No.1 was fully synthesized and connected to the pET 28a empty plasmid, and then double-digested with restriction endonucleases BamHI and HindIII overnight, and then purified by agarose gel electrophoresis and recovered by a DNA kit. The recovered enzyme-digested target fragment and the empty vector were connected at 4°C for 12 hours under the action of T4 DNA ligase to obtain the recombinant plasmid pET28a-RasADH, which was further transformed into E.coli BL21 (DE3), and the positive clones were picked to obtain the recombinant expression transformant E.coli BL21 (DE3) / pET28a-RasADH.

[0211] Example 2 Construction of carbonyl reductase RasADH mutant

[0212] Construction of a mutation library of carbonyl reductase RasADH: Based on the crystal structure of RasADH, non-conserved residues and substrate channel amino acids in its substrate binding pocket were selected and grouped for combinatorial saturation mutation. Mutation primers were designed using degenerate codons NDT. pET28a-RasADH was used as a template and PCR was performed using high-fidelity polymerase PrimeSTAR. The PCR reaction conditions were as follows: 0.5-20 ng of template, 10 μL 2× PrimeSTAR (Premix), 0.4 μL of each pair of mutation primers (10 μM), and sterile distilled water were added to a PCR reaction system with a total volume of 20 μL. PCR reaction procedure: (1) denaturation at 98°C for 10 sec, (2) annealing at 55°C for 5 sec, (3) extension at 72°C for 60 sec, steps (1) to (3) were performed for a total of 30 cycles, and the product was stored at 4°C. After verification by agarose gel electrophoresis, the PCR product was digested with the restriction enzyme DpnI at 37°C for 1 h. The digestion product was transferred into E.coli BL21 (DE3) competent cells and spread on a plate containing kanamycin, and then placed in a 37°C incubator for about 12 hours. The obtained monoclonal colonies were picked into 96-well deep-well plates for culture, and the expressed proteins were screened for high-throughput activity, the mutants with higher activity were purified and characterized, and the corresponding genes were sequenced.

[0213] Among them, the protein sequence of the representative mutant 2 is shown in SEQ ID NO.:6, and its nucleotide sequence is shown in SEQ ID NO.:2.

[0214] The protein sequence of mutant 3 is shown in SEQ ID NO.:7, and the nucleotide sequence thereof is shown in SEQ ID NO.:3.

[0215] The protein sequence of mutant 5 is shown in SEQ ID NO.:8, and the nucleotide sequence thereof is shown in SEQ ID NO.:4.

[0216] The protein sequence of mutant 6 is shown in SEQ ID NO.:9, and the nucleotide sequence thereof is shown in SEQ ID NO.:5.

[0217] Table 2 provides a list of carbonyl reductase RasADH mutants of the present invention having relevant activities and specific sequences with higher proportions (%) of 2R,3S chiral products.

[0218] In the following table, the sequence numbers refer to a series of sequences following Table 1. In the activity column, one plus sign "+" indicates that the mutant protein has a 1 to 5-fold increase in specific activity over the protein consisting of the amino acid sequence shown in SEQ ID No.1 (wild-type carboxyl reductase) in the sequence listing; two plus signs "++" indicate that the mutant protein has a 5 to 10-fold increase in specific activity over the protein consisting of the amino acid sequence shown in SEQ ID No.1; and three plus signs "+++" indicate that the mutant protein has a more than 10-fold increase in specific activity over the protein consisting of the amino acid sequence shown in SEQ ID No.1.

[0219] Table 2

[0220]

[0221]

[0222] Example 3: Inducible expression and purification of carbonyl reductase RasADH mutant

[0223] Prepare 50 mL of seed solution, the culture medium is LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L), pick a single colony of genetically engineered bacteria with an inoculation loop and inoculate it into the culture medium, and culture it at 37°C, 200 rpm overnight. Transfer the overnight cultured seed solution to the fermentation medium (LB medium) at a 1% inoculum amount, and culture it at 37°C, 200 rpm until A 600 0.6-1.0, add 0.25mM IPTG, and place at 37℃, 200rpm for induction for 10-12h. Collect the cells by centrifugation at 4℃, 6000rpm, wash twice with sodium phosphate buffer (50mM, pH 7.5), and crush with a high-pressure homogenizer, centrifuge at 13000rpm to retain the supernatant, and then use metal affinity chromatography (nickel column) to purify and recover the target protein. After the target protein is dialyzed to remove imidazole, the pure enzyme solution of RasADH mutant is obtained. The SDS-PAGE electrophoresis pattern shows that the purified protein band is single, such as Figure 1 shown.

[0224] The results showed that the method of this example could obtain a relatively pure protein mutant with a molecular weight of 27 KD and a purity of >95%.

[0225] Example 4 Substrate spectrum of RasADH mutants

[0226] Construct the reaction to detect substrate conversion and product stereoselectivity: 1 mL, pH 7.0-7.5, 100 mM sodium phosphate buffer, 0.1 mM NADP +and 10mM of different cyclopentanedione substrates, 30mM glucose, 0.2U glucose dehydrogenase, add appropriate amount of pure enzyme solution, react overnight and then detect by HPLC. Product determination method: Aglient high performance liquid chromatograph, chromatographic column: Daicel OD-H (3μm, 4.6×150mm), mobile phase: n-hexane-isopropanol (V / V=95:5), detection wavelength: 254nm, column temperature: 30℃, flow rate: 0.8mL / min.

[0227] The results showed that the RasADH mutant could efficiently convert cyclopentanedione compounds (such as ethyl condensates) into 2R,3S-cyclopentanone alcohol, and the ee and de values ​​of the conversion products were both >98%.

[0228] Example 5 Method for converting cyclopentanedione by recombinant bacteria of RasADH mutant

[0229] The RasADH mutant of the present invention was induced to express according to the method of Example 2, the bacterial cells were collected by centrifugation (8000 rpm), and the bacterial cells were washed twice with potassium phosphate buffer (pH 7.0, 100 mM), and the bacterial cells were used as biocatalysts.

[0230] (1) Take the bacterial cell and resuspend it in 10mL potassium phosphate buffer (pH 7.0, 100mM) to a bacterial cell concentration of 20g / L. Add substrate cyclopentanedione (10% v / v ethanol or DMF) to a final concentration of 10g / L, and auxiliary substrate glucose 30g / L. React on a shaker at 30℃ and 200r / min for 8h. After the reaction, extract the reaction solution several times with ethyl acetate, combine the organic phases, dry them over anhydrous sodium sulfate, and remove the solvent under reduced pressure. HPLC detection shows that the yield (equivalent to the output) is 95%, the time-space yield is 2.40g / (Lh), and the ee and de values ​​are both >98%.

[0231] (2) The bacterial cell was resuspended in 10 mL potassium phosphate buffer (pH 7.0, 100 mM) to a bacterial cell concentration of 50 g / L. The substrate cyclopentanedione (10% v / v ethanol or DMF) was added to a final concentration of 40 g / L, and the auxiliary substrate glucose was 60 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min. The reaction was stopped after 12 hours. After the reaction was completed, the reaction solution was extracted several times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the time-space yield was 3.96 g / (Lh), and the ee and de values ​​were both >98%.

[0232] (3) The bacterial cell was resuspended in 10 mL potassium phosphate buffer (pH 7.0, 100 mM) to a bacterial cell concentration of 50 g / L. The substrate ethyl condensate (10% v / v ethanol or DMF) was added to a final concentration of 20 g / L, and the auxiliary substrate glucose was added at 40 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min for 12 hours. After the reaction was completed, the reaction solution was extracted several times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the space-time yield was 2.40 g / (Lh), and the ee and de values ​​were both >98%.

[0233] (4) The bacterial cells were resuspended in 10 mL potassium phosphate buffer (pH 7.0, 100 mM) to a bacterial concentration of 50 g / L. The substrate ethyl condensate (10% v / v ethanol or DMF) was added to a final concentration of 40 g / L, and the auxiliary substrate glucose was 60 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min for 12 hours. After the reaction was completed, the reaction solution was extracted several times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the time-space yield was 3.96 g / (Lh), and the ee and de values ​​were both >98%.

[0234] (5) The bacterial cells were resuspended in 1L potassium phosphate buffer (pH 7.0, 100mM) to a bacterial concentration of 50g / L. The substrate ethyl condensate (10% v / v ethanol or DMF) was added to a final concentration of 40g / L, and the auxiliary substrate glucose was 60g / L. The reaction was carried out on a shaker at 30°C and 200r / min. The reaction was stopped after 12h. After the reaction was completed, the reaction solution was extracted with ethyl acetate several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the time-space yield was 3.96g / (Lh), and the ee and de values ​​were both >98%.

[0235] (6) The bacterial cell was resuspended in 10 mL potassium phosphate buffer (pH 7.0, 100 mM) to a bacterial cell concentration of 50 g / L. The substrate cyclopentanedione (R substituent is vinyl) (10% v / v ethanol or DMF) was added to a final concentration of 40 g / L, and the auxiliary substrate glucose was 60 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min. The reaction was stopped after 12 h. After the reaction was completed, the reaction solution was extracted several times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the space-time yield was 3.96 g / (Lh), and the ee and de values ​​were both >98%.

[0236] (7) The bacterial cell was resuspended in 10 mL potassium phosphate buffer (pH 7.0, 100 mM) to a bacterial cell concentration of 50 g / L. The substrate cyclopentanedione (R substituent is ethynyl) (10% v / v ethanol or DMF) was added to a final concentration of 40 g / L, and the auxiliary substrate glucose was 60 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min. The reaction was stopped after 12 h. After the reaction was completed, the reaction solution was extracted several times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the space-time yield was 3.96 g / (Lh), and the ee and de values ​​were both >98%.

[0237] (8) The bacterial cell was resuspended in 10 mL potassium phosphate buffer (pH 7.0, 100 mM) to a bacterial cell concentration of 50 g / L. The substrate cyclopentanedione (R substituent is phenyl) (10% v / v ethanol or DMF) was added to a final concentration of 40 g / L, and the auxiliary substrate glucose was 60 g / L. The reaction was carried out on a shaker at 30°C and 200 r / min. The reaction was stopped after 12 h. After the reaction was completed, the reaction solution was extracted several times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. HPLC detection showed that the yield was 95%, the time-space yield was 3.96 g / (Lh), and the ee and de values ​​were both >98%.

[0238] See Table 3 for specific results.

[0239] Table 3

[0240]

[0241]

[0242] The results show that compared with the wild-type carbonyl reductase, the mutant protein of the carbonyl reductase of the present invention can significantly improve the yield, space-time yield, ee and de value of 2R, 3S-cyclopentanone alcohol.

[0243] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A carbonyl reductase mutant protein, characterized in that The mutant protein is a non-natural protein, and the mutant protein has catalytic activity of catalyzing cyclopentanedione compounds to generate cyclopentanone alcohol compounds, and the mutant protein is mutated in the core amino acid related to the enzyme catalytic activity corresponding to SEQ ID NO.: 1 of the wild-type carbonyl reductase selected from the following group: Isoleucine (I) at position 91; Isoleucine (I) at position 187; Isoleucine (I) at position 188; and Phenylalanine (F) at position 205; The amino acid sequence of the mutant protein is shown in SEQ ID NO.: 7-9.

2. A polynucleotide, characterized in that The polynucleotide encodes the mutant protein according to claim 1.

3. A carrier, characterized in that The vector contains the polynucleotide according to claim 2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3, or the polynucleotide according to claim 2 is integrated into its genome, and the host cell does not include a plant cell.

5. A method for producing a mutant protein of the carbonyl reductase according to claim 1, characterized in that: Includes steps: Cultivating the host cell according to claim 4 under conditions suitable for expression, thereby expressing a mutant protein of carbonyl reductase; and / or A mutant protein of the carbonyl reductase is isolated.

6. An enzyme preparation, characterized in that The enzyme preparation comprises the mutant protein of the carbonyl reductase according to claim 1.

7. A method for preparing a cyclopentanone alcohol compound, characterized in that: Includes steps: (i) contacting the mutant protein of the carbonyl reductase according to claim 1 with a reaction substrate to carry out a catalytic reaction, thereby obtaining the cyclopentanone alcohol compound; and (ii) optionally, isolating and purifying the cyclopentanone alcohol compound; The reaction substrate is a cyclopentanedione compound; The cyclopentanedione compound is selected from the following group: Wherein, R is vinyl, ethynyl, phenyl or 8. A use of the mutant protein according to claim 1, characterized in that: The mutant protein is used to catalyze cyclopentanedione compounds to generate cyclopentanone alcohol compounds, or is used to prepare a catalytic preparation for catalyzing cyclopentanedione compounds to generate cyclopentanone alcohol compounds; The cyclopentanedione compound is selected from the following group: Wherein, R is vinyl, ethynyl, phenyl or 9. Use of the mutant protein according to claim 1 or the host cell according to claim 4, characterized in that: Used to prepare cyclopentanone alcohol compounds.

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