A ketoreductase mutant and its application

Abstract

The present invention relates to a ketoreductase mutant and its application. The ketoreductase mutant is obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes at least one of the following mutation sites: the 113th position is mutated from N to R or K, the 141st position is mutated from S to A, D, E, G or V, the 149th position is mutated from D to A or F, the 152nd position is mutated from W to H, L, M or Y, the 197th position is mutated from A to M or F, and the 207th position is mutated from S to A or T. The ketoreductase mutant can be applied to the green chemical synthesis of chiral alcohol compounds, has high catalytic activity and good stability. Compared with the chemical synthesis method, the reaction catalyzed by the ketoreductase mutant is simple and mild, has high reaction selectivity, low preparation cost, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a ketoreductase mutant and application thereof. Background Art

[0002] At present, bioenzymatic reactions are widely used in the chemical industry to produce drugs or chiral intermediates on a large scale. Enzyme-catalyzed reactions have many advantages, such as being able to be carried out in benign solvents and under mild reaction conditions, and having good specificity and selectivity. However, enzyme-catalyzed reactions also have their own disadvantages, including: lack of stability under production reaction conditions, product inhibition, and the need to carry out the reaction in an aqueous medium, while many substrates of interest have poor water solubility and cannot react effectively. In order to overcome the above defects, various technical modifications have been made to enzymes, such as directed evolution, random mutagenesis, and protein engineering, to overcome the above shortcomings, so that enzymes can be used efficiently and environmentally friendly for the synthesis of chemical molecules, such as active pharmaceutical ingredients.

[0003] Ketoreductases (KREDs) (EC 1.1.1) are part of the aldehyde-ketoreductase family, which catalyzes the reduction of carbonyl groups to chiral alcohols. Examples of KRED-catalyzed reduction of prochiral ketones (carbonyl groups) to chiral alcohols have been reported, including some applications on an industrial scale. The synthesis of montelukast is a typical example, in which KREDs were used to replace the existing and expensive chemical reducing agent (DIP-Cl) during the synthesis of the drug. The prior art also reports the use of ketoreductases to synthesize chiral alcohols in the synthesis of drugs such as sulfenem, atorvastatin, rosuvastatin, and ticagrelor. Ketoreductases are increasingly used as stereoselective reduction catalysts in the synthesis of active drug molecules or their intermediates. However, the catalytic activity of ketoreductases on different non-natural substrates varies greatly. For example, the same ketoreductase has a good effect on the catalytic reduction of some non-natural carbonyl substrates to chiral alcohols, but has no catalytic effect or very poor catalytic effect on other specific non-natural carbonyl substrates, and also has large differences in stability and selectivity. Therefore, for many carbonyl substrates, the problem in reducing them to chiral alcohols using ketoreductases is that there are no suitable ketoreductases in the prior art because these ketoreductases have poor catalytic effects in most cases. It is necessary to find suitable ketoreductases, or to improve the activity and specificity of known ketoreductases through rational design, so as to promote the application of ketoreductases in the synthesis and industrial production of chiral alcohols. Summary of the invention

[0004] In order to solve the problems of low efficiency and low selectivity in the process of chemical synthesis of specific chiral alcohols, as well as low activity and poor stability of existing ketoreductases in catalyzing the reduction of specific carbonyl substrates to chiral alcohols, one aspect of the present invention aims to provide a ketoreductase mutant with high catalytic activity and good stereoselectivity for specific carbonyl substrates. To achieve the purpose of the present invention, the present invention adopts the following technical scheme:

[0005] A ketoreductase mutant, the amino acid sequence of the ketoreductase mutant is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes at least one of the following mutation sites: position 113 mutates from N to R or K, position 141 mutates from S to A, D, E, G or V, position 149 mutates from D to A or F, position 152 mutates from W to H, L, M or Y, position 197 mutates from A to M or F, and position 207 mutates from S to A or T, or the amino acid sequence of the ketoreductase mutant has the mutation site in the mutated amino acid sequence, has at least 80% or more, or at least 85% or more, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence.

[0006] In some preferred embodiments of the present invention, the above mutations include at least one of the following mutation sites: position 113 mutates from N to R (N113R), position 141 mutates from S to A (S114A), position 149 mutates from D to F (D149F), position 152 mutates from W to Y (W152Y), position 197 mutates from A to M (A197M), and position 207 mutates from S to A (S207A), or the amino acid sequence of the ketoreductase mutant has the mutation site in the mutated amino acid sequence, has at least 80%, or at least 85%, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence.

[0007] In some more preferred embodiments of the present invention, the above mutations include at least a mutation from N to R at position 113, a mutation from S to A at position 141, a mutation from D to F at position 149, and a mutation from W to Y at position 152. In some preferred embodiments of the present invention, the above mutations include at least a mutation from N to R at position 113, a mutation from S to A at position 141, a mutation from D to F at position 149, a mutation from W to Y at position 152, and a mutation from A to M at position 197. In some preferred embodiments of the present invention, the above mutations include at least mutation of position 113 from N to R, mutation of position 141 from S to A, mutation of position 149 from D to F, mutation of position 152 from W to Y, mutation of position 197 from A to M and mutation of position 207 from S to A, or the amino acid sequence of the ketoreductase mutant has the mutation site in the mutated amino acid sequence, has at least 80% or more, or at least 85% or more, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence.

[0008] Another object of the present invention is to provide a gene encoding the above-mentioned ketoreductase mutant.

[0009] In some preferred embodiments of the present invention, the nucleotide sequence of the above gene is the sequence shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

[0010] In some more preferred embodiments of the present invention, the nucleotide sequence of the above gene is the sequence shown in SEQ ID NO.8.

[0011] Another aspect of the present invention is to provide a recombinant expression vector comprising a gene encoding the above-mentioned ketoreductase mutant.

[0012] In some preferred embodiments of the present invention, the recombinant expression vector is selected from pET-28a, pET-dute1, and pRSF-dute1. In some more preferred embodiments of the present invention, the recombinant expression vector is selected from pET-28a.

[0013] Another aspect of the present invention is to provide a genetically engineered bacterium for producing the above-mentioned ketoreductase mutant, which comprises the above-mentioned recombinant expression vector.

[0014] In some preferred embodiments of the present invention, the genetically engineered bacteria are selected from Escherichia coli MG1655, Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS.

[0015] In some more preferred embodiments of the present invention, the genetically engineered bacteria are selected from Escherichia coli BL21 (DE3).

[0016] Another aspect of the present invention is to provide the use of the above-mentioned ketoreductase mutant in the preparation of chiral alcohol compounds using carbonyl compounds as raw materials.

[0017] In some preferred embodiments of the present invention, the structural formula of the carbonyl compound is

[0018] Wherein, R1 and R2 are each independently C1-C8 alkyl, C5-C 10 Cycloalkyl,

[0019] C5~C 10 Aryl, C5~C 10 a heteroaryl group or a group containing an amine group, an ether group, a thioether group or an ester group, or R1 and R2 together with the carbon on the carbonyl group form a 5-10 membered ring, the 5-10 membered ring is entirely composed of carbon, or the 5-10 membered ring has heteroatoms in addition to carbon atoms participating in the formation of the 5-10 membered ring, the heteroatoms are selected from N, O, S, and the 5-10 membered ring is unsubstituted or substituted by at least one group selected from halogen, oxygen, sulfur, hydroxyl, alkoxy or alkyl,

[0020] The structural formula of the chiral alcohol compound is

[0021] In some preferred embodiments of the present invention, the carbonyl compound is a compound of formula I, and the alcohol compound is a compound of formula II.

[0022]

[0023] The above-mentioned ketoreductase mutant of the present invention is mutated by site-directed mutagenesis on the basis of the ketoreductase with the amino acid sequence shown in SEQ ID NO: 1, thereby changing its amino acid sequence, achieving changes in protein structure and function, and then obtaining the ketoreductase with the above-mentioned mutation site by a directed screening method. Compared with the wild-type ketoreductase, the ketoreductase mutant of the present invention has greatly improved substrate selectivity, and the ee value of the chiral alcohol product obtained by catalytic reduction under the same conditions is increased from 0 to 99.5% relative to the wild-type ketoreductase, and the enzyme activity is also greatly improved. When used for chiral alcohol production, the cost of chiral alcohol industrial production is greatly reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Figure 1 is a diagram of the ketoreductase recombinant expression vector of the present invention.

[0025] Figure 2The lanes shown from left to right are Marker, BL21(DE3)-iii protease (whose amino acid sequence is Marker, Seq ID No: 5), BL21(DE3)-ii protease (whose amino acid sequence is Seq ID No: 4), BL21(DE3)-i protease (whose amino acid sequence is Seq ID No: 3), wild-type protease (whose amino acid sequence is Seq ID No: 1), and blank polyacrylamide gel electrophoresis results.

[0026] Figure 3 The polyacrylamide gel electrophoresis results of Marker and BL21(DE3)-iii protease.

[0027] Figure 4 This is the liquid chromatography spectrum of the chiral alcohol product obtained in Example 7.

[0028] Figure 5 The chiral alcohol product obtained in Example 7 1 HNMR spectrum. DETAILED DESCRIPTION

[0029] The ketoreductase derived from Arthrobacter sp.TS-15 can selectively reduce carbonyl compounds, but its reduction activity for the compound shown in the above formula I is low and its selectivity is poor. The inventors of the present invention improve the activity and selectivity of the ketoreductase derived from Arthrobacter sp.TS-15 by a rational design method. A mutation site is introduced into the ketoreductase derived from Arthrobacter sp.TS-15 by means of whole plasmid PCR, and the activity and stability of the mutant are tested to select mutants with improved activity and stability.

[0030] The ketoreductase mutant gene provided by the present invention is derived from the wild-type gene of Arthrobacter sp.TS-15. The nucleotide sequence of the wild-type gene is shown in SEQ ID NO:1, and the gene sequence after codon optimization for Escherichia coli is shown in SEQ ID NO.2. Wherein, "wild type" 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 separated from a natural source and has not been intentionally modified by artificial manipulation. The enzymes obtained after the expression of these genes have low catalytic activity for certain substrates and poor thermal stability.

[0031] The amino acid sequence of the ketoreductase from Arthrobacter sp. TS-15 is shown in SEQ ID NO: 1:

[0032] MINMRNRVAIVTGGAMGMGNGCARKLAEAGAKVYLIDRSNLVAAAAQDMREAG

[0033] LNANHVQVDITDQESLTSAYDGIAAESGRLDVLVNAAGVGDSRMFLDVDDAHYK

[0034] KVIDVNVRGTWNSCRAAVPHMLSNKHGRIINFGSISGPIVADPGWTVYALSKGAIF

[0035] GFTKALASEFAGQNILVNAILPGSMDTPMMRAAAADTNPADPQSVIDEIAAAVPLK

[0036] RLGTIDDAGNLALFLASDLASYLTGQAIVLDGAFTLAEYQSGGLEAPAETPALVN

[0037] The cDNA of the ketoreductase from Arthrobacter sp.TS-15, after codon optimization for Escherichia coli, is shown in SEQ ID NO.2:

[0038] ATGATTAACATGCGCAACCGCGTGGCGATTGTGACCGGCGGCGCGATGGGCAT

[0039] GGGCAACGGCTGCGCGCGCAAACTGGCGGAAGCGGGCGCGAAAGTGTATCTGA

[0040] TTGATCGCAGCAACCTGGTGGCGGCCGCGGCCCAAGATATGCGCGAAGCGGGC

[0041] CTGAACGCGAACCATGTGCAAGTGGATATTACCGATCAAGAAAGCCTGACGAG

[0042] CGCGTATGATGGCATTGCGGCGGAAAGCGGCCGCCTGGATGTGCTGGTGAACG

[0043] CCCGGGTGTGGGCGATAGCCGCATGTTTCTGGATGTGGATGATGCGCATTATA

[0044] AAAAAGTGATTGATGTGAACGTGCGCGGCACCTGGAACAGCTGCCGCGCGGCG

[0045] GTGCCGCACATGCTGAGCAACAAACATGGCCGCATTATTAACTTTGGCAGCATT

[0046] AGCGGCCCGATTGTGGCGGATCCGGGCTGGACCGTGTATGCGCTGAGCAAAGG

[0047] CGCGATTTTTGGCTTTACCAAAGCGCTGGCGAGCGAATTTGCGGGTCAGAACAT

[0048] TCTGGTGAACGCGATTCTGCCGGGCAGCATGGATACCCCGATGATGCGCGCGGC

[0049] CGCGGCGGATACCAACCCGGCGGATCCGCAGAGCGTGATTGATGAAATTGCGG

[0050] CCGCGGTGCCGCTGAAACGCCTGGGCACCATTGATGATGCGGGCAACCTGGCGC

[0051] TGTTTCTGGCGAGCGATCTGGCGAGCTATCTGACCGGCCAAGCGATTGTGCTGG

[0052] ATGGCGCGTTTACCCTGGCGGAATATCAGAGCGGCGGCCTGGAAGCGCCGGCG

[0053] GAAACCCCGGCGCTGGTGAAC

[0054] The present invention obtains the three-dimensional structure (6QHE_A) of the ketoreductase derived from Arthrobacter sp.TS-15 through PDB, then performs a combination simulation of the substrate of formula I and the three-dimensional structure of the ketoreductase protein through AutoDock, and finally selects amino acids that may be related to substrate binding as mutant amino acids through Pymol analysis. According to the Pymol analysis results, multiple pairs of site-directed mutation (N113R / K; S114A / D / E / G / V; D149A / F; W152H / L / M / Y; A197M / F; S207A / T) primers are designed, and a mutant plasmid with a target gene is obtained by using a site-directed mutation method and using pET-28a, pET-dute1 or pRSF-dute1 as an expression vector. Wherein, site-directed mutation refers to the introduction of a desired change (usually a change that characterizes a favorable direction) into a target DNA fragment (which can be a genome or a plasmid) through methods such as polymerase chain reaction (PCR), including addition, deletion, point mutation, etc. of bases. Site-directed mutagenesis can rapidly and efficiently improve the properties and characteristics of the target protein expressed by DNA, and is a very useful method in genetic research.

[0055] The ketoreductase mutant provided by the present invention is an amino acid sequence obtained by mutation of the amino acid sequence shown in SEQ ID NO: 1, wherein the mutation includes at least one of the following mutation sites: position 113 is mutated from N to R or K (N113R / K); position 141 is mutated from S to A, D, E, G or V (S114A / D / E / G / V); position 149 is mutated from D to A or F (D149A / F); position 152 is mutated from W to H, L, M or Y (W152H / L / M / Y); position 197 ... A mutates to M or F (A197M / F); position 207 mutates from S to A or T (S207A / T), or the amino acid sequence of the ketoreductase mutant has the mutation site in the mutated amino acid sequence, has at least 80% or more, or at least 85% or more, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence. In a preferred embodiment of the present invention, the mutation includes at least one of the following mutation sites: position 113 mutates from N to R (N113R), position 141 mutates from S to A (S114A), position 149 mutates from D to F (D149F), position 152 mutates from W to Y (W152Y), position 197 mutates from A to M (A197M), position 207 mutates from S to A (S207A), or the amino acid sequence of the ketoreductase mutant has the mutation site in the mutated amino acid sequence, and has at least 80% or more, or at least 85% or more, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence. Wherein "homology" refers to the identity between two amino acid sequences. The sequences defined by different degrees of homology in the present invention must also have improved ketoreductase activity at the same time. Wherein, "same function" means that it has the same function of catalyzing the reduction of carbonyl substrate to chiral alcohol as the mutated amino acid sequence. A person skilled in the art can obtain the amino acid sequence of a ketoreductase mutant under the guidance of the disclosure of the present application, which has the mutation site in the mutated amino acid sequence and has at least 80% or more, or at least 85% or more, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence.

[0056] In a more preferred embodiment of the present invention, the ketoreductase mutant comprises mutation sites N113R, S114A, D149F and W152Y, and its amino acid sequence is shown in SEQ ID NO: 3. In a more preferred embodiment of the present invention, the ketoreductase mutant comprises mutation sites N113R, S114A, D149F, W152Y and A197M, and its amino acid sequence is shown in SEQ ID NO: 4. In a more preferred embodiment of the present invention, the ketoreductase mutant comprises mutation sites N113R, S114A, D149F, W152Y, A197M and S207A, and its amino acid sequence is as shown in SEQ ID NO: 5, or the amino acid sequence of the ketoreductase mutant has the mutation site in the mutated amino acid sequence, and has at least 80%, or at least 85%, or at least 90%, 93%, 95%, 96%, 97%, 98%, 99% or more homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence.

[0057] The amino acid sequence shown in SEQ ID NO: 3 is as follows:

[0058] MINMRNRVAIVTGGAMGMGNGCARKLAEAGAKVYLIDRSNLVA

[0059] AAAQDMREAGLNANHVQVDITDQESLTSAYDGIAAAESGRLDVLVNAA

[0060] GVGDSRMFLDVDDAHYKKVIDVRVRGTWNSCRAAVPHMLSNKHGRII

[0061] NFGAISGPIVAFPGYTVYALSKGAIFGFTKALASEFAGQNILVNAILPGS

[0062] MDTPMMRAAAADTNPADPQSVIDEIAAAVPLKRLGTIDDAGNLALFLA

[0063] SDLASYLTGQAIVLDGAFTLAEYQSGGLEAPAETPALVN

[0064] The amino acid sequence shown in SEQ ID NO: 4 is as follows:

[0065] MINMRNRVAIVTGGAMGMGNGCARKLAEAGAKVYLIDRSNLVAAAAQDMREAGLNANHVQVDITDQESLTSAYDGIAAESGRLDVLVNAAGVGDSRMFLDVDDAHYKKVIDVRVRGTWNSCRAAVPHMLSNKHGRIINFGAISGPIVAFPGYTVYALSKGAIFGFTKALASEFAGQNILVNAILPGSMDTPMMRAAMADTNPADPQSVIDEIAAAVPLKRLGTIDDAGNLALFLASDLASYLTGQAIVLDGAFTLAEYQSGGLEAPAETPALVN

[0066] The amino acid sequence shown in SEQ ID NO: 5 is as follows:

[0067] MINMRNRVAIVTGGAMGMGNGCARKLAEAGAKVYLIDRSNLVA

[0068] AAAQDMREAGLNANHVQVDITDQESLTSAYDGIAAESGRLDVLVNAA

[0069] GVGDSRMFLDVDDAHYKKVIDVRVRGTWNSCRAAVPHMLSNKHGRII

[0070] NFGAISGPIVAFPGYTVYALSKGAIFGFTKALASEFAGQNILVNAILPGS

[0071] MDTPMMRAAMADTNPADPQAVIDEIAAAVPLKRLGTIDDAGNLALFL

[0072] ASDLASYLTGQAIVLDGAFTLAEYQSGGLEAPAETPALVN

[0073] The present invention provides a gene encoding the above-mentioned ketoreductase mutant. The present invention mutates the gene of the wild-type ketoreductase by rational design (site-directed mutagenesis or other methods to change individual amino acids in the protein molecule) and overlap extension PCR, seamless cloning and other methods to obtain the target gene of the above-mentioned ketoreductase mutant. In a preferred embodiment of the present invention, the nucleotide sequence of the gene is the sequence shown in SEQ ID NO.6. In a preferred embodiment of the present invention, the nucleotide sequence of the gene is the sequence shown in SEQ ID NO.7. In a preferred embodiment of the present invention, the nucleotide sequence of the gene is the sequence shown in SEQ ID NO.8, or the nucleotide sequence of the gene has the same identity as the sequence shown in SEQ ID NO.8. Wherein "homology" refers to the degree of identity between two nucleotide sequences.

[0074] The nucleotide sequence shown in SEQ ID NO: 6 is as follows:

[0075] ATGATTAACATGCGCAACCGCGTGGCGATTGTGACCGGCGGCGCGATGGGC

[0076] ATGGGCAACGGCTGCGCGCGCAAACTGGCGGAAGCGGGCGCGAAAGTGTATCT

[0077] GATTGATCGCAGCAACCTGGTGGCGGCCGCGGCGCAAGATATGCGCGAAGCGG

[0078] GCCTGAACGCGAACCATGTGCAAGTGGATATTACCGATCAAGAAAGCCTGACG

[0079] AGCGCGTATGATGGCATTGCGGCGGAAAGCGGCCGCCTGGATGTGCTGGTTAAC

[0080] GCCGCGGGCGTGGGCGATAGCCGCATGTTTCTGGATGTGGATGATGCGCATTAT

[0081] AAAAAAGTGATTGATGTGCGCGTGCGCGGCACCTGGAACAGCTGCCGCGCCGC

[0082] GGTGCCGCACATGCTGAGCAACAAACATGGCCGCATTATTAACTTTGGCGCGAT

[0083] TAGCGGCCCGATTGTGGCGTTTCCGGGCTATACCGTGTATGCGCTGAGCAAAGG

[0084] CGCGATTTTTGGCTTTACCAAAGCGCTGGCGAGCGAATTTGCGGGTCAGAACAT

[0085] TCTGGTTAACGCGATTCTGCCGGGCAGCATGGATACCCCGATGATGCGCGCGGC

[0086] GGCGGCGGATACCAACCCGGCGGATCCGCAAAGCGTGATTGATGAAATTGCGG

[0087] CCGCGGTGCCACTGAAGCGCCTGGGCACCATTGATGATGCGGGCAACCTGGCGC

[0088] TGTTTCTGGCGAGCGATCTGGCGAGCTATCTGACCGGCCAAGCGATTGTGCTGG

[0089] ATGGCGCGTTTACCCTGGCGGAATATCAGAGCGGCGGCCTGGAAGCGCCGGCG

[0090] GAAACCCCGGCGCTGGTGAAC

[0091] The nucleotide sequence shown in SEQ ID NO: 7 is as follows:

[0092] ATGATTAACATGCGCAACCGCGTGGCGATTGTGACCGGCGGCGCGATGGGCATGGGCAACGGCTGCGCGCGCAAACTGGCGGAAGCGGGCGCGAAAGTGTATCTGATTGATCGCAGCAACCTGGTGGCGGCCGCGGCGCAAGATATGCGCGAAGCGGGCCTGAACGCGAACCATGTGCAAGTGGATATTACCGATCAAGAAAGCCTGACGAGCGCGTATGATGGCATTGCGGCGGAAAGCGGCCGCCTGGATGTGCTGGTTAACGCCGCGGGCGTGGGCGATAGCCGCATGTTTCTGGATGTGGATGATGCGCATTATAAAAAAGTGATTGATGTGCGCGTGCGCGGCACCTGGAACAGCTGCCGCGCCGCGGTGCCGCACATGCTGAGCAACAAACATGGCCGCATTATTAACTTTGGCGCGATTAGCGGCCCGATTGTGGCGTTTCCGGGCTATACCGTGTATGCGCTGAGCAAAGGCGCGATTTTTGGCTTTACCAAAGCGCTGGCGAGCGAATTTGCGGGTCAGAACATTCTGGTTAACGCGATTCTGCCGGGCAGCATGGATACCCCGATGATGCGCGCGGCGATGGCGGATACCAACCCGGCGGATCCGCAAAGCGTGATTGATGAAATTGCGGCCGCGGTGCCACTGAAGCGCCTGGGCACCATTGATGATGCGGGCAACCTGGCGCTGTTTCTGGCGAGCGATCTGGCGAGCTATCTGACCGGCCAAGCGATTGTGCTGGATGGCGCGTTTACCCTGGCGGAATATCAGAGCGGCGGCCTGGAAGCGCCGGCGGAAACCCCGGCGCTGGTGAAC

[0093] The nucleotide sequence shown in SEQ ID NO: 8 is as follows:

[0094] ATGATTAACATGCGCAACCGCGTGGCGATTGTGACCGGCGGCGCGATGGGC

[0095] ATGGGCAACGGCTGCGCGCGCAAACTGGCGGAAGCGGGCGCGAAAGTGTATCT

[0096] GATTGATCGCAGCAACCTGGTGGCGGCCGCGGCGCAAGATATGCGCGAAGCGG

[0097] GCCTGAACGCGAACCATGTGCAAGTGGATATTACCGATCAAGAAAGCCTGACG

[0098] AGCGCGTATGATGGCATTGCGGCGGAAAGCGGCCGCCTGGATGTGCTGGTTAAC

[0099] GCCGCGGGCGTGGGCGATAGCCGCATGTTTCTGGATGTGGATGATGCGCATTAT

[0100] AAAAAAGTGATTGATGTGCGCGTGCGCGGCACCTGGAACAGCTGCCGCGCCGC

[0101] GGTGCCGCACATGCTGAGCAACAAACATGGCCGCATTATTAACTTTGGCGCGAT

[0102] TAGCGGCCCGATTGTGGCGTTTCCGGGCTATACCGTGTATGCGCTGAGCAAAGG

[0103] CGCGATTTTTGGCTTTACCAAAGCGCTGGCGAGCGAATTTGCGGGTCAGAACAT

[0104] TCTGGTTAACGCGATTCTGCCGGGCAGCATGGATACCCCGATGATGCGCGCGGC

[0105] GATGGCGGATACCAACCCGGCGGATCCGCAAGCGGTGATTGATGAAATTGCGG

[0106] CCGCGGTGCCACTGAAGCGCCTGGGCACCATTGATGATGCGGGCAACCTGGCGC

[0107] TGTTTCTGGCGAGCGATCTGGCGAGCTATCTGACCGGCCAAGCGATTGTGCTGG

[0108] ATGGCGCGTTTACCCTGGCGGAATATCAGAGCGGCGGCCTGGAAGCGCCGGCG

[0109] GAAACCCCGGCGCTGGTGAAC

[0110] The ketoreductase obtained by the gene encoding of the present invention improves the enzyme activity and enzyme stability, making the industrial production efficiency of chiral alcohol higher and the cost lower.

[0111] The recombinant expression vector provided by the present invention contains a gene encoding the ketoreductase mutant of the present invention. The gene is located at an appropriate position of the recombinant expression vector so that the gene can be correctly and smoothly replicated, transcribed or expressed. In order to meet the requirements of the recombination operation, in the recombinant expression vector, both ends of the gene sequence can be added with suitable restriction endonuclease sites, or additional start codons, stop codons, etc. can be added. The recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. In the present invention, the recombinant expression vector includes but is not limited to pET-28a, pET-dute1 or pRSF-dute1.

[0112] The genetically engineered bacteria provided by the present invention are used to produce the above-mentioned ketoreductase mutant, which comprises the above-mentioned recombinant expression vector. In the present invention, the genetically engineered bacteria include but are not limited to Escherichia coli MG1655 or Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS.

[0113] The ketoreductase mutant can be prepared by fermenting the genetically engineered bacteria. For example, the ketoreductase mutant can be prepared industrially under certain production tank fermentation conditions. The production tank fermentation conditions are preferably: DO 20% or more, temperature 20-30°C.

[0114] In a preferred embodiment of the present invention, when the ketoreductase gene is mutated, a seamless cloning method is adopted, and the primers on the pET28a plasmid are respectively located at the upstream and downstream of the ketoreductase gene. Primers with 15bp homologous arms at both ends are set at the mutation site, and the PCR reaction conditions are: 95℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 53-60℃ annealing for 15s and 72℃ extension for 50s for 30 cycles; continue to extend at 72℃ for 10min and cool to 4℃. According to the above method, the fragment after PCR amplification is connected with the pET28a plasmid vector using a seamless cloning kit, and the connected vector is transferred into Escherichia coli BL21 (DE3) to establish a ketoreductase gene mutant. Escherichia coli BL21 (DE3) is used as a host and pET28a plasmid is used as a vector to express the extended mutant ketoreductase.

[0115] In the process of using the ketoreductase mutant of the present invention to catalyze the carbonyl compound to prepare chiral alcohol, the participation of coenzyme NADH (reduced coenzyme I) or NADPH (reduced coenzyme II) is required. Compared with the chemical method, their combined use has high stereochemical selectivity for the carbonyl substrate and one-step reduction. Wherein NAD (P) H is cyclically regenerated during the reaction process, and the regeneration method can be achieved by the process of converting isopropanol into acetone by ketoreductase, or by the process of converting glucose into gluconic acid by glucose dehydrogenase (glucose dehydrogenase, GDH), or by the process of converting formic acid into carbon dioxide by formate dehydrogenase (formate dehydrogenase, FDH). In the reaction system of the ketoreductase mutant provided by the present invention to catalyze the carbonyl compound to prepare chiral alcohol, NADP + is preferentially added.

[0116] In the description of the present invention, the term "room temperature" or "normal temperature" refers to a temperature of 4-40°C, preferably, 35±5°C.

[0117] In the description of the present invention, the amine group in the group containing amine group, ether group, thioether group or ester group is a primary amine group, a secondary amine group or a tertiary amine group. The amine group, ether group, thioether group or ester group may be located at one end of the group but not directly connected to the carbonyl group, or may be located in the middle of the group.

[0118] Compared with the prior art, the present invention has the following advantages and effects:

[0119] 1. The ketoreductase mutant provided by the present invention, in particular, the ketoreductase mutant with an amino acid sequence as shown in SEQ ID NO.5, makes the conversion rate of the compound shown in Formula I greater than 99% in 10 hours; while under the same conditions, the conversion rate of the wild-type enzyme catalytic reduction of such substrates is about 10%; in addition, the ee value of the chiral alcohol product obtained by catalytic reduction of the compound shown in Formula I by the ketoreductase mutant of the present invention can reach 99.5%, while the ee value of the chiral alcohol product obtained by catalytic reduction of the compound shown in Formula I by the wild-type enzyme under the same conditions is 0. Therefore, the catalytic activity of the ketoreductase mutant provided by the present invention on carbonyl substrates is greatly improved compared with the wild-type ketoreductase. The ketoreductase of the present invention is used to catalyze the synthesis of chiral alcohol compounds from carbonyl substrates, thereby realizing the green chemical synthesis of chiral alcohol compounds shown in Formula II.

[0120] 2. The ketoreductase mutant provided by the present invention has high catalytic activity and good stereoselectivity for carbonyl substrates, and can be applied to the biosynthesis of chiral alcohols; compared with chemical synthesis methods, the reduction reaction catalyzed by it has simple and mild conditions, high reaction selectivity, low preparation cost, and good application prospects.

[0121] The present invention is further described in detail below in conjunction with the examples, which are explanations of the present invention and are not limited to the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0122] The composition of the LB liquid (per liter) used in the following examples is: peptone 1 g ± 0.1; yeast extract 0.5 g ± 0.1; NaCl 1 g ± 0.1.

[0123] Example 1: Establishment of wild ketoreductase genetic engineering bacteria

[0124] The whole gene fragment (nucleotide sequence as shown in SEQ ID NO.8) was artificially synthesized after sequence optimization according to the wild-type gene sequence of Arthrobacter sp.TS-15 ketoreductase collected by NCBI (PDB: 6QHE_A), and the gene was inserted into the pET-28a plasmid by NdeI and XhoI endonucleases by a gene synthesis company ( Figure 1 ), the ligated vector was transferred into Escherichia coli BL21 (DE3) to establish a wild-type ketoreductase genetic engineering bacterium, and the kanamycin resistance was screened and then sequenced for verification.

[0125] Example 2: Design of ketoreductase mutants

[0126] The three-dimensional structure (6QHE_A) of Arthrobacter sp. TS-15 ketoreductase (whose amino acid sequence is shown in SEQ ID NO. 1) was obtained through PDB, and then the binding simulation of the substrate of formula I and the three-dimensional structure of the ketoreductase protein was performed through AutoDock. Finally, amino acids that may be related to substrate binding were selected as mutant amino acids through Pymol analysis.

[0127] According to the above Pymol analysis results, at least one site in the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO.1 is mutated: position 113, position 141, position 149, position 152, position 197, position 207. And position 113 is mutated from N to R or K; position 141 is mutated from S to A, D, E, G or V; position 149 is mutated from D to A or F; position 152 is mutated from W to H, L, M or Y; position 197 is mutated from A to M or F; position 207 is mutated from S to A or T. According to the docking results, the substrate binding region is analyzed, and preferably at least one site in the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO:1 is mutated: N113R, S114A, D149F, W152Y, A197M, S207A. According to Pymol analysis, the final design was to screen highly active and highly selective mutants from the combined mutation sites: (i) N113R, S114A, D149F, W152Y (corresponding to the amino acid sequence shown in SEQ ID NO.3); (ii) N113R, S114A, D149F, W152Y, A197M (corresponding to the amino acid sequence shown in SEQ ID NO.4); (iii) N113R, S114A, D149F, W152Y, A197M, S207A (corresponding to the amino acid sequence shown in SEQ ID NO.5).

[0128] Example 3 Construction of recombinant expression vector

[0129] Expression vector pET28a(+) (see Figure 1 ) were double digested with restriction endonucleases NdeI and XhoI, respectively, and the digestion system was: pET28a(+) 84uL (about 30μg), NdeII 3μL, XhoI 3μL, and agarose gel electrophoresis was performed after digestion at 37℃ for 1.5h. The target band was at the position of 0.8kb, and the digested fragment was recovered with a DNA recovery kit.

[0130] Seq ID No: 6, Seq ID No: 7, and Seq ID No: 8 were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. Using the synthetic gene of Seq ID No: 6 as a template, PCR amplification was performed with the mutation point primers P1 / P2 (N113R, S114A, D149F and W152Y in Table 1), and a band of about 0.8k was separated after agarose gel electrophoresis; the PCR amplification product was recovered using a DNA recovery kit. The vector linearized by double restriction digestion of NdeI and XhoI was connected with the PCR amplification product using a seamless cloning kit to construct a recombinant expression vector containing the ketoreductase gene (nucleotide sequence shown in Seq ID No: 6). The connection was performed according to the following reaction system: 50 ng of linearized vector pET28a (+), 100 ng of amplification product of mutation point primers P1 / P2, 5 μL of 2* seamless cloning buffer, incubated at 50°C for 20 min to obtain the recombinant expression vector, and PCR verification was performed using T7 / T7t universal primers (see Figure 2 ) and sequenced and verified, and named pET28a-i (containing the nucleotide sequence shown in SEQ ID NO: 6) after being correct. The same method was used to obtain the recombinant expression vectors pET28a-ii (containing the nucleotide sequence shown in SEQ ID NO: 7) and pET28a-iii (containing the nucleotide sequence shown in SEQ ID NO: 8).

[0131] Table 1

[0132]

[0133]

[0134] Example 4 Obtaining recombinant expression transformants

[0135] The aforementioned recombinant expression vectors pET28a-i, pET28a-ii, and pET28a-iii were transformed into BL21 (DE3) to obtain recombinant expression transformants. The transformation method used the heat shock method: the competent state was taken out from the -80°C refrigerator and placed on ice, and about 200ng to 500ng of the expression vector was added and then ice-bathed for 15min, heat-shocked at 42°C for 90s, and ice-bathed for 3min, and 500μLLB culture solution was added, and incubated at 37°C and 220rpm for 45min. 50uL was evenly spread on LB solid culture medium containing 50μg / ml, and inverted and cultured overnight to obtain a single clone. After expansion, it was stored at -80°C with 20% final concentration of glycerol. After sequencing analysis and verification, it was a recombinant expression transformant, named BL21 (DE3) -i, BL21 (DE3) -ii, and BL21 (DE3) -iii.

[0136] Example 5 Shake flask culture and fermentation of recombinant expression transformants

[0137] The recombinant expression transformants BL21(DE3)-i, BL21(DE3)-ii, and BL21(DE3)-iii prepared in Example 4 were inoculated into LB liquid medium (added with 100 μg / ml kanamycin) and cultured overnight at 37°C and 220 rpm; the culture was transferred into 50 mL fresh LB medium (1 g±0.1 peptone per liter; 0.5 g±0.1 yeast extract; 1 g±0.1 NaCl per liter, 250 mL shake flask) at a ratio of 1:100 and grown at 37°C. When the optical density at 600 nm (OD 600 ) reached about 0.6, isopropylthiogalactoside (IPTG) was added to a final concentration of 1 mM, and the cells were grown at 25°C for 16 hours. Centrifuged at 12000rpm and 4°C for 10 min, the supernatant was discarded, and the cell pellet was the whole cell. It was resuspended in pre-cooled 100mM Tris-HCl buffer (pH7.5) at 200g / L, ultrasonically disrupted, and then centrifuged at 12000rpm and 4°C for 30 min. The supernatant was collected, i.e. the crude enzyme solution, and stored at -20°C. Shake flask expression polyacrylamide gel electrophoresis, the results are shown in Figure 2 and Figure 3 .in Figure 2 The lanes from left to right are Marker, protease of BL21(DE3)-iii (whose amino acid sequence is Seq ID No: 5), protease of BL21(DE3)-ii (whose amino acid sequence is Seq ID No: 4), protease of BL21(DE3)-i (whose amino acid sequence is Seq ID No: 3), wild type, and blank; Figure 3 The polyacrylamide gel electrophoresis results of Marker and BL21(DE3)-iii protease.

[0138] The three recombinant expression transformants (BL21 (DE3) -i, BL21 (DE3) -ii, BL21 (DE3) -iii) obtained by fermentation in this embodiment, and the wild-type ketoreductase genetic engineering bacteria obtained in Example 1 were catalyzed to reduce the compound shown in the above formula I to a chiral alcohol in the following manner: 9.75ml 0.1M Tris-Hcl, 250ul NADP+ (20g / L), 1g substrate (compound shown in formula I), 0.12g glucose, 0.01g glucose dehydrogenase (Shanghai Shangke Biopharmaceutical Co., Ltd., ES-GDH-109, 42U / mg), 0.4g whole cells. 40℃ / 200rpm reaction for 10 hours and sampling for determination. The reaction solutions of the reduction reaction of the four recombinant expression transformants were respectively subjected to HPLC detection, and the results are shown in Table 2 below.

[0139] Table 2.

[0140] type Conversion rate (%) ee value (%) wild type 10 0 BL21(DE3)-i 90 ++ BL21(DE3)-ii 95 +++ BL21(DE3)-iii 99 ++++

[0141] Note: In the above table, ++ represents that the ee value is greater than or equal to 40% and less than 60%, +++ represents that the ee value is greater than or equal to 60% and less than 80%, and ++++ represents that the ee value is greater than or equal to 80% and less than 100%.

[0142] Example 6: High-density fermentation preparation of recombinant expression transformants

[0143] The recombinant mutant strain (BL21(DE3)-iii) obtained in Example 4 was inoculated into 3 mL of liquid LB medium, and cultured overnight at 37°C and 220 rpm, and then inoculated into 400 mL of liquid LB medium at a ratio of about 1%, and cultured until OD 600 When it reaches 4, it is used as seed liquid and inoculated into 2L fermentation medium for high-density fermentation. The initial temperature is 37°C, the stirring speed is 300rpm, the ventilation volume is 1.5vvm / L / min, and the pH is 6.8. Then the stirring speed is continuously increased to a maximum of 1000rpm. The fermentation culture is divided into two stages. After the first stage is inoculated, the carbon source in the culture medium is completely consumed after about 4 hours of culture, and the feeding is fed back according to the DO. After feeding, the temperature drops to 25°C, and the dissolved oxygen is maintained above 30%. After 8 hours of feeding, isopropylthiogalactoside (IPTG) is added for induction, and the tank is released after 12 hours of induction. The wet cells of the bacteria obtained by centrifuging at 8000rpm for 10 minutes and discarding the supernatant are named KRED-APTI-116.

[0144] Example 7: Whole-cell catalytic reaction system of ketoreductase

[0145] At room temperature, 97.5 ml 0.1 M Tris-Hcl, 2.5 ml NADPNa (BiDe Pharmaceuticals, BD116591, 20 g / L), 10 g substrate (the compound shown in the above formula I), 1.2 g glucose, 0.1 g glucose dehydrogenase (Shanghai Suntech Biopharmaceuticals Co., Ltd., ES-GDH-109, 42 U / mg), and 4 g KRED-APTI-116 were mixed evenly, reacted at 40°C / 200 rpm for 10 hours, and samples were taken for determination. During the reaction, the pH was monitored and controlled at 7.5, and the mixture was sampled for HPLC analysis. After 8 hours, the complete conversion rate reached 95.9%; after 10 hours, the complete conversion rate reached 99.9%. The target product was detected by HPLC, and the ee value was >99% ( Figure 4 ). The target product was characterized by hydrogen spectrum, and the results are shown in Figure 5 .

[0146] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A ketoreductase mutant, characterized in that The amino acid sequence of the ketoreductase mutant is the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

2. The ketoreductase mutant according to claim 1, characterized in that The amino acid sequence of the ketoreductase mutant is the amino acid sequence shown in SEQ ID NO:

5.

3. A gene encoding the ketoreductase mutant according to claim 1.

4. The gene according to claim 3, characterized in that The nucleotide sequence of the gene is the nucleotide sequence shown in SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.

8.

5. The gene according to claim 4, characterized in that The nucleotide sequence of the gene is the nucleotide sequence shown in SEQ ID NO.

8.

6. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the gene according to claim 3 or 4.

7. The recombinant expression vector according to claim 6, characterized in that The recombinant expression vector is selected from pET-28a, pET-dute1 or pRSF-dute1.

8. The recombinant expression vector according to claim 7, characterized in that: The recombinant expression vector is selected from pET-28a.

9. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria is used to produce the ketoreductase mutant according to claim 1, and comprises the recombinant expression vector according to claim 6 or 7.

10. The genetically engineered bacterium according to claim 9, characterized in that: The genetically engineered bacteria are selected from Escherichia coli MG1655, Escherichia coli BL21 (DE3), or Escherichia coli BL21 (DE3) pLysS.

11. The genetically engineered bacterium according to claim 10, characterized in that: The genetically engineered bacteria is selected from Escherichia coli BL21 (DE3).

12. Use of the ketoreductase mutant according to claim 1 in preparing chiral alcohol compounds using carbonyl compounds as raw materials.

13. The use according to claim 12, characterized in that: The structural formula of the carbonyl compound is Wherein, R1 and R2 are each independently C1-C8 alkyl, C5-C 10 Cycloalkyl, C5~C 10 Aryl, C5~C 10 a heteroaryl group or a group containing an amine group, an ether group, a thioether group or an ester group, or R1 and R2 together with the carbon on the carbonyl group form a 5-10 membered ring, the 5-10 membered ring is entirely composed of carbon, or the 5-10 membered ring contains heteroatoms other than carbon atoms, the heteroatoms are selected from N, O and S, and the 5-10 membered ring is unsubstituted or substituted with at least one group selected from halogen, oxygen, sulfur, hydroxyl, alkoxy or alkyl, The structural formula of the chiral alcohol compound is 14. The use according to claim 13, wherein the carbonyl compound is a compound of formula I, and the alcohol compound is a compound of formula II,

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