A ketoreductase mutant

Abstract

The present invention relates to the field of applied microbiology and enzyme engineering, and specifically includes a ketoreductase mutant. The ketoreductase mutant is obtained by mutating SEQ ID NO:1 as a template, and the mutation sites are V14E+I42L+A97H+G170R+A242L+I262A. The obtained mutant can use ketone compounds as raw materials to efficiently produce chiral alcohols through asymmetric reduction, and has high stereoselectivity, which is conducive to industrial production.

Description

Technical Field

[0001] The present invention relates to the fields of applied microbiology and enzyme engineering, and specifically includes a recombinant ketoreductase and its mutants. Background Art

[0002] Ketoreductase is a versatile catalyst that enantioselectively reduces aldehydes or ketones to the corresponding alcohols. (R)-specific ketoreductase and (S)-specific ketoreductase have different characteristics, and these catalysts are frequently used in the synthesis of optically active alcohols. In the reaction catalyzed by ketoreductase, the participation of cofactors is required, including reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), oxidized nicotinamide adenine dinucleotide (NAD+), and oxidized nicotinamide adenine dinucleotide phosphate (NADP+).

[0003] (R)-Ethyl 4-chloro-3-hydroxybutanoate ((R)-CHBE), as an important organic intermediate, has the following structure:

[0004]

[0005] It can be used in the synthesis of many pharmaceuticals, such as L-carnitine and R-γ-amino-β-hydroxybutyric acid (GABOB), etc.

[0006] Currently, there are mainly two methods for the asymmetric reduction of (R)-CHBE by ketoreductase, namely the chemical method and the biological method. The chemical method uses metals such as rhodium and ruthenium as catalysts, and requires a certain hydrogen pressure for asymmetric reduction, and the optical purity of the product is relatively low.

[0007] The biological method has the advantages of mild reaction conditions, strong specificity, and high conversion rate, and thus has received extensive attention. Japanese scholars Kataoka et al. conducted relevant research on the ketoreductase derived from Sporobolomyces salmonicolor, heterologously expressed the enzyme, and finally the concentration of ethyl 4-chloroacetoacetate catalyzed was as high as 0.3 kg / L, and the chiral purity was between 91% and 93%. Chinese patent application CN103160547A used the alcohol dehydrogenase derived from Candida albicans to asymmetrically reduce ethyl 4-chloroacetoacetate, with resting cells as the catalyst and NADH as the cofactor, to catalyze the preparation of (R)-CHBE, and the concentration of the catalytic substrate was 25 - 50 g / L, and the substrate conversion rate was relatively low. Summary of the Invention

[0008] To solve the problems existing in the prior art, the present invention provides a ketoreductase mutant with improved enzyme activity and its application.

[0009] According to one aspect of the present invention, a mutant of ketoreductase is provided. The ketoreductase mutant is:

[0010] A mutant with the amino acid sequence shown in MKALQYTEIGSVPVVVDVPTPAPGPGEILLKVTAAGLCHSDIFVMDMPAEQYIYGLPLTLGHEGVGRVAELGAGVTGFETGDAVAVYGPWGCGACHACARGRENYCTRAAELGITPPGLGSPGSMAEYMIVDSARHLVPIGDLDPVAAVPLTDAGLTPYHAISRVLPLLGPGSTAVVIGVGGLGHVGIQILRAVSAARVIAVDLDDDRLALAREVGADAAVKSGAGAADAIRELTGGEGATAVFDFVGAQSTIDTAQQVVAIDGHISVVGIHAGAHAKVGFFMIPFGASVVTPYWGTRSELMDVVDLARAGRLDIHTETFTLDEGPTAYRRLREGSIRGRGVVVPG (SEQ ID NO:1), and the mutation sites include T67. Further, the mutation of the ketoreductase includes an amino acid sequence having more than 80% identity with SEQ ID NO:1, and the identical sequence contains the mutation site T67; preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 85% identity with SEQ ID NO:1, and the identical sequence contains the mutation site T67; more preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 90% identity with SEQ ID NO:1, and the identical sequence contains the mutation site T67; even more preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 95% identity with SEQ ID NO:1, and the identical sequence contains the mutation site T67; most preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 98% identity with SEQ ID NO:1, and the identical sequence contains the mutation site T67.

[0011] The mutant obtained by mutation of the present invention can use a ketone compound as a raw material and efficiently produce chiral alcohol through stereoselective reduction, and is suitable for popularization in the industrial production of chiral alcohol.

[0012] Furthermore, the mutated site(s) also at least include any one of the following sites or a combination of two or more sites: V14, I42, A97, G170, A242, I262, and F286; or the amino acid sequence of the ketoreductase mutant has the mutated site(s) in the mutated amino acid sequence and has an amino acid sequence with an identity of more than 95% to the mutated amino acid sequence. Further, the mutation of the ketoreductase includes an amino acid sequence with an identity of more than 80% to Y (for convenience of description, the mutant with the mutated site including T67 and including any one of the following sites or a combination of two or more sites: V14, I42, A97, G170, A242, I262, F286 is defined as Y), and the identity sequence contains the mutated site T67 and a composition of one or two or more mutated sites among V14, I42, A97, G170, A242, I262, F286; preferably, the mutation of the ketoreductase includes an amino acid sequence with an identity of more than 85% to Y, and the identity sequence contains the mutated site T67 and a composition of one or two or more mutated sites among V14, I42, A97, G170, A242, I262, F286; more preferably, the mutation of the ketoreductase includes an amino acid sequence with an identity of more than 90% to Y, and the identity sequence contains the mutated site T67 and a composition of one or two or more mutated sites among V14, I42, A97, G170, A242, I262, F286; more preferably still, the mutation of the ketoreductase includes an amino acid sequence with an identity of more than 95% to Y, and the identity sequence contains the mutated site T67 and a composition of one or two or more mutated sites among V14, I42, A97, G170, A242, I262, F286; most preferably, the mutation of the ketoreductase includes an amino acid sequence with an identity of more than 98% to Y, and the identity sequence contains the mutated site T67 and a composition of one or two or more mutated sites among V14, I42, A97, G170, A242, I262, F286.

[0013] Preferably, the mutated sites further include at least one or a combination of two or more of the following mutations: V14E, I42L, A97H, G170R, A242L, I262A, and F286S. Further preferably, for the convenience of description, the mutated site includes T67, and a mutant including any one of the following sites or a combination of two or more sites: V14E, I42L, A97H, G170R, A242L, I262A, and F286S is defined as Y'. Preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 80% identity with Y', and the identical sequence contains the mutated site T67, and a composition of one or two or more mutated sites among V14E, I42L, A97H, G170R, A242L, I262A, and F286S; preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 85% identity with Y', and the identical sequence contains the mutated site T67, and a composition of one or two or more mutated sites among V14E, I42L, A97H, G170R, A242L, I262A, and F286S; preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 90% identity with Y', and the identical sequence contains the mutated site T67, and a composition of one or two or more mutated sites among V14E, I42L, A97H, G170R, A242L, I262A, and F286S; more preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 95% identity with Y', and the identical sequence contains the mutated site T67, and a composition of one or two or more mutated sites among V14E, I42L, A97H, G170R, A242L, I262A, and F286S; most preferably, the mutation of the ketoreductase includes an amino acid sequence having more than 98% identity with Y', and the identical sequence contains the mutated site T67, and a composition of one or two or more mutated sites among V14E, I42L, A97H, G170R, A242L, I262A, and F286S.

[0014] Preferably,

[0015] Exemplarily, the mutations of the ketoreductase further include any one of the following site combination mutations: V14E+I42L, V14E+A97H, V14E+G170R, V14E+A242L, V14E+I262A, V14E+F286S, V14E+I42L+A97H, V14E+I42L+G170R, V14E+I42L+A242L, V14E+I42L+I262A, V14E+I42L+F286S, V14E+I42L+A97H+G170R, V14E+I42L+A97H+A242L, V14E+I42L+A97H+I262A, V14E+I42L+A97H+F286S, V14E+I42L+A97H+G170R+A242L, V14E+I42L+A97H+G170R+I262A, V14E+I42L+A97H+G170R+F286S, V14E+I42L+A97H+G170R+A242L+I262A, V14E+I42L+A97H+G170R+A242L+F286S, V14E+I42L+A97H+G170R+A242L+I262A+F286S.

[0016] According to another aspect of the present invention, there is provided a DNA molecule. This DNA molecule encodes the above-mentioned ketoreductase mutant or an identical sequence.

[0017] According to another aspect of the present invention, there is provided a recombinant plasmid. This recombinant plasmid is ligated with the above-mentioned DNA molecule.

[0018] The term "plasmid" used in the present invention includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid, which can be a prokaryotic expression plasmid or a eukaryotic expression plasmid, but preferably a prokaryotic expression plasmid.

[0019] Preferably, the recombinant plasmid is selected from pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18 and pUC-19.

[0020] According to another aspect of the present invention, there is provided a host cell. The host cell contains any one of the above recombinant plasmids.

[0021] Furthermore, the host cell includes prokaryotic cells, yeast or eukaryotic cells.

[0022] The preferred prokaryotic cell is a bacterium, such as a Gram-negative bacterium or a Gram-positive bacterium. More preferably, the prokaryotic cell is Escherichia coli BL21(DE3), BL21 Star(DE3), TunerTM(DE3), RosettaTM 2(DE3), BLR(DE3), NovaBlue(DE3), OrigamiTM(DE3), OrigamiB(DE3).

[0023] According to another aspect of the present invention, there is provided a method for producing chiral alcohols. The method includes the step of using a ketoreductase to catalyze the reduction reaction of prochiral ketone compounds to produce chiral alcohols, and the ketoreductase is any one of the above-mentioned ketoreductase mutants. Since the above-mentioned ketoreductase mutants of the present invention have good activity characteristics, the chiral alcohols prepared by using the ketoreductase mutants of the present invention can increase the reaction rate, increase the substrate concentration, reduce the enzyme dosage, and reduce the difficulty of post-treatment.

[0024] Further, the chiral ketone compound has the following structure of formula (I):

[0025]

[0026] Wherein R' and R" are each independently an alkyl group, an alkaryl group, an alkheteroaryl group, a cycloalkyl group, an aryl group or a heteroaryl group, or R' and R" together with the carbon on the carbonyl group form a heterocyclic group, a carbocyclic group or a heteroaryl group, and the heteroatoms in the heterocyclic group and the heteroaryl group are each independently selected from at least one of nitrogen, oxygen and sulfur, and the aryl group in the alkaryl group, the aryl group in the aryl group, the heteroaryl group in the alkheteroaryl group, the heteroaryl group in the heteroaryl group, the carbocyclic group in the carbocyclic group or the heterocyclic group in the heterocyclic group are each independently unsubstituted or substituted by at least one group selected from a halogen, an alkoxy group, a nitro group or an alkyl group.

[0027] Preferably, R' and R" are each independently a C1-C8 alkyl group, a C5-C10 cycloalkyl group, a C5-C10 aryl group or a C5-C10 heteroaryl group, or R' and R" together with the carbon on the carbonyl group form a C5-C10 heterocyclic group, a C5-C10 carbocyclic group or a C5-C10 heteroaryl group, and the heteroatoms in the C5-C10 heterocyclic group and the C5-C10 heteroaryl group are each independently selected from at least one of nitrogen, oxygen and sulfur, and the aryl group in the C5-C10 aryl group, the heteroaryl group in the C5-C10 heteroaryl group, the carbocyclic group in the C5-C10 carbocyclic group or the heterocyclic group in the C5-C10 heterocyclic group are each independently unsubstituted or substituted by at least one group selected from a halogen, an alkoxy group, a nitro group or an alkyl group.

[0028] Preferably, the structural formula of the ketone compound is as shown in formula (I-1):

[0029]

[0030] Wherein R1 or R2 is selected from hydrogen, a halogen, a C1-C8 alkyl group, a C5-C10 cycloalkyl group, a C5-C10 aryl group or a C5-C10 heteroaryl group, wherein the alkyl group, cycloalkyl group, aryl group or heteroaryl group is each independently unsubstituted or substituted by at least one group selected from a halogen, an alkoxy group, a nitro group or an alkyl group; R3 is selected from hydrogen, a halogen, a C1-C3 alkyl group.

[0031] More preferably, the ketone compound is

[0032] Furthermore, in the reaction system for producing chiral alcohols by reducing ketone compounds using ketoreductase, a coenzyme, a coenzyme regeneration system, and a buffer solution are also included.

[0033] Furthermore, the concentration of the ketone compound in the reaction system is 1 g / L to 200 g / L.

[0034] Furthermore, the pH value of the reaction system is 5.0 to 9.0, and the reaction temperature of the reaction system is 4 to 60 °C.

[0035] Furthermore, the coenzyme is NADH, NADPH, or NAD⁺.

[0036] Furthermore, the coenzyme regeneration system is selected from, but not limited to, the following: one is isopropanol, coenzyme NADH or NAD⁺; the second is glucose (exemplarily D-glucose), coenzyme NADH or NAD⁺, glucose dehydrogenase (GDH); the third is formate compounds (exemplarily formate), coenzyme NADH or NAD⁺, formate dehydrogenase (FDH). In some embodiments using purified ketoreductase, such cofactors and optionally such cofactor regeneration systems are generally added to the reaction medium together with the substrate and ketoreductase. Similar to ketoreductase, any enzyme containing a cofactor regeneration system can be in the form of an extract or lysate of such cells, or added to the reaction mixture as a purified enzyme. In embodiments using cell extracts or cell lysates, the cells used to produce the extracts or lysates can be cells expressing only the cofactor regeneration system or containing both the cofactor regeneration system and ketoreductase. In embodiments using whole cells, the cells can express an enzyme containing both the cofactor regeneration system and ketoreductase.

[0037] More preferably, regardless of using whole cells, cell extracts, or purified ketoreductase, a single ketoreductase can be used, or alternatively, a mixture of two or more ketoreductases can be used.

[0038] Furthermore, the buffer solution is a phosphate buffer solution, a Tris-HCl buffer solution, a sodium barbital-HCl buffer solution, or a citric acid-sodium citrate buffer solution.

[0039] The mutant obtained by mutation of the present invention can use ketone compounds as raw materials and efficiently produce chiral alcohols through stereoselective reduction, and is suitable for popularization in the industrial production of chiral alcohols. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0041] Figure 1 Shows the conversion rate results of the wild-type enzyme and mutant enzyme catalyzing the substrate ethyl 4-chloroacetoacetate in Example 4;

[0042] Figure 2 Shows the ee value results of the enzyme-catalyzed products of the wild-type at different temperatures in Example 4;

[0043] Figure 3 Shows the ee value results of the enzyme-catalyzed products of the mutant at different temperatures in Example 4;

[0044] Figure 4 Shows the ee value map of the mutant in Example 4. Detailed implementation mode

[0045] To better understand the technical solution of the present invention, the technical solution of the present invention will be further described below in conjunction with specific examples. The examples are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0046] It should be noted that, without conflict, the examples in this application and the features in the examples can be combined with each other. The present invention will be described in detail below in conjunction with the examples.

[0047] Explanation of names:

[0048] Ketoreductase refers to a polypeptide that can reduce a ketone group to its corresponding alcohol. Specifically, the ketoreductase polypeptide of the present application can stereoselectively reduce a ketone compound to a corresponding alcohol product. This polypeptide generally uses the cofactor-reduced nicotinamide adenine dinucleotide (NADH) or oxidized nicotinamide adenine dinucleotide (NAD+) as a reducing agent. In the present application, ketoreductase includes naturally occurring (wild-type) ketoreductase and non-naturally occurring ketoreductase mutants produced by artificial treatment.

[0049] "Naturally occurring" or "wild-type" is opposite to "mutant" and refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism, which can be isolated from a natural source and has not been deliberately modified or changed by humans.

[0050] In the present application, when referring to, for example, a cell, nucleic acid or polypeptide "recombinant", it means that it has been modified in a way that does not exist in nature, or is the same as the form existing in nature, but is prepared or derived by using synthetic materials and / or through treatment using recombinant technology, or corresponds to a natural or inherent form of a cell, nucleic acid or polypeptide. Among them, non-limiting examples include recombinant cells that express genes other than the inherent (non-recombinant) form or express inherent genes at different levels.

[0051] "Percent sequence identity" refers to the comparison between polynucleotides and is determined by comparing two optimally aligned sequences over a comparison window, where a portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated as follows: by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optionally, the percentage can be calculated as follows: by determining the number of positions at which the same nucleic acid base or amino acid residue occurs aligned with a blank position in the two sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Herein, "reference sequence" refers to a designated sequence used as a basis for sequence comparison. The reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence.

[0052] Site-directed mutagenesis: It refers to introducing the desired changes (usually changes representing a favorable direction), including addition, deletion, point mutation, etc. of bases, into a target DNA fragment (which can be a genome or a plasmid) by methods such as polymerase chain reaction (PCR). Site-directed mutagenesis can rapidly and efficiently improve the traits and characteristics of the target protein expressed by DNA and is a very useful means in gene research work.

[0053] Saturation mutagenesis technology: It is to transform the coding gene of the target protein to obtain mutants in which the amino acids at the target site are respectively replaced by 19 other natural amino acids in a short time. Using site-saturation mutagenesis to identify protein functional sites can improve enzyme specific activity and improve various properties such as enzyme thermal stability, substrate binding specificity, and stereospecificity.

[0054] The ketoreductase mutant derived from Rhodococcus ruber, such as T67R for example (in the present invention, taking "T67R" as an example, which means "original amino acid + site + mutated amino acid", that is, T at the 67th position is changed to R) can catalyze the target substrate to obtain the product, but its stability needs to be further improved. The present invention attempts to improve the substrate conversion rate and stereoselectivity by the method of directed evolution.

[0055] In the present application, first, mutation sites are introduced into the ketoreductase by site-directed mutagenesis, the activities of the mutants are detected, and mutants with improved activities are selected. Exemplarily, among them, the enzyme activity of the mutant T67R is improved compared with the starting template.

[0056] Introducing site-directed mutations using whole plasmid PCR is simple and effective and is currently a widely used method. Its principle is as follows: A pair of primers (forward and reverse) containing the mutation site anneal to the template plasmid, and then the polymerase performs "cycle extension" (cycle extension means that the polymerase extends the primer according to the template, terminates at the 5' end of the primer after one round, and then goes through repeated cycles of heating, annealing, and extension. This reaction is different from rolling circle amplification and does not form multiple tandem copies). The extension products of the forward and reverse primers anneal and pair to form a nicked open circular plasmid. The DpnI enzyme digests the extension products. Since the original template plasmid is derived from conventional Escherichia coli and has been modified by dam methylation, it is sensitive to Dpn I and is chopped up, while the plasmid with the mutated sequence synthesized in vitro is not cut because it has no methylation. Therefore, it can be successfully transformed in the subsequent transformation, and clones of the mutated plasmid can be obtained. The mutated plasmid is transformed into the host cell, and the target protein is induced to be expressed. Then, crude enzyme solution is obtained by the method of ultrasonic cell disruption. The optimal conditions for inducing the expression of ketoreductase are: 25 °C, induced with 0.1 mM IPTG for 16 h.

[0057] The DNA molecule according to the present invention may also exist in the form of an "expression cassette". An "expression cassette" refers to a linear or circular nucleic acid molecule that encompasses DNA and RNA sequences capable of directing the expression of a specific nucleotide sequence in a suitable host cell. Generally, it includes a promoter effectively linked to the target nucleotide, which is optionally effectively linked to a termination signal and / or other regulatory elements. The expression cassette may also include sequences required for the correct translation of the nucleotide sequence. The coding region usually encodes the target protein, but also encodes the target functional RNA in the sense or antisense direction, such as antisense RNA or non-translated RNA. The expression cassette containing the target polynucleotide sequence may be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. The expression cassette may also be naturally occurring, but obtained in an effective recombinant form for heterologous expression.

[0058] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any one of the above DNA molecules. The DNA molecule in the above recombinant plasmid is placed at an appropriate position in the recombinant plasmid so that the above DNA molecule can be correctly and smoothly replicated, transcribed, or expressed.

[0059] Although the qualifier used when defining the above DNA molecule in the present invention is "containing", it does not mean that other sequences unrelated to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombinant operations, appropriate restriction enzyme cleavage sites need to be added to both ends of the DNA sequence, or promoters, termination codons, etc. need to be additionally added. Therefore, if a closed expression is used to define it, these situations cannot be truly covered.

[0060] Example 1

[0061] Construction of Recombinant Escherichia coli E.coli Rosetta(pET-28a-RR)

[0062] 1.1 Acquisition of Ketoreductase Gene

[0063] The strain Rhodococcus ruber (purchased from the China General Microbiological Culture Collection Center, No. CGMCC 1.10360) was amplified in culture. It was cultured at 30 °C for 1 day. The seed medium (g / L): glycerol 10, peptone 5, malt extract powder 3, yeast powder 3, pH 7.0.

[0064] The Rhodococcus ruber in the logarithmic growth phase was centrifuged, and genomic DNA was extracted using a genomic DNA extraction kit (Beijing Tianwei Biotechnology Co., Ltd.) according to the instructions.

[0065] Based on the ketoreductase gene information in the NCBI database, upstream and downstream primers were designed. The primer sequences are as follows:

[0066] Upstream primer (containing NdeI site):

[0067] 5’-GGAATTCCATATGAAAGCCCTCCAGTACACCGAGA-3’ (SEQ NO:2)

[0068] Downstream primer (containing XhoI site):

[0069] 5’-CCCCTCGAGTCAACCCGGAACCACAACGCCGCG-3’ (SEQ NO:3)

[0070] All primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0071] Gene PCR amplification conditions:

[0072] Denaturation at 98 °C for 3 min, and cycled 30 times according to the following parameters: denaturation at 98 °C for 10 sec, annealing at 58 °C for 5 sec, extension at 72 °C for 1.5 min. Finally, extension at 72 °C for 4 min.

[0073] 1.2 Construction of Strains

[0074] The expression vector pET-28a (purchased from Novagen (Merck China)) and the amplified target gene containing two restriction enzyme sites were digested with Nde I and Xho I. The digested target fragment and expression vector were recovered by gel extraction respectively. The digested expression vector pET-28a and the target gene were ligated overnight with T4 ligase. 10 μL of the ligation product pET-28a-RR was added to Rosetta(DE3) competent cells, placed on ice for 30 min, heat shocked at 42 °C for 90 sec, and then placed on ice for 2 min. 1 mL of LB medium was added, and the cells were cultured with shaking at 37 °C and 200 rpm for 0.5 h. The bacterial solution was aspirated and spread on an LB solid plate containing 50 mM kanamycin, and cultured overnight at 37 °C to obtain the recombinant bacterium E. coli Rosetta(pET-28a-RR).

[0075] 1.3 Protein sequence alignment

[0076] The recombinant bacterium E. coli Rosetta(pET-28a-RR) constructed was used to extract plasmid, which was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The analysis of the sequencing results showed that the amplified sequence of the target gene was highly homologous to the sequence with the accession number MW808991.1 in the GenBanK database, and a mutation occurred at positions 199 to 201, where the codon ATT mutated to CGC.

[0077] 1.4 Enzyme activity detection

[0078] In this application, the enzyme activity detection method is as follows:

[0079] 1.4.1 Reagent preparation:

[0080] Substrate mother liquor 100 mM: Weigh 6.76 mg of ethyl 4-chloroacetoacetate, dissolve it in isopropanol, stir and mix well until completely dissolved;

[0081] NADH mother liquor 10 mM: Weigh 33.17 mg of NADH and dissolve it in 5 mL of 0.1 M PB pH7.0 buffer.

[0082] 1.4.2 Enzyme activity system:

[0083] First, add the enzyme, then add the mixture of the substrate ethyl 4-chloroacetoacetate, NADH and buffer, and place it in an enzyme-labeling instrument to detect the enzyme activity at 30 °C and a wavelength of 340 nM.

[0084] The preparation of the detection system is shown in Table 1

[0085] Table 1

[0086] system dosage final concentration enzyme mutant 20 μL N / A substrate 150 μL 50 mM NADH 10 μL 0.33 mM pH 7.0 buffer 120 μL 0.1M

[0087] The ketoreductase mutant T67R, abbreviated as "template" in the present invention, and the listed mutation sites are mutations based on this "template".

[0088] Method for preparing enzyme solution in high-throughput screening: Centrifuge the 96-well plate to remove the supernatant medium, add 200 μL of enzymatic hydrolysis solution (lysozyme 2 mg / mL, polymyxin 0.5 mg / mL, pH 7.0) to each well, and treat at 37 °C for 2 h.

[0089] Enzyme catalysis and detection method: First add the enzyme, then add the substrate ethyl 4-chloroacetoacetate, the mixture of NADH and buffer, and catalyze for a certain time. Transfer the catalytic sample to an enzyme detection plate, place it in an enzyme-labeling instrument, and detect the enzyme activity at 30 °C and a wavelength of 340 nM.

[0090] Example 2

[0091] Irrational engineering of recombinant bacterium E. coli Rosetta (pET-28a-RR)

[0092] 2.1 Codon optimization of Rhodococcus ruber

[0093] The ketoreductase derived from Rhodococcus ruber was submitted to Sangon Biotech (Shanghai) Co., Ltd. for codon optimization. The optimization host was Escherichia coli, and the gene sequence of DNA2 was the codon-optimized result of the original sequence DNA1.

[0094] 2.2 Primer design

[0095] Design upstream primers for the ketoreductase gene information. The primer sequences are as follows:

[0096] Upstream primer (containing NdeI site):

[0097] 5’-GGAATTCCATATGAAAGCACTGCAGTACACTGAA-3’ (SEQ NO:4)

[0098] Downstream primer (containing XhoI site):

[0099] 5’-CCCCTCGAGTCAACCCGGAACCACAACGCCGCG-3’ (SEQ NO:5)

[0100] 2.3 Construction and screening of strain mutant library

[0101] 2.3.1 Construction of strain mutation library: The codon-optimized recombinant E. coli Rosetta (pET-28a-RR) was amplified and cultured, and the plasmid was extracted using the plasmid extraction kit of Sangon Biotech (Shanghai) Co., Ltd. The obtained plasmid was used as a mutation amplification template. The base mutation was introduced while amplifying using the ready-to-use error-prone PCR kit of Beijing Tianenze Biotechnology Co., Ltd. The strain was constructed according to 1.2 in Example 1, and all the mutant strains finally obtained were the mutation library of the strain.

[0102] 2.3.2 Screening of strain mutant library: The mutant library obtained above was amplified and cultured in a 96-deep-well plate, protein expression was induced, and enzyme activity was determined according to 1.4 in Example 1. Mutants with higher enzyme activity than that of the recombinant bacterium E. coli Rosetta (pET-28a-RR) were selected for shake flask culture and protein expression, and enzyme activity was determined.

[0103] Example 3

[0104] Rational transformation of recombinant E. coli Rosetta (pET-28a-RR)

[0105] 3.1 Site-directed mutagenesis

[0106] Plasmids were extracted from the mutant strains with improved enzyme activity in Example 2 and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed and compared with the original sequences, and primers were designed for the mutated amino acid sites and sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. Site-directed mutagenesis was performed according to Example 2 to construct mutant strains. It was finally determined that the mutation sites V14, I42, T67, A97, G170, A242, I262 and F286 significantly improved the enzyme activity.

[0107] Table 2

[0108] mutant enzyme activity (%) V14E + I42M ++ T67R + A97H + G170R + A242S + I262S ++ F286S +

[0109] + represents enzyme activity. The more + there are, the higher the enzyme activity.

[0110] 3.2 Saturation mutation

[0111] Saturation mutagenesis was carried out on the mutation sites V14, I42, T67, A97, G170, A242, I262 and F286. Degenerate bases provided by Sangon Biotech (Shanghai) Co., Ltd. were used to design primers and synthesize them. According to Example 2, saturation mutagenesis was carried out to construct mutant strains and screen mutant strains. The results showed that there was no significant change in enzyme activity after saturation mutagenesis at the amino acid sites of V14, T67, A97, G170, A242 and F286, while after mutation of I42 and I262, I42L, I42V, I262A and I262G were obtained by screening, and the enzyme activities of the four mutant strains were improved.

[0112] 3.3 Mutation combinations

[0113] Furthermore, corresponding combinations were made for the mutation sites to screen mutant strains with high enzyme activity: The "template" and mutants were respectively subjected to catalytic reactions at 30 °C, and then their activities were measured. The enzyme activity results of all mutants are shown in Table 3.

[0114] Table 3

[0115]

[0116]

[0117] + represents enzyme activity, and the more +, the higher the enzyme activity.

[0118] Combined saturation mutagenesis can obtain mutants with synergistic effects between several mutation sites, and the amino acid composition can be optimized and combined. Using T67R as a template, mutation combination was carried out. At this time, the enzyme solution was detected at 30 °C for 17 h, then the reaction was terminated and the activity was measured.

[0119] Example 4

[0120] 4.1 Enzyme catalysis of the best mutant

[0121] The mutant V14E+I42L+A97H+G170R+A242L+I262A was used to verify different substrate reactions, and the results are shown in Table 4.

[0122] 1) Add 0.5 g of ethyl acetoacetate as the substrate to a 100 mL reaction flask, add 0.1 M PB pH 7.0, 0.2 g of isopropanol, 20 mg of NAD+, and 0.05 g of the ketoreductase mutant, mix well, and the total volume is 20 mL. React at 30 °C and 200 rpm on a shaker for 1 h;

[0123] 2) Add 0.5 g of ethyl 2-chloroacetoacetate as the substrate into a 100 mL reaction flask, add 0.1 M PB pH 7.0, 0.2 g of isopropanol, 20 mg of NAD⁺, and 0.05 g of ketoreductase mutant. Mix well. The total volume is 20 mL. React at 30 °C on a shaker at 200 rpm for 1 h;

[0124] 3) Add 0.5 g of methyl 4-chloroacetoacetate as the substrate into a 100 mL reaction flask, add 0.1 M PB pH 7.0, 0.2 g of isopropanol, 20 mg of NAD⁺, and 0.05 g of ketoreductase mutant. Mix well. The total volume is 20 mL. React at 30 °C on a shaker at 200 rpm for 1 h.

[0125] Table 4

[0126]

[0127]

[0128] 4.2 Optimal mutant enzyme catalysis

[0129] Taking the recombinant bacterium E. coli Rosetta (pET-28a-RR) as a control, the conversion rates of the mutants V14E + G170R, V14E + I42L + I262A, and V14E + I42L + A97H + G170R + F286S for the substrate ethyl 4-chloroacetoacetate within 25 °C - 35 °C are as Figure 1 , and the conversion rates and product ee values of the mutant V14E + I42L + A97H + G170R + A242L + I262A (SEQ NO: 6) for the substrate ethyl 4-chloroacetoacetate at different temperatures. The catalytic results are as Figure 1 , Figure 2 , Figure 3 and Figure 4 . Compared with the recombinant bacterium E. coli Rosetta (pET-28a-RR), the catalytic time of the mutant V14E + I42L + A97H + G170R + A242L + I262A is shortened from 17 h to 1 h, and the catalytic conversion rate is greater than 99%. Within 25 °C - 35 °C, for the recombinant bacterium E. coli Rosetta (pET-28a-RR), the ee value decreases significantly as the temperature increases, while for the mutant V14E + I42L + A97H + G170R + A242L + I262A, the increase in temperature has no obvious effect on the ee value.

[0130] In summary, the mutant V14E+I42L+A97H+G170R+A242L+I262A has the following advantages: First, it can catalyze 0.8 - 1 mol / L of substrate within a certain catalytic time, with an ee value higher than 99%, a space-time conversion rate of 1440 g / L / d, and extremely high catalytic efficiency. Second, the catalytic system has mild operating conditions, is basically completed in an environment of normal temperature, neutrality, water, etc., uses less organic solvent, has low risk during the reaction process, is environmentally friendly, meets current production requirements, and has great commercial value potential.

[0131] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Sequence Listing <110> Anhui Puli Pharmaceutical Co., Ltd. <120> A ketoreductase mutant <130> hnpoly002 <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 346 <212> PRT <213> 2 Ambystoma laterale x Ambystoma jeffersonianum <220> <223> Ketoreductase mutant T67R derived from Rhodococcus ruber <400> 1 Met Lys Ala Leu Gln Tyr Thr Glu Ile Gly Ser Val Pro Val Val Val 1 5 10 15 Asp Val Pro Thr Pro Ala Pro Gly Pro Gly Glu Ile Leu Leu Lys Val 20 25 30 Thr Ala Ala Gly Leu Cys His Ser Asp Ile Phe Val Met Asp Met Pro 35 40 45 Ala Glu Gln Tyr Ile Tyr Gly Leu Pro Leu Thr Leu Gly His Glu Gly 50 55 60 Val Gly Arg Val Ala Glu Leu Gly Ala Gly Val Thr Gly Phe Glu Thr 65 70 75 80 Gly Asp Ala Val Ala Val Tyr Gly Pro Trp Gly Cys Gly Ala Cys His 85 90 95 Ala Cys Ala Arg Gly Arg Glu Asn Tyr Cys Thr Arg Ala Ala Glu Leu 100 105 110 Gly Ile Thr Pro Pro Gly Leu Gly Ser Pro Gly Ser Met Ala Glu Tyr 115 120 125 Met Ile Val Asp Ser Ala Arg His Leu Val Pro Ile Gly Asp Leu Asp 130 135 140 Pro Val Ala Ala Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His 145 150 155 160 Ala Ile Ser Arg Val Leu Pro Leu Leu Gly Pro Gly Ser Thr Ala Val 165 170 175 Val Ile Gly Val Gly Gly Leu Gly His Val Gly Ile Gln Ile Leu Arg 180 185 190 Ala Val Ser Ala Ala Arg Val Ile Ala Val Asp Leu Asp Asp Asp Arg 195 200 205 Leu Ala Leu Ala Arg Glu Val Gly Ala Asp Ala Ala Val Lys Ser Gly 210 215 220 Ala Gly Ala Ala Asp Ala Ile Arg Glu Leu Thr Gly Gly Glu Gly Ala 225 230 235 240 Thr Ala Val Phe Asp Phe Val Gly Ala Gln Ser Thr Ile Asp Thr Ala 245 250 255 Gln Gln Val Val Ala Ile Asp Gly His Ile Ser Val Val Gly Ile His 260 265 270 Ala Gly Ala His Ala Lys Val Gly Phe Phe Met Ile Pro Phe Gly Ala 275 280 285 Ser Val Val Thr Pro Tyr Trp Gly Thr Arg Ser Glu Leu Met Asp Val 290 295 300 Val Asp Leu Ala Arg Ala Gly Arg Leu Asp Ile His Thr Glu Thr Phe 305 310 315 320 Thr Leu Asp Glu Gly Pro Thr Ala Tyr Arg Arg Leu Arg Glu Gly Ser 325 330 335 Ile Arg Gly Arg Gly Val Val Val Pro Gly 340 345 <210> 2 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer (containing Nde I site) <400> 2 ggaattccat atgaaagccc tccagtacac cgaga 35 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer (containing Xho I site) <400> 3 cccctcgagt caacccggaa ccacaacgcc gcg 33 <210> 4 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Upstream primer (containing Nde I site) <400> 4 ggaattccat atgaaagcac tgcagtacac tgaa 34 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Downstream primer (containing Xho I site) <400> 5 cccctcgagt caacccggaa ccacaacgcc gcg 33 <210> 6 <211> 346 <212> PRT <213> Artificial Sequence <220> <223> Containing V14E+I42L+A97H+G170R+A242L+I262A mutations <400> 6 Met Lys Ala Leu Gln Tyr Thr Glu Ile Gly Ser Val Pro Glu Val Val 1 5 10 15 Asp Val Pro Thr Pro Ala Pro Gly Pro Gly Glu Ile Leu Leu Lys Val 20 25 30 Thr Ala Ala Gly Leu Cys His Ser Asp Leu Phe Val Met Asp Met Pro 35 40 45 Ala Glu Gln Tyr Ile Tyr Gly Leu Pro Leu Thr Leu Gly His Glu Gly 50 55 60 Val Gly Arg Val Ala Glu Leu Gly Ala Gly Val Thr Gly Phe Glu Thr 65 70 75 80 Gly Asp Ala Val Ala Val Tyr Gly Pro Trp Gly Cys Gly Ala Cys His 85 90 95 His Cys Ala Arg Gly Arg Glu Asn Tyr Cys Thr Arg Ala Ala Glu Leu 100 105 110 Gly Ile Thr Pro Pro Gly Leu Gly Ser Pro Gly Ser Met Ala Glu Tyr 115 120 125 Met Ile Val Asp Ser Ala Arg His Leu Val Pro Ile Gly Asp Leu Asp 130 135 140 Pro Val Ala Ala Val Pro Leu Thr Asp Ala Gly Leu Thr Pro Tyr His 145 150 155 160 Ala Ile Ser Arg Val Leu Pro Leu Leu Arg Pro Gly Ser Thr Ala Val 165 170 175 Val Ile Gly Val Gly Gly Leu Gly His Val Gly Ile Gln Ile Leu Arg 180 185 190 Ala Val Ser Ala Ala Arg Val Ile Ala Val Asp Leu Asp Asp Asp Arg 195 200 205 Leu Ala Leu Ala Arg Glu Val Gly Ala Asp Ala Ala Val Lys Ser Gly 210 215 220 Ala Gly Ala Ala Asp Ala Ile Arg Glu Leu Thr Gly Gly Glu Gly Ala 225 230 235 240 Thr Leu Val Phe Asp Phe Val Gly Ala Gln Ser Thr Ile Asp Thr Ala 245 250 255 Gln Gln Val Val Ala Ala Asp Gly His Ile Ser Val Val Gly Ile His 260 265 270 Ala Gly Ala His Ala Lys Val Gly Phe Phe Met Ile Pro Phe Gly Ala 275 280 285 Ser Val Val Thr Pro Tyr Trp Gly Thr Arg Ser Glu Leu Met Asp Val 290 295 300 Val Asp Leu Ala Arg Ala Gly Arg Leu Asp Ile His Thr Glu Thr Phe 305 310 315 320 Thr Leu Asp Glu Gly Pro Thr Ala Tyr Arg Arg Leu Arg Glu Gly Ser 325 330 335 Ile Arg Gly Arg Gly Val Val Val Pro Gly 340 345

Claims

1. A ketoreductase mutant, which is obtained by mutating using SEQ ID NO:1 as a template, and the mutation sites are V14E+I42L+A97H+G170R+A242L+I262A.

2. A DNA molecule, which encodes the ketoreductase mutant according to claim 1.

3. A recombinant plasmid, which is ligated with the DNA molecule according to claim 2.

4. The recombinant plasmid according to claim 3, which is a recombinant expression plasmid.

5. The recombinant plasmid according to claim 4, which is a prokaryotic recombinant expression plasmid or a eukaryotic recombinant expression plasmid.

6. The recombinant plasmid according to claim 3, wherein the vector of the recombinant plasmid is selected from pET-22a(+), pET-22b(+), pET-3a(+), pET-3d(+), pET-11a(+), pET-12a(+), pET-14b(+), pET-15b(+), pET-16b(+), pET-17b(+), pET-19b(+), pET-20b(+), pET-21a(+), pET-23a(+), pET-23b(+), pET-24a(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-31b(+), pET-32a(+), pET-35b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a(+), pET-41b(+), pET-42a(+), pET-43a(+), pET-43b(+), pET-44a(+), pET-49b(+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18 or pUC-19.

7. A host cell, which contains the recombinant plasmid according to any one of claims 3-6.

8. The host cell according to claim 7, which is a prokaryotic cell.

9. The host cell according to claim 7, which is a eukaryotic cell.

10. The host cell according to claim 8, wherein the prokaryotic cell is a bacterium.

11. The host cell according to claim 10, wherein the bacterium is a Gram-negative bacterium or a Gram-positive bacterium.

12. The host cell according to claim 8, wherein the prokaryotic cell is Escherichia coli BL21(DE3), BL21 Star(DE3), TunerTM(DE3), RosettaTM 2(DE3), BLR(DE3), NovaBlue(DE3), OrigamiTM(DE3) or Origami B(DE3).

Images