Ketoreductase mutants and uses thereof

Abstract

The present application relates to a kind of ketoreductase mutant and its application, the ketoreductase mutant is obtained by the amino acid sequence shown in SEQ ID NO:1 Mutations, the mutation at least includes one in following mutation site: the 94th by H mutation F or M, the 146th by T mutation A, G or V, the 161st by Y mutation A or T, the 190th by C mutation H, L or Q, the 195th by A mutation K or E.The ketoreductase mutant can be applied to the green chemical synthesis of chiral alcohol compound, catalytic activity is high, and stability is good.Compared with chemical synthesis method, the reaction catalyzed by it is simple and mild, reaction selectivity is high, preparation cost is low, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a ketone reductase mutant and its applications. Background Technology

[0002] Currently, enzymatic synthesis is widely used in the chemical industry for the large-scale production of pharmaceuticals or their chiral intermediates. Reactions catalyzed by enzymes have many advantages, such as being able to proceed in benign solvents under mild reaction conditions, exhibiting good specificity and stereoselectivity. However, enzyme-catalyzed reactions also have their drawbacks, including: lack of stability under production reaction conditions, product inhibition, the need for aqueous media, and the poor water solubility of many substrates of interest, preventing efficient reaction. To overcome these shortcomings, various techniques have been employed to modify enzymes, such as directed evolution, random mutagenesis, and protein engineering, enabling the efficient and environmentally friendly use of enzymes in the synthesis of chemical molecules, such as active pharmaceutical ingredients.

[0003] Ketoreductases (KREDs) (EC 1.1.1) are a type of aldehyde-ketone reductase that catalyzes the reduction of carbonyl groups to chiral alcohols. Examples of KRED-catalyzed reductions of prochiral ketones (carbonyl groups) to chiral alcohols have been reported, including some industrial-scale applications. The synthesis of montelukast is a prime example, where KREDs were used to replace existing and expensive chemical reducing agents (DIP-Cl) in the synthesis of this drug. The prior art has also reported the use of ketone reductases to synthesize chiral alcohols in the synthesis of drugs such as sulfopenem, 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 ketone reductases varies greatly for different non-natural substrates. For example, the same ketone reductase may be very effective at catalyzing the reduction of some non-natural carbonyl substrates to chiral alcohols, but may have no catalytic effect or very poor catalytic effect for other specific non-natural carbonyl substrates, and there are also significant differences in stability and selectivity. Therefore, for many carbonyl compounds, the problem in using ketone reductases to catalyze their reduction to chiral alcohols is that there are no suitable ketone reductases in the existing technology because these ketone reductases are mostly ineffective in catalysis. It is necessary to find suitable ketone reductases or improve the activity and specificity of known ketone reductases through rational design to promote the application of ketone reductases in the synthesis and industrial production of chiral alcohols. Summary of the Invention

[0004] To address the low efficiency and selectivity in the chemical synthesis of specific chiral alcohols, as well as the low activity and poor stability of existing ketone reductases catalyzing the reduction of specific carbonyl bases to chiral alcohols, one objective of this invention is to provide a ketone reductase mutant with high catalytic activity and good stereoselectivity for specific carbonyl bases. To achieve this objective, the invention employs the following technical solution:

[0005] A ketone reductase mutant, wherein the amino acid sequence of the ketone reductase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO:1, wherein the mutation includes at least one of the following mutation sites: H to F or M at position 94, T to A, G or V at position 146, Y to A or T at position 161, C to H, L or Q at position 190, and A to K or E at position 195; or the amino acid sequence of the ketone reductase 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%, or 99% 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-mentioned mutation includes at least one of the following mutation sites: H to F at position 94, T to A at position 146, Y to T at position 161, C to Q at position 190, and A to K at position 195; or the amino acid sequence of the ketone reductase 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%, or 99% homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence.

[0007] In some preferred embodiments of the present invention, the above-mentioned ketone reductase mutant includes the following mutation sites: position 94 is mutated from H to F, position 146 is mutated from T to A, and position 161 is mutated from Y to T. In some preferred embodiments of the present invention, the above-mentioned ketone reductase mutant includes the following mutation sites: position 94 is mutated from H to F, position 146 is mutated from T to A, position 161 is mutated from Y to T, and position 190 is mutated from C to Q. In some preferred embodiments of the present invention, position 94 is mutated from H to F, position 146 from T to A, position 161 from Y to T, position 190 from C to Q, and position 195 from A to K; or the amino acid sequence of the ketone reductase 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%, or 99% homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence.

[0008] Another objective of the present invention is to provide a gene encoding the above-mentioned ketone reductase mutant.

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

[0011] Another aspect of the present invention aims to provide a recombinant expression vector containing a gene encoding the aforementioned ketone reductase mutant.

[0012] In some preferred embodiments of the present invention, the recombinant expression vector is selected from pET-28a, pET-dute1, or 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 aims to provide a genetically engineered bacterium for producing the above-mentioned ketone reductase mutant, which comprises the above-mentioned recombinant expression vector.

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

[0016] Another objective of this invention is to provide the application of the above-mentioned ketone reductase mutant in the catalytic preparation of chiral alcohol compounds from carbonyl compounds.

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

[0018] Wherein, R1 and R2 are each independently C1-C8 alkyl, C5-C6 alkyl, C6-C7 alkyl, C6-C8 alkyl, C6-C7 ... 10 cycloalkyl, C5-C 10 Aryl, C5~C 10 The group consists of heteroaryl groups or groups containing amino, ether, thioether, or ester groups, or R1 and R2 together with the carbonyl group to form a 5- to 10-membered ring.

[0019] The 5- to 10-membered rings are entirely composed of carbon, or the 5- to 10-membered rings contain heteroatoms in addition to carbon atoms, wherein the heteroatoms are selected from N, O, S, and

[0020] The 5- to 10-membered ring is either unsubstituted or substituted with at least one group selected from halogen, oxygen, sulfur, hydroxyl, alkoxy, or alkyl groups.

[0021] The structural formula of the chiral alcohol compound is as follows:

[0022] 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:

[0023]

[0024] The ketone reductase mutant of the present invention is based on the ketone reductase shown in SEQ ID NO:1, and is mutated by site-directed mutagenesis to change its amino acid sequence, thereby altering the protein structure and function. Ketoreductase with the aforementioned mutation site is then obtained through targeted screening. Compared with wild-type ketone reductase, the ketone reductase mutant of the present invention exhibits significantly improved selectivity. Under the same conditions, the ee value of the product obtained from reduction is increased by 99.7% compared to 0 for wild-type ketone reductase, and the enzyme activity is also significantly improved. When used in the production of chiral alcohols, it significantly reduces the cost of industrial production of chiral alcohols. Attached Figure Description

[0025] Figure 1 This is a diagram of the recombinant expression vector for ketone reductase of the present invention.

[0026] Figure 2The lanes shown from left to right are the polyacrylamide gel electrophoresis results for the marker, wild type (amino acid sequence SEQ ID NO:1), BL21(DE3)-i protease (amino acid sequence Seq ID No.3), BL21(DE3)-ii protease (amino acid sequence Seq ID No.4), and BL21(DE3)-iii protease (amino acid sequence Seq ID No.5).

[0027] Figure 3 The results of polyacrylamide gel electrophoresis for the marker and BL21(DE3)-iii protease.

[0028] Figure 4 The image shows the liquid chromatogram of the chiral alcohol product obtained in Example 7. Detailed Implementation

[0029] Ketoreductases derived from Arthrobacter sp. TS-15 can selectively reduce carbonyl compounds, but their activity and selectivity in catalyzing the reduction of compounds shown in Formula I to chiral alcohols are low. The inventors of this invention have improved the activity and selectivity of ketone reductases derived from Arthrobacter sp. TS-15 through a rationally designed method. Mutation sites were introduced into the ketone reductase derived from Arthrobacter sp. TS-15 using whole-plasmid PCR, and the activity and stability of the mutants were tested to select mutants with improved activity and stability.

[0030] The ketone reductase mutant gene provided by this invention is derived from the wild-type gene of *Arthrobacter* sp. TS-15. The nucleotide sequence of this wild-type gene is shown in SEQ ID NO:1, and the codon-optimized gene sequence for *E. coli* is shown in SEQ ID NO:2. Here, "wild-type" refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences present in organisms, which can be isolated from natural sources and have not been intentionally modified by human intervention. Enzymes obtained after expression of these genes exhibit low catalytic activity and poor thermal stability for certain substrates.

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

[0032] MNGRIMLVEGKNGIVTGAAGGIGRASAVAFAREGANVVIGDLQARLEDLEETARQ

[0033] VKKAGGEARVLVMDVTALADQEHAVRTVIENFGTLDDFAHNNAGIELQKTALETTE

[0034] EEWDRVHDVNLKGVFLGMKAQLRAMIDNGGGSIVNTASAAGILGLPGYSGYASSK

[0035] HGVVGLTKSVAVEAADTGVRINAVCPASIATPMLLSLPAEEQETLLSWQAIKRLGK

[0036] PDEVAQAVVWLASDRASFITGVSLPVDAGYSAF

[0037] The cDNA of ketone reductase derived from Arthrobacter sp. TS-15, with codon optimization for E. coli, is shown in SEQ ID NO.2:

[0038] ATGAACGGCCGCATTATGCTGGTGGAAGGCAAAAACGGCATTGTGACCGGCGC

[0039] GGCGGGCGGTATCGGTCGTGCGAGCGCCGTGGCGTTTGCGCGCGAAGGCGCGA

[0040] ACGTGGTGATTGGCGATCTGCAAGCGCGCCTGGAAGATCTGGAAGAAACCGCG

[0041] CGCCAAGTGAAAAAAGCGGGCGGCGAAGCGCGCGTGCTGGTGATGGATGTGAC

[0042] CGCGCTGGCGGATCAAGAACATGCGGTGCGCACCGTGATTGAAAACTTTGGCAC

[0043] CCTGGATTTTGCGCATAACAACGCGGGCATTGAACTGCAGAAAACCGCGCTGGA

[0044] AACCACCGAAGAGGAATGGGATCGCGTGCATGATGTGAACCTGAAAGGCGTGT

[0045] TTCTGGGCATGAAAGCGCAGCTGCGCGCGATGATTGATAACGGCGGTGGCAGC

[0046] ATTGTGAACACCGCGAGCGCGGCGGGCATTCTGGGCCTGCCGGGCTACAGCGGT

[0047] TACGCGAGCAGCAAACATGGCGTGGTGGGCCTGACCAAAAGCGTGGCGGTGGA

[0048] AGCGGCGGATACCGGCGTGCGCATTAACGCGGTGTGCCCGGCGAGCATTGCGA

[0049] CCCCGATGCTGCTGAGCCTGCCGGCGGAAGAACAAGAAACCCTGCTGAGCTGG

[0050] CAAGCGATTAAACGCCTGGGCAAACCGGATGAAGTGGCGCAAGCGGTGGTGTG

[0051] GCTGGCGAGCGATCGCGCGAGCTTTATTACCGGCGTGAGCCTGCCGGTGGACGC

[0052] CGGTTATAGCGCGTTT

[0053] This invention utilizes an online protein structure prediction tool to obtain the three-dimensional structure of ketoreductase derived from Arthrobacter sp. TS-15. The three-dimensional structure of cyclohexanol dehydrogenase (4ure.1.A, 46.75% homology), which has the highest structural similarity to Arthrobacter sp. TS-15, is then obtained via PDB for structural comparison. AutoDock is used to simulate the binding of the Formula I substrate to the ketoreductase protein. Finally, Pymol analysis is used to select amino acids potentially related to substrate binding as mutant amino acids. Based on the Pymol analysis results, multiple pairs of site-directed mutagenesis primers (H94F / M, T146A / G / V, Y161A / T, C190H / L / Q, A195K / E) are designed. Using site-directed mutagenesis with pET-28a as the expression vector, a mutant plasmid carrying the target gene is obtained. Site-directed mutagenesis refers to the introduction of desired changes (usually changes that characterize a favorable direction) into a target DNA fragment (which can be a genome or a plasmid) using methods such as polymerase chain reaction (PCR). These changes include base addition, deletion, and point mutations. Site-directed mutagenesis can rapidly and efficiently improve the traits and characterization of the target protein expressed by the DNA, making it a very useful tool in gene research.

[0054] The ketone reductase mutant provided by the present invention is an amino acid sequence obtained by mutating the amino acid sequence shown in SEQ ID NO:1. The mutation includes at least one of the following mutation sites: H at position 94 is mutated to F or M, T at position 146 is mutated to A, G or V, Y at position 161 is mutated to A or T, C at position 190 is mutated to H, L or Q, and A at position 195 is mutated to K or E. In a preferred embodiment of the present invention, the mutation includes at least one of the following mutation sites: H to F at position 94 (H94F), T to A at position 146 (T146A), Y to T at position 161 (Y161T), C to Q at position 190 (C190Q), and A to K at position 195 (A195K); or the amino acid sequence of the ketone reductase 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%, or 99% homology with the mutated amino acid sequence, and has the same function as the mutated amino acid sequence. Here, "homology" refers to the degree of similarity between two amino acid sequences. The sequences defined by different degrees of homology in the present invention must also simultaneously possess improved ketone reductase activity. The phrase "same function" refers to the ability to catalyze the reduction of carbonyl substrates to chiral alcohols, similar to the mutated amino acid sequence. Those skilled in the art can obtain the amino acid sequence of the ketone reductase mutant under the guidance of this disclosure, which possesses the mutation sites in the aforementioned mutated amino acid sequence and exhibits at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% homology with the mutated amino acid sequence.

[0055] In a more preferred embodiment of the present invention, the above-mentioned ketone reductase mutant includes mutation sites H94F, T146A, and Y161T, and its amino acid sequence is shown in SEQ ID NO: 3. In a more preferred embodiment of the present invention, the above-mentioned ketone reductase mutant includes mutation sites H94F, T146A, Y161T, and C190Q, and its amino acid sequence is shown in SEQ ID NO: 4. In a more preferred embodiment of the present invention, the above-mentioned ketone reductase mutant includes mutation sites H94F, T146A, Y161T, C190Q, and A195K, and its amino acid sequence is shown in SEQ ID NO: 5.

[0056] The amino acid sequence shown in SEQ ID NO: 3 is as follows:

[0057] MNGRIMLVEGKNGIVTGAAGGIGRASAVAFAREGANVVIGDLQARLEDLEETARQ

[0058] VKKAGGEARVLVMDVTALADQEHAVRTVIENFGTLDFAFNNAGIELQKTALETTE

[0059] EEWDRVHDVNLKGVFLGMKAQLRAMIDNGGGSIVNAASAAGILGLPGYSGTASSK

[0060] HGVVGLTKSVAVEAADTGVRINAVCPASIATPMLLSLPAEEQETLLSWQAIKRLGK

[0061] PDEVAQAVVWLASDRASFITGVSLPVDAGYSAF

[0062] The amino acid sequence shown in SEQ ID NO: 4 is as follows:

[0063] MNGRIMLVEGKNGIVTGAAGGIGRASAVAFAREGANVVIGDLQARLEDLEETARQ

[0064] VKKAGGEARVLVMDVTALADQEHAVRTVIENFGTLDFAFNNAGIELQKTALETTE

[0065] EEWDRVHDVNLKGVFLGMKAQLRAMIDNGGGSIVNAASAAGILGLPGYSGTASSK

[0066] HGVVGLTKSVAVEAADTGVRINAVQPASIATPMLLSLPAEEQETLLSWQAIKRLGK

[0067] PDEVAQAVVWLASDRASFITGVSLPVDAGYSAF

[0068] The amino acid sequence shown in SEQ ID NO: 5 is as follows:

[0069] MNGRIMLVEGKNGIVTGAAGGIGRASAVAFAREGANVVIGDLQARLEDLEETARQ

[0070] VKKAGGEARVLVMDVTALADQEHAVRTVIENFGTLDFAFNNAGIELQKTALETTE

[0071] EEWDRVHDVNLKGVFLGMKAQLRAMIDNGGGSIVNAASAAGILGLPGYSGTASSK

[0072] HGVVGLTKSVAVEAADTGVRINAVQPASIKTPMLLSLPAEEQETLLSWQAIKRLGK

[0073] PDEVAQAVVWLASDRASFITGVSLPVDAGYSAF

[0074] This invention provides a gene encoding the aforementioned ketone reductase mutant. This invention obtains the target gene of the aforementioned ketone reductase mutant by mutating the wild-type ketone reductase gene through rational design (site-directed mutagenesis or other methods to alter individual amino acids in the protein molecule) and methods such as overlap extension PCR and seamless cloning. In a preferred embodiment of this invention, the nucleotide sequence of the gene is the sequence shown in SEQ ID NO. 6. In a preferred embodiment of this invention, the nucleotide sequence of the gene is the sequence shown in SEQ ID NO. 7. In a preferred embodiment of this invention, the nucleotide sequence of the gene is the sequence shown in SEQ ID NO. 8, or the nucleotide sequence of the gene has at least 80%, at least 85%, or at least 90%, 93%, 95%, 96%, 97%, 98%, or 99% homology to the sequence shown in SEQ ID NO. 8.

[0075] The nucleotide sequence shown in SEQ ID NO.6 is as follows:

[0076] ATGAACGGCCGCATTATGCTGGTGGAAGGCAAAAACGGCATTGTGACGGGCGCGGCGGGT

[0077] GGCATTGGTCGCGCGAGTGCGGTGGCGTTTGCGCGCGAAGGCGCGAACGTGGTGATTGGCG

[0078] ATCTGCAAGCGCGCCTGGAAGATCTGGAAGAAACCGCGCGCCAAGTGAAAAAAGCGGGCG

[0079] GCGAAGCGCGCGTGCTGGTGATGGATGTGACCGCGCTGGCGGATCAAGAACATGCGGTGCG

[0080] CACCGTGATTGAAAACTTTGGCACCCTGGATTTTGCGTTTAACAACGCGGGCATTGAACTGC

[0081] AGAAAACCGCGCTGGAAACCACCGAAGAGGAATGGGATCGCGTGCATGATGTGAACCTGA

[0082] AAGGCGTGTTTCTGGGCATGAAAGCGCAGCTGCGCGCGATGATTGATAACGGTGGCGGTAG

[0083] TATTGTGAACGCGGCGAGCGCGGCGGGCATCCTGGGCCTGCCGGGCTACAGCGGCACCGCG

[0084] AGCAGCAAACATGGCGTGGTGGGCCTGACCAAAAGCGTGGCGGTGGAAGCGGCGGATACC

[0085] GGCGTGCGCATTAACGCGGTGTGTCCGGCGAGCATTGCGACCCCGATGCTGCTGAGCCTGC

[0086] CGGCCGAGGAACAAGAAACCCTGCTGAGCTGGCAAGCGATTAAACGCCTGGGCAAACCGG

[0087] ATGAAGTGGCGCAAGCGGTGGTGTGGCTGGCGAGCGATCGCGCGAGCTTTATTACCGGCGT

[0088] TAGCCTGCCGGTTGATGCGGGCTATAGCGCGTTT

[0089] The nucleotide sequence shown in SEQ ID NO.7 is as follows:

[0090] ATGAACGGCCGCATTATGCTGGTGGAAGGCAAAAACGGCATTGTGACGGGCGC

[0091] GGCGGGTGGCATTGGTCGCGCGAGTGCGGTGGCGTTTGCGCGCGAAGGCGCGA

[0092] ACGTGGTGATTGGCGATCTGCAAGCGCGCCTGGAAGATCTGGAAGAAACCGCG

[0093] CGCCAAGTGAAAAAAGCGGGCGGCGAAGCGCGCGTGCTGGTGATGGATGTGAC

[0094] CGCGCTGGCGGATCAAGAACATGCGGTGCGCACCGTGATTGAAAACTTTGGCAC

[0095] CCTGGATTTTGCGTTTAACAACGCGGGCATTGAACTGCAGAAAACCGCGCTGGA

[0096] AACCACCGAAGAGGAATGGGATCGCGTGCATGATGTGAACCTGAAAGGCGTGT

[0097] TTCTGGGCATGAAAGCGCAGCTGCGCGCGATGATTGATAACGGTGGCGGTAGTA

[0098] TTGTGAACGCGGCGAGCGCGGCGGGCATCCTGGGCCTGCCGGGCTACAGCGGC

[0099] ACCGCGAGCAGCAAACATGGCGTGGTGGGCCTGACCAAAAGCGTGGCGGTGGA

[0100] AGCGGCGGATACCGGCGTGCGCATTAACGCGGTGCAGCCGGCGAGCATTGCGA

[0101] CCCCGATGCTGCTGAGCCTGCCGGCCGAGGAACAAGAAACCCTGCTGAGCTGG

[0102] CAAGCGATTAAACGCCTGGGCAAACCGGATGAAGTGGCGCAAGCGGTGGTGTG

[0103] GCTGGCGAGCGATCGCGCGAGCTTTATTACCGGCGTTAGCCTGCCGGTTGATGC

[0104] GGGCTATAGCGCGTTT

[0105] The nucleotide sequence shown in SEQ ID NO.8 is as follows:

[0106] ATGAACGGCCGCATTATGCTGGTGGAAGGCAAAAACGGCATTGTGACGGGCGCGGC

[0107] GGGTGGCATTGGTCGCGCGAGTGCGGTGGCGTTTGCGCGCGAAGGCGCGAACGTGGT

[0108] GATTGGCGATCTGCAAGCGCGCCTGGAAGATCTGGAAGAAACCGCGCGCCAAGTGA

[0109] AAAAAGCGGGCGGCGAAGCGCGCGTGCTGGTGATGGATGTGACCGCGCTGGCGGAT

[0110] CAAGAACATGCGGTGCGCACCGTGATTGAAAACTTTGGCACCCTGGATTTTGCGTTTA

[0111] ACAACGCGGGCATTGAACTGCAGAAAACCGCGCTGGAAACCACCGAAGAGGAATGG

[0112] GATCGCGTGCATGATGTGAACCTGAAAGGCGTGTTTCTGGGCATGAAAGCGCAGCTG

[0113] CGCGCGATGATTGATAACGGTGGCGGTAGTATTGTGAACGCGGCGAGCGCGGCGGGC

[0114] ATCCTGGGCCTGCCGGGCTACAGCGGCACCGCGAGCAGCAAACATGGCGTGGTGGGC

[0115] CTGACCAAAAGCGTGGCGGTGGAAGCGGCGGATACCGGCGTGCGCATTAACGCGGT

[0116] GCAGCCGGCGAGCATTAAAACCCCGATGCTGCTGAGCCTGCCGGCCGAGGAACAAG

[0117] AAACCCTGCTGAGCTGGCAAGCGATTAAACGCCTGGGCAAACCGGATGAAGTGGCGC

[0118] AAGCGGTGGTGTGGCTGGCGAGCGATCGCGCGAGCTTTATTACCGGCGTTAGCCTGC

[0119] CGGTTGATGCGGGCTATAGCGCGTTT

[0120] The ketone reductase encoded by the gene of this invention improves enzyme activity and stability, resulting in higher efficiency and lower cost in the industrial production of chiral alcohols.

[0121] The recombinant expression vector provided by this invention contains a gene encoding the ketoreductase mutant of this invention. This gene is located at an appropriate position in the recombinant expression vector, enabling the gene to be correctly and smoothly replicated, transcribed, or expressed. To meet the requirements of recombination operations, suitable restriction endonuclease sites, or additional start codons, stop codons, etc., can be added to both ends of the gene sequence in this recombinant expression vector. This recombinant expression vector can be a prokaryotic expression vector or a eukaryotic expression vector. In this invention, the recombinant expression vector includes, but is not limited to, pET-28a, pET-dute1, or pRSF-dute1.

[0122] The genetically engineered bacteria provided by this invention are used to produce the above-mentioned ketone reductase mutant, which contains the above-mentioned recombinant expression vector. In this invention, the genetically engineered bacteria include, but are not limited to, Escherichia coli MG1655, Escherichia coli BL21(DE3), and Escherichia coli BL21(DE3)pLysS.

[0123] The aforementioned ketone reductase mutant can be prepared by fermenting the genetically engineered bacteria. For example, the ketone reductase mutant can be prepared industrially under specific fermentation conditions in a production tank. The preferred fermentation conditions in the production tank are: DO ≥ 20% and room temperature.

[0124] In a preferred embodiment of the present invention, a seamless cloning method is used when mutating the ketone reductase gene. The primers on the pET28a plasmid are located upstream and downstream of the ketone reductase gene, respectively. Primers with 15 bp homologous arms at both ends are set at the mutation site. The PCR reaction conditions are: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 53-60℃ annealing for 15 s and 72℃ extension for 50 s for 30 cycles; further extension at 72℃ for 10 min, and cooling to 4℃. After PCR amplification according to the above method, the fragment is ligated to the pET28a plasmid vector using a seamless cloning kit. The ligated vector is then transformed into E. coli BL21(DE3) to establish a ketone reductase gene mutant. Using E. coli BL21(DE3) as the host and the pET28a plasmid as the vector, the extended mutant ketone reductase is expressed.

[0125] The ketone reductase mutant provided by this invention can catalyze the reduction of carbonyl compounds to chiral alcohols. It requires the participation of coenzymes NADH (reduced coenzyme I), NADP+, or NADPH (reduced coenzyme II). Compared with chemical methods, their combined use exhibits high stereoselectivity and chemoselectivity for carbonyl compounds, achieving one-step catalysis. NAD(P)H is cyclically regenerated during the reaction. Regeneration can be achieved through the conversion of isopropanol to acetone by ketone reductase, the conversion of glucose to gluconic acid by glucose dehydrogenase (GDH), or the conversion of formic acid to carbon dioxide by formate dehydrogenase (FDH). In the reaction system for the preparation of chiral alcohols from carbonyl compounds catalyzed by the ketone reductase mutant provided by this invention, NADP+ is preferentially added.

[0126] In the description of this invention, the terms "room temperature" or "normal temperature" refer to a temperature of 4-40°C, preferably 35±5°C.

[0127] Compared with the prior art, the present invention has the following advantages and effects:

[0128] 1. The ketone reductase mutant provided by this invention, particularly the ketone reductase mutant with the amino acid sequence shown in SEQ ID NO. 5, achieves a 10-hour conversion rate greater than 99% for the carbonyl substrate shown in Formula I; while the wild-type enzyme achieves a 10-hour conversion rate of approximately 30% for the same substrate. Furthermore, the ee value of the chiral alcohol product obtained by the reduction of the carbonyl substrate shown in Formula I by this enzyme mutant can reach 99.7%, while the ee value of the chiral alcohol product obtained by the reduction of the carbonyl substrate shown in Formula I by the wild-type enzyme under the same conditions is 0. The ketone reductase mutant provided by this invention exhibits significantly improved catalytic activity for carbonyl substrates compared to the wild type, realizing a green chemical synthesis of chiral alcohol compounds using ketone reductase catalysis. Moreover, using the enhanced-activity ketone reductase for the reduction reaction easily yields highly active and selective chiral alcohol compounds.

[0129] 2. The ketone reductase mutant provided by this 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 it catalyzes is simple and mild, has high reaction selectivity, low preparation cost, and has good application prospects.

[0130] The present invention will be further described in detail below with reference to the embodiments. These embodiments are illustrative of the invention, and the invention is not limited to them. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0131] The LB liquid used in the following examples consisted of: 1 g ± 0.1 g peptone; 0.5 g ± 0.1 g yeast extract; and 1 g ± 0.1 g NaCl.

[0132] Example 1: Establishment of a genetically engineered strain of wild-type ketone reductase

[0133] The complete gene fragment (nucleotide sequence shown in SEQ ID NO. 8) was artificially synthesized after sequence optimization based on the wild-type gene sequence of Arthrobacter sp. TS-15 ketoreductase (WP_142940120.1) indexed by NCBI. The gene was then inserted into the pET-28a plasmid by a gene synthesis company using NdeI and XhoI restriction enzymes. Figure 1 The ligated vector was transferred into Escherichia coli BL21(DE3) to establish a wild-type ketone reductase genetically engineered bacterium, which was then screened for kanamycin resistance and sequenced for verification.

[0134] Example 2: Design of Ketoreductase Mutants

[0135] The three-dimensional structure of ketone reductase from Arthrobacter sp. TS-15 was obtained using an online protein structure prediction tool, and the three-dimensional structure of cyclohexanol dehydrogenase (4ure.1.A), which has the highest structural similarity to it, was obtained using PDB. Then, the binding simulation of the three-dimensional structure of ketone reductase protein with substrate of formula I was performed using AutoDock. Finally, Pymol analysis was used to select amino acids that may be related to substrate binding as mutant amino acids.

[0136] Based on the Pymol analysis results above, at least one site in the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO:1 should be mutated: positions 94, 146, 161, 190, and 195. Specifically, position 94 should be mutated from H to F or M, position 146 from T to A, G, or V, position 161 from Y to A or T, position 190 from C to H, L, or Q, and position 195 from A to K or E. Based on the docking results and analysis of the substrate binding region, it is preferable to mutate at least one site in the amino acid sequence of the wild-type ketoreductase shown in SEQ ID NO:1: H94F, T146A, Y161T, C190Q, and A195K. Based on Pymol analysis, the final design selected highly active and selective mutants from the following combined mutation sites: (i) H94F, T146A, Y161T (corresponding to the amino acid sequence shown in SEQ ID NO.3); (ii) H94F, T146A, Y161T, C190Q (corresponding to the amino acid sequence shown in SEQ ID NO.4); and (iii) H94F, T146A, Y161T, C190Q, A195K (corresponding to the amino acid sequence shown in SEQ ID NO.5).

[0137] Example 3: Construction of recombinant expression vector:

[0138] Expression vector pET28a(+) (see) Figure 1 The sample was double-digested with restriction endonucleases NdeI and XhoI, respectively. The digestion system consisted of 84 μL (approximately 30 μg) of pET28a(+), 3 μL of NdeI, and 3 μL of XhoI. After digestion at 37°C for 1.5 h, agarose gel electrophoresis was performed. The target band was located at 0.76 kb, and the digested fragments were recovered using a DNA recovery kit.

[0139] The coding sequences for the three ketone reductases, Seq ID No:6, Seq ID No:7, and Seq ID No:8, were synthesized by Suzhou Genewiz Biotechnology Co., Ltd. Using the synthesized gene of Seq ID No:6 as a template, PCR amplification was performed using primers P1 / P2 (H94F, T146A, and Y161T in Table 1). A band of approximately 0.76 kb was separated after agarose gel electrophoresis. The PCR amplification product was recovered using a DNA recovery kit. The linearized vector, double-digested with NdeI and XhoI, was ligated to the PCR amplification product using a seamless cloning kit to construct a recombinant expression vector containing the ketone reductase gene (nucleotide sequence shown in Seq ID No:6). Ligation was performed according to the following reaction system: 50 ng of linearized vector pET28a(+), 100 ng of primer P1 / P2 amplification product, and 5 μL of 2* seamless cloning buffer. Incubation was performed at 50°C for 20 min to obtain the recombinant expression vector, which was verified using T7 / T7t universal primers (see [link to relevant documentation]). Figure 2 The sequence was then sequenced and verified. Once confirmed, it was named pET28a-i (containing the nucleotide sequence shown in SEQ ID NO:6). 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).

[0140] Table 1

[0141]

[0142] Example 4: Obtaining the recombinant expression transformant

[0143] The recombinant expression vectors pET28a-i, pET28a-ii, and pET28a-iii were transformed into BL21(DE3) to obtain recombinant expression transformants. The transformation method employed heat shock: competent cells were removed from a -80°C freezer and placed on ice. Approximately 200-500 ng of expression vector was added, followed by an ice bath for 15 min, a heat shock at 42°C for 90 s, an ice bath for 3 min, the addition of 500 μL of LB medium, and incubation at 37°C and 220 rpm for 45 min. 50 μL of the transformed cells were then evenly spread onto LB solid medium containing 50 μg / ml, incubated upside down overnight to obtain single clones. After expansion, the transformed cells were stored at -80°C with a final concentration of 20% glycerol. Sequencing analysis confirmed their correctness, and these were the recombinant expression transformants, named BL21(DE3)-i, BL21(DE3)-ii, and BL21(DE3)-iii.

[0144] Example 5: Shake-flask culture and fermentation of recombinant expression transformants

[0145] The recombinant expression transformants (BL21(DE3)-i, BL21(DE3)-ii, and BL21(DE3)-iii) obtained in Example 4 were inoculated into LB liquid medium (with 100 μg / ml kanamycin) and cultured overnight at 37°C and 220 rpm. The cultures were then transferred at a 1:100 ratio into 50 mL of fresh LB medium (1 g ± 0.1 peptone, 0.5 g ± 0.1 yeast extract, and 1 g ± 0.1 NaCl per liter, in a 250 mL shake flask) and grown at 37°C. The optical density (OD) at 600 nm was measured. 600 When the concentration reached approximately 0.6, isopropyl galactothioglycoside (IPTG) was added to bring the final concentration to 1 mM. Cells were grown at 25°C for 16 hours. After centrifugation at 12000 rpm and 4°C for 10 min, the supernatant was discarded. The cell pellet was resuspended in pre-chilled 100 mM Tris-HCl buffer (pH 7.5) at 200 g / L, sonicated, and then centrifuged at 12000 rpm and 4°C for 30 min. The supernatant, i.e., the crude enzyme solution, was collected and stored at -20°C. Polyacrylamide gel electrophoresis was performed on shake-flask expression cells; the results are shown below. Figure 2 and Figure 3 .in Figure 2 The lanes from left to right are: Marker, BL21(DE3)-iii protease (its amino acid sequence is Seq ID No:5), BL21(DE3)-ii protease (its amino acid sequence is Seq ID No:4), BL21(DE3)-i protease (its amino acid sequence is Seq ID No:3), wild type, and blank. Figure 3 The results of polyacrylamide gel electrophoresis for the Marker and BL21(DE3)-iii protease.

[0146] Three recombinant expression transformants (BL21(DE3)-i, BL21(DE3)-ii, and BL21(DE3)-iii) obtained by fermentation in this example, and the wild-type ketone reductase genetically engineered bacteria obtained in Example 1, were used to catalyze the reduction of the compound shown in Formula I to chiral alcohols in the following manner: 9.75 ml 0.1 M Tris-HCl, 250 μL NADPNa (Biode Pharmaceuticals, BD116591, 20 g / L), 1 g substrate (compound shown in Formula I), 0.12 g glucose, 0.01 g glucose dehydrogenase (Shanghai Shangke Biopharmaceutical Co., Ltd., ES-GDH-109, 42 U / mg), and 0.4 g whole cells. The reaction was carried out at 40 °C / 200 rpm for 10 hours, and samples were taken for analysis. The reaction solutions of the reduction reactions of the four recombinant expression transformants were analyzed by HPLC, and the results are shown in Table 2 below.

[0147] Table 2.

[0148] Type Conversion rate (%) ee value (%) Wild type 30 0 BL21(DE3)-i 90 ++ BL21(DE3)-ii 95 +++ BL21(DE3)-iii 99 ++++

[0149] Note: In the table above, ++ represents an ee value greater than or equal to 40% and less than 60%, +++ represents an ee value greater than or equal to 60% and less than 80%, and ++++ represents an ee value greater than or equal to 80% and less than 100%.

[0150] Example 6: High-density fermentation preparation of recombinant expression transformant (BL21(DE3)-iii)

[0151] 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 with shaking at 220 rpm. Then, it was inoculated into 400 mL of liquid LB medium at a ratio of approximately 1% and cultured until OD. 600 When the culture reaches pH 4, it is used as seed culture and inoculated into 2L of fermentation medium for high-density fermentation. The initial temperature is 37℃, the stirring speed is 300rpm, the aeration rate is 1.5vvm / L / min, and the pH is 6.8. Subsequently, the stirring speed is continuously increased to a maximum of 1000rpm. The fermentation culture is divided into two stages. In the first stage, after inoculation, the culture is incubated for about 4 hours until the carbon source in the medium is completely consumed, and then fed back according to DO (dissolved oxygen) guidelines. After feeding back, the temperature is reduced to 25℃, and dissolved oxygen is maintained above 30%. Eight hours after feeding back, isopropyl thiogalactoside (IPTG) is added for induction. After 12 hours of induction, the culture is transferred to the fermentation tank. The bacterial cells obtained by centrifugation at 8000rpm for 10min and discarding the supernatant are KRED-APTI-115.

[0152] Example 7: Whole-cell catalytic reaction system of ketone reductase

[0153] At room temperature, 97.5 ml of 0.1 M Tris-HCl, 2.5 ml of NADPNa (Biode Pharmaceuticals, BD116591, 20 g / L), 10 g of substrate (compound I mentioned above), 1.2 g of glucose, 0.1 g of glucose dehydrogenase (Shanghai Shangke Biopharmaceutical Co., Ltd., ES-GDH-109, 42 U / mg), and 4 g of KRED-APTI-115 were mixed thoroughly and reacted at 40 °C / 200 rpm for 10 hours before sampling and analysis. The pH was monitored and maintained at 7.5 during the reaction, and the mixture was sampled for HPLC analysis. After 8 hours, the complete conversion rate reached 97.5%; after 10 hours, the complete conversion rate reached 99.9%. The target product, as determined by HPLC, had an ee value > 99%. Figure 4 .

[0154] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within 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 of claim 1, wherein, 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 of claim 1.

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

8.

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

7. The recombinant expression vector of claim 6, wherein, The recombinant expression vector is selected from pET-28a, pET-dute1 or pRSF-dute1.

8. The recombinant expression vector of claim 7, wherein, The recombinant expression vector is selected from pET-28a.

9. A genetically engineered bacterium, characterized by, The genetically engineered bacteria for producing the ketoreductase mutant of claim 1 or 2 comprises the recombinant expression vector of claim 6 or 7. 10.The genetically engineered bacterium of claim 9, characterized in that, The genetically engineered bacteria is selected from Escherichia coli MG1655 or Escherichia coli BL21 (DE3) or Escherichia coli BL21 (DE3) pLysS.

11. The genetically engineered bacteria of claim 10, wherein, The genetically engineered bacteria is selected from Escherichia coli BL21 (DE3).

12. Use of the ketoreductase mutant of claim 1 or 2 in preparing a chiral alcohol compound from a carbonyl compound, The carbonyl compound is a compound shown in formula I, and the chiral alcohol compound is a compound shown in formula II, 。

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