A process for the production of chiral alcohols

Abstract

The present application relates to the technical field of medicine and chemical industry, and particularly relates to a method for producing chiral alcohol, which comprises using a ketoreductase mutant to catalyze a reduction reaction of a prochiral ketone compound to produce the chiral alcohol, wherein the ketoreductase mutant is SEQ ID NO:1 or an amino acid sequence having more than 80% identity with SEQ ID NO:1. The method of the present application can efficiently produce chiral alcohol, and has high stereoselectivity, which is beneficial to industrial production.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical and chemical technology, and specifically to a method for producing chiral alcohols. Background Technology

[0002] Ketoreductases are versatile catalysts that selectively reduce aldehydes or ketones to their corresponding alcohols via enantiomeric reduction. (R)-specific ketone reductases differ from (S)-specific ketone reductases, and these catalysts are frequently used in the synthesis of optically active alcohols. The reactions catalyzed by ketone reductases require the participation of cofactors, 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] 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 RY-amino-β-hydroxybutyric acid (GABOB).

[0006] Currently, there are two main methods for the asymmetric reduction of ketone reductase to prepare (R)-CHBE: chemical and biological methods. The chemical method uses catalysts such as rhodium and ruthenium, requires a certain hydrogen pressure for asymmetric reduction, and the optical purity of the product is relatively low.

[0007] Biological methods have attracted widespread attention due to their advantages of mild reaction conditions, high specificity, and high conversion rates. Japanese scholars, including Kataoka et al., conducted research on ketone reductase derived from *Sporobolomyces salmonicolor*, heterologously expressing the enzyme and ultimately catalyzing a high concentration of 4-chloro-acetoacetate at 0.3 kg / L with a chiral purity of 91%-93%. Chinese patent application CN103160547A describes the asymmetric reduction of 4-chloro-acetoacetate by an alcohol dehydrogenase derived from *Candida albicans*, using resting cells as a catalyst and NADH as a cofactor, to catalyze the preparation of (R)-CHBE. However, the substrate concentration was only 25-50 g / L, and the substrate conversion rate was relatively low. Summary of the Invention

[0008] To address the problems existing in the prior art, the present invention provides a ketone reductase mutant with enhanced enzyme activity and its application.

[0009] According to one aspect of the present invention, a method for producing chiral alcohols is provided. The method includes the step of using a ketone reductase to catalyze the reduction reaction of a prochiral ketone compound to produce a chiral alcohol, wherein the ketone reductase is a mutant of any of the aforementioned ketone reductases. Because the ketone reductase mutants of the present invention possess excellent activity characteristics, the chiral alcohols prepared using these mutants can increase the reaction rate, increase the substrate concentration, reduce the amount of enzyme used, and simplify post-processing.

[0010] Furthermore, the prochiral ketone compounds have the following structure: Formula I:

[0011] ,

[0012] R' and R" are each independently alkyl, alkylaryl, alkylheteroaryl, cycloalkyl, aryl, or heteroaryl, or R' and R" together with the carbon on the carbonyl group to form a heterocyclic group, carbocyclic group, or heteroaryl group. The heteroatoms in the heterocyclic group and heteroaryl group are each independently selected from at least one of nitrogen, oxygen, and sulfur. The aryl group in the alkylaryl group, the aryl group in the aryl group, the heteroaryl group in the alkylheteroaryl 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 halogen, alkoxy, nitro, or alkyl.

[0013] Preferably, R' and R" are each independently C1-C8 alkyl, C5-C10 cycloalkyl, C5-C10 aryl, or C5-C10 heteroaryl, or R' and R" together with the carbon on the carbonyl group to form a C5-C10 heterocyclic group, a C5-C10 carbocyclic group, or a C5-C10 heteroaryl group, wherein 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 halogen, alkoxy, nitro, or alkyl.

[0014] Preferably, the structural formula of the prochiral ketone compound is shown in Formula I-1:

[0015] ,

[0016] R1 or R2 is selected from hydrogen, halogen, C1-C8 alkyl, C5-C10 cycloalkyl, C5-C10 aryl or C5-C10 heteroaryl, wherein the alkyl, cycloalkyl, aryl or heteroaryl group is independently unsubstituted or substituted by at least one group selected from halogen, alkoxy, nitro or alkyl; R3 is selected from hydrogen, halogen, C1-C3 alkyl.

[0017] More preferably, the prochiral ketone compound is or .

[0018] Furthermore, the reaction system for producing chiral alcohols by reducing prochiral ketone compounds with ketone reductase also includes a coenzyme, a coenzyme regeneration system, and a buffer solution.

[0019] Furthermore, the concentration of the prochiral ketone compound in the reaction system is 1 g / L to 200 g / L;

[0020] Furthermore, the pH value of the reaction system is 5.0–9.0, and the reaction temperature of the reaction system is 4–60℃.

[0021] Furthermore, the coenzyme is NADH, NADPH, or NAD+.

[0022] Further, the coenzyme regeneration system is selected from, but not limited to, the following: (i) isopropanol, coenzyme NADH or NAD+; (ii) glucose (e.g., D-glucose), coenzyme NADH or NAD+, glucose dehydrogenase (GDH); and (iii) formate compounds (e.g., formate), coenzyme NADH or NAD+, formate dehydrogenase (FDH). In some embodiments using purified ketone reductase, such cofactors and optionally such cofactor regeneration systems are typically added to the reaction medium along with the substrate and ketone reductase. Similar to ketone reductase, 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 extract or lysate can express an enzyme containing only a cofactor regeneration system or containing both a cofactor regeneration system and ketone reductase. In embodiments using whole cells, the cells can express an enzyme containing both a cofactor regeneration system and ketone reductase.

[0023] Further preferably, a single ketone reductase can be used, whether whole cell, cell extract or purified ketone reductase is used, or alternatively, a mixture of two or more ketone reductases can be used.

[0024] Further, the buffer solution is phosphate buffer, Tris-HCl buffer, sodium barbital-HCl buffer, or citrate-sodium citrate buffer.

[0025] According to another aspect of the present invention, a mutant of ketone reductase is provided. The ketone reductase mutant is:

[0026] The mutant of the amino acid sequence shown in SEQ ID NO:1 has a mutation site including T67. Further, the ketone reductase mutation includes an amino acid sequence with more than 80% identity to SEQ ID NO:1, and the identity sequence contains the mutation site T67; preferably, the ketone reductase mutation includes an amino acid sequence with more than 85% identity to SEQ ID NO:1, and the identity sequence contains the mutation site T67; more preferably, the ketone reductase mutation includes an amino acid sequence with more than 90% identity to SEQ ID NO:1, and the identity sequence contains the mutation site T67; more preferably, the ketone reductase mutation includes an amino acid sequence with more than 95% identity to SEQ ID NO:1, and the identity sequence contains the mutation site T67; most preferably, the ketone reductase mutation includes an amino acid sequence with more than 98% identity to SEQ ID NO:1, and the identity sequence contains the mutation site T67.

[0027] The mutant obtained by the mutation of this invention can efficiently produce chiral alcohols from prochiral ketone compounds through stereoselective reduction, and is suitable for industrial production of chiral alcohols.

[0028] Furthermore, the mutation site also includes at least one or a combination of two or more of the following sites: V14, I42, A97, G170, A242, I262 and F286; or the amino acid sequence of the ketone reductase mutant has the mutation site in the mutated amino acid sequence and has more than 95% identity with the mutated amino acid sequence. Further, the mutation of the ketone reductase includes an amino acid sequence having more than 80% identity with Y (for ease of description, the mutation site includes T67, and includes any one or a combination of two or more of the following sites: V14, I42, A97, G170, A242, I262, F286, defined as Y), and the identity sequence contains a combination of mutation site T67 and one or more of the mutation sites V14, I42, A97, G170, A242, I262, F286; preferably, the mutation of the ketone reductase includes an amino acid sequence having more than 85% identity with Y, and the identity sequence contains a combination of mutation site T67 and one or more of the mutation sites V14, I42, A97, G170, A242, I262, F286; more preferably, the ketone reductase... The mutation comprises an amino acid sequence having more than 90% identity with Y, wherein the identity sequence contains a mutation site T67 and a combination of one or more mutation sites of V14, I42, A97, G170, A242, I262, and F286; more preferably, the ketone reductase mutation comprises an amino acid sequence having more than 95% identity with Y, wherein the identity sequence contains a mutation site T67 and a combination of one or more mutation sites of V14, I42, A97, G170, A242, I262, and F286; most preferably, the ketone reductase mutation comprises an amino acid sequence having more than 98% identity with Y, wherein the identity sequence contains a mutation site T67 and a combination of one or more mutation sites of V14, I42, A97, G170, A242, I262, and F286.

[0029] Preferably, the mutation site further includes at least one or more combinations of the following mutations: V14E, I42L, A97H, G170R, A242L, I262A, and F286S. More preferably, for ease of description, the mutation site includes T67, and includes any one or more of the following sites or combinations of two or more sites: mutants of V14E, I42L, A97H, G170R, A242L, I262A, and F286S, defined as Y'. Preferably, the ketone reductase mutation comprises an amino acid sequence having more than 80% identity with Y', and the identity sequence contains a mutation site T67, and a combination of one or more mutation sites of V14E, I42L, A97H, G170R, A242L, I262A, and F286S; preferably, the ketone reductase mutation comprises an amino acid sequence having more than 85% identity with Y', and the identity sequence contains a mutation site T67, and a combination of one or more mutation sites of V14E, I42L, A97H, G170R, A242L, I262A, and F286S; preferably, the ketone reductase mutation comprises an amino acid sequence having more than 90% identity with Y', and the identity sequence contains a mutation site T67, and a combination of V14E, I42L, A97H, G170R, A242L, I262A, and F286S. A composition comprising one or more mutation sites of V14E, I42L, A97H, G170R, A242L, I262A, and F286S; more preferably, the mutation of the ketone reductase comprises an amino acid sequence having more than 95% identity with Y', and the identity sequence contains the mutation site T67, and a composition comprising one or more mutation sites of V14E, I42L, A97H, G170R, A242L, I262A, and F286S; most preferably, the mutation of the ketone reductase comprises an amino acid sequence having more than 98% identity with Y', and the identity sequence contains the mutation site T67, and a composition comprising one or more mutation sites of V14E, I42L, A97H, G170R, A242L, I262A, and F286S.

[0030] Preferred,

[0031] Exemplarily, the mutation of the ketoreductase further includes any combination of the following site 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+I 42L+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.

[0032] According to another aspect of the invention, a DNA molecule is provided. This DNA molecule encodes the aforementioned ketoreductase mutant or identity sequence.

[0033] According to another aspect of the present invention, a recombinant plasmid is provided. This recombinant plasmid is linked to the aforementioned DNA molecule.

[0034] The term "plasmid" as used in this invention includes any plasmid, granule, bacteriophage, 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 is preferred to be a prokaryotic expression plasmid.

[0035] 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, pQE3 2. pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-1, pGEX-6 p-2, pBV220, pBV221, pBV222, pTrc99A, pTwinl, pEZZ18, pKK232-18, pUC-18 and pUC-19.

[0036] According to another aspect of the present invention, a host cell is provided. The host cell contains any of the recombinant plasmids described above.

[0037] Furthermore, the host cell includes prokaryotic cells, yeast, or eukaryotic cells.

[0038] Preferred prokaryotic cells are bacteria, such as Gram-negative or Gram-positive bacteria. More preferred prokaryotic cells are Escherichia coli BL21(DE3), BL21 Star(DE3), Turner™(DE3), Rosetta™ 2(DE3), BLR(DE3), NovaBlue(DE3), Origami™(DE3), and Origami B(DE3).

[0039] The mutant obtained by the present invention can efficiently produce chiral alcohols from prochiral ketone compounds through stereoselective reduction, and is suitable for industrial production of chiral alcohols. Attached Figure Description

[0040] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

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

[0042] Figure 2 The results of ee values ​​of the enzyme-catalyzed products of the wild type in Example 4 at different temperatures are shown;

[0043] Figure 3 The results of ee values ​​of the enzyme-catalyzed products of the mutant in Example 4 at different temperatures are shown;

[0044] Figure 4 The ee value spectrum of the mutant in Example 4 is shown. Detailed Implementation

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

[0046] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0047] Explanation of the name:

[0048] Ketoreductases are polypeptides capable of reducing ketone groups to their corresponding alcohols. Specifically, the ketone reductase polypeptide of this application can stereoselectively reduce ketone compounds to their corresponding alcohol products. This polypeptide typically utilizes either reduced nicotinamide adenine dinucleotide (NADH) or oxidized nicotinamide adenine dinucleotide (NAD+) as a reducing agent. In this application, ketone reductases include naturally occurring (wild-type) ketone reductases and non-naturally occurring ketone reductase mutants generated through artificial treatment.

[0049] "Naturally occurring" or "wild-type" is the opposite of "mutant" and refers to the form found in nature. For example, naturally occurring or wild-type polypeptide or polynucleotide sequences are sequences that exist in organisms, can be isolated from natural sources, and have not been intentionally modified or altered artificially.

[0050] In this application, when referring to, for example, cells, nucleic acids, or polypeptides as "recombinant," it means cells, nucleic acids, or polypeptides that have been modified in a manner not found in nature, or that are identical to those found in nature but are prepared or derived from synthetic materials and / or through the use of recombinant technology, or that correspond to the natural or inherent form of the cell, nucleic acid, or polypeptide. Non-limiting embodiments include recombinant cells that express genes other than their inherent (non-recombinant) forms or express inherent genes at different levels.

[0051] The "percentage of sequence identity" refers to the comparison between polynucleotides and is determined by comparing two best-aligned sequences across a comparison window. The portion of the polynucleotide sequence in the comparison window compared to the reference sequence may include additions or deletions (i.e., vacancies) for optimal alignment of the two sequences. The percentage can be calculated as follows: by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue appears to generate a 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. Alternatively, the percentage can be calculated as follows: by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue aligns with a vacancy position to generate a 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. Here, the "reference sequence" refers to the designated sequence used as the basis for sequence comparison. The reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence.

[0052] 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 genomic or 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 DNA, making it a very useful tool in gene research.

[0053] Saturation mutagenesis is a technique that modifies the gene encoding a target protein to quickly generate mutants where the target amino acid is replaced by one of 19 other natural amino acids. Point saturation mutagenesis can be used to identify protein functional sites, improve enzyme specific activity, and enhance various properties such as enzyme thermostability, substrate binding specificity, and stereoisomer specificity.

[0054] Ketoreductase mutants derived from *Rhodococcus ruber*, such as T67R (in this invention, "T67R" represents "original amino acid + site + mutated amino acid," i.e., T at position 67 is changed to R), can catalyze the target substrate to the product, but their stability needs further improvement. This invention aims to improve substrate conversion and stereoselectivity through directed evolution.

[0055] In this application, a mutation site is first introduced into the ketoreductase via site-directed mutagenesis, and the mutants are then subjected to activity testing to select those with enhanced activity. For example, the mutant T67R exhibits increased enzyme activity compared to the starting template.

[0056] Introducing site-directed mutagenesis using whole-plasmid PCR is a simple and effective method, and is currently widely used. The principle is as follows: a pair of primers (forward and reverse) containing the mutation site, and the template plasmid, after annealing, are extended using polymerase in a "cyclic extension" process (cyclic extension refers to the polymerase extending the primers according to the template, returning to the 5' end of the primer after one cycle, and then repeating the extension cycle with heating and annealing; this reaction differs from rolling circle amplification and does not form multiple tandem copies). The extension products of the forward and reverse primers are paired after annealing to form a notched open circular plasmid. The extension products are digested with Dpn I. Since the original template plasmid is derived from conventional E. coli and is modified with dam methylation, it is sensitive to Dpn I and is cleaved. However, the in vitro synthesized plasmid with the mutation sequence is not methylated and is not cleaved. Therefore, it is successfully transformed in subsequent transformations, resulting in a clone of the mutant plasmid. The mutant plasmid is transformed into host cells, inducing the expression of the target protein. Then, crude enzyme solution is obtained by sonicating the cells. The optimal conditions for ketone reductase expression induction were: 25℃, 0.1mM IPTG induction for 16h.

[0057] The DNA molecule described in this invention can also exist in the form of an "expression cassette." An "expression cassette" refers to a linear or circular nucleic acid molecule encompassing DNA and RNA sequences capable of directing the expression of a specific nucleotide sequence in an appropriate host cell. Generally, it includes a promoter effectively linked to the target nucleotide, optionally linked to a termination signal and / or other regulatory elements. The expression cassette may also include the sequence required for the correct translation of the nucleotide sequence. The coding region typically encodes the target protein, but may also encode the target functional RNA in the sense or antisense direction, such as antisense RNA or untranslated RNA. Expression cassettes containing the target polynucleotide sequence can be chimeric, meaning that at least one of its components is heterologous to at least one of its other components. Expression cassettes can also be naturally occurring, but are obtained through efficient recombination for heterologous expression.

[0058] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the aforementioned DNA molecules. The DNA molecules in the recombinant plasmid are positioned at appropriate locations within the plasmid, enabling the DNA molecules to be correctly and smoothly replicated, transcribed, or expressed.

[0059] Although the present invention uses the qualifier "contains" when defining the aforementioned DNA molecule, this 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 will know that, in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cleavage sites to both ends of the DNA sequence, or to add additional promoters, stop codons, etc. Therefore, using a closed-form expression to define it will not truly cover these situations.

[0060] Example 1

[0061] Construction of recombinant Escherichia coli Rosetta (pET-28a-RR)

[0062] 1.1 Obtaining the ketoreductase gene

[0063] Rhodococcus ruber (purchased from China General Microbiological Culture Collection Center, No. CGMCC 1.10360) was cultured at 30℃ for 1 day. Seed culture medium (g / L): glycerol 10, peptone 5, malt extract 3, yeast extract 3, pH 7.0.

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

[0065] Upstream and downstream primers were designed using ketone reductase gene information from the NCBI database. The primer sequences are as follows:

[0066] Upstream primer (containing Nde I site):

[0067] 5'-GGAATTCCATATGAAAGCCCTCCAGTACACCGAGA-3' (SEQ ID NO: 2);

[0068] Downstream primer (containing Xho I site):

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

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

[0071] Gene PCR amplification conditions:

[0072] Denature at 98℃ for 3 minutes, then cycle 30 times with the following parameters: denature at 98℃ for 10 seconds, anneal at 58℃ for 5 seconds, extend at 72℃ for 1.5 minutes, and finally extend at 72℃ for 4 minutes.

[0073] 1.2 Construction of strains

[0074] The expression vector pET-28a (purchased from Novagen (Merck China)) and the amplified target gene containing both restriction enzyme sites were double-digested with Nde I and Xho I. The double-digested target fragment and expression vector were recovered separately using gel electrophoresis. The double-digested expression vector pET-28a and the target gene were ligated overnight using T4 ligase. 10 μL of the ligation product pET-28a-RR was added to Rosetta (DE3) competent cells, incubated on ice for 30 min, and then heat-shocked at 42°C for 90 sec. The cells were then incubated on ice for 2 min. 1 mL of LB medium was added, and the cells were cultured at 37°C with shaking at 200 rpm for 0.5 h. The bacterial culture was then plated onto LB agar plates containing 50 mM kanamycin and incubated overnight at 37°C to obtain recombinant E. coli Rosetta (pET-28a-RR).

[0075] 1.3 Protein sequence alignment

[0076] Plasmids were extracted from the constructed recombinant E. coli Rosetta (pET-28a-RR) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results showed that the amplified target gene sequence was highly homologous to sequence MW808991.1 in the GenBanK library, with a mutation occurring at positions 199 to 201, where the codon ATT was 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] 100mM substrate stock solution: Weigh 6.76 mg of ethyl 4-chloroacetoacetate, dissolve it in isopropanol, stir and mix until completely dissolved;

[0081] NADH stock solution 10mM: Weigh 33.17 mg of NADH and dissolve it in 5 mL of 0.1 M PB pH 7.0 buffer.

[0082] 1.4.2 Enzyme activation system:

[0083] First, add the enzyme, then add the substrate ethyl 4-chloroacetoacetate, a mixture of NADH and buffer, place it in a microplate reader, and detect the enzyme activity at 30℃ and 340nM wavelength.

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

[0085] Table 1

[0086]

[0087] The ketone reductase mutant T67R, referred to as the "template" in this invention, and the listed mutation sites are mutations performed on the basis of the "template".

[0088] Preparation method of enzyme solution in high-throughput screening: Centrifuge a 96-well plate to remove the supernatant culture medium, add 200 μL of enzyme digestion solution (lysozyme 2 mg / mL, polymyxin 0.5 mg / mL, pH 7.0) to each well, and treat at 37℃ for 2 h.

[0089] Enzyme catalysis and detection method: First, add the enzyme, then add the substrate ethyl 4-chloroacetoacetate, a mixture of NADH and buffer, and catalyze for a certain period of time. Transfer the catalyzed sample to an enzyme detection plate, place it in a microplate reader, and detect enzyme activity at 30℃ and 340 nm wavelength.

[0090] Example 2

[0091] Irrationally modified recombinant E. coli Rosetta (pET-28a-RR)

[0092] 2.1 Codon Optimization for Rhodococcus ruber

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

[0094] 2.2 Primer Design

[0095] Upstream primers were designed based on the ketone reductase gene information. The primer sequences are as follows:

[0096] Upstream primer (containing Nde I site):

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

[0098] Downstream primer (containing Xho I site):

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

[0100] 2.3 Construction and Screening of Strain Mutation Libraries

[0101] 2.3.1 Construction of the mutant library: The codon-optimized recombinant E. coli Rosetta (pET-28a-RR) was amplified and cultured. Plasmid extraction was performed using a plasmid extraction kit from Sangon Biotech (Shanghai) Co., Ltd., and the obtained plasmids were used as templates for mutant amplification. Base mutations were introduced during amplification using a ready-to-use error-prone PCR kit from Beijing Tianenze Biotechnology Co., Ltd. The strain was constructed according to section 1.2 of Example 1, and all the resulting mutant strains constituted the mutant library.

[0102] 2.3.2 Screening of mutant libraries: The mutant libraries obtained above were amplified and cultured in 96-well plates, and protein expression was induced. Enzyme activity was measured according to 1.4 in Example 1. Mutant strains with higher enzyme activity than recombinant E. coli Rosetta (pET-28a-RR) were selected for shake-flask culture and protein expression, and enzyme activity was measured.

[0103] Example 3

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

[0105] 3.1 Site-directed mutagenesis

[0106] Plasmids were extracted from the mutant strain with increased 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 sequence. Primers were designed for the mutated amino acid sites and synthesized at Sangon Biotech (Shanghai) Co., Ltd. Site-directed mutagenesis was performed according to Example 2 to construct the mutant strain. Finally, it was determined that the mutation sites V14, I42, T67, A97, G170, A242, I262, and F286 significantly increased enzyme activity.

[0107] Table 2

[0108]

[0109] + indicates enzyme activity; the more +s, the higher the enzyme activity.

[0110] 3.2 Saturation Mutation

[0111] Saturation mutations were performed on amino acid sites V14, I42, T67, A97, G170, A242, I262, and F286. Primers were designed and synthesized using degenerate bases provided by Sangon Biotech (Shanghai) Co., Ltd. Saturation mutations were performed according to Example 2 to construct mutant strains, which were then screened. Results showed that saturation mutations at amino acid sites V14, T67, A97, G170, A242, and F286 did not significantly alter enzyme activity. However, mutations at I42 and I262 resulted in four mutant strains (I42L, I42V, I262A, and I262G) with increased enzyme activity.

[0112] 3.3 Mutation Combinations

[0113] Furthermore, the mutation sites were combined accordingly to screen for mutants with high enzyme activity: the template and the mutant were catalytically reacted at 30℃, and their activities were then measured. The enzyme activity results of all mutants are shown in Table 3.

[0114] Table 3

[0115]

[0116] + indicates enzyme activity; the more +s, the higher the enzyme activity.

[0117] Combinatorial saturation mutagenesis can yield mutants with synergistic effects among several mutation sites, and the amino acid composition can be optimized. Using T67R as a template, mutagenesis combinations were performed. The enzyme solution was reacted at 30℃ for 17 hours, then the reaction was terminated, and the activity was measured.

[0118] Example 4

[0119] 4.1 Optimal mutant enzyme catalysis

[0120] Different substrate reactions were validated using the mutant V14E+I42L+A97H+G170R+A242L+I262A, and the results are shown in Table 4.

[0121] Add 0.5 g of substrate (I-1), 0.1 M PB pH 7.0, 0.2 g isopropanol, 20 mg NAD+, and 0.05 g ketone reductase mutant to a 100 mL reaction flask, mix well, and the total volume is 20 mL. Incubate at 30 °C and 200 rpm on a shaker for 1 h.

[0122] ,

[0123] R1 or R2 is selected from hydrogen, halogen, C1-C8 alkyl, C5-C10 cycloalkyl, C5-C10 aryl or C5-C10 heteroaryl, wherein the alkyl, cycloalkyl, aryl or heteroaryl group is independently unsubstituted or substituted by at least one group selected from halogen, alkoxy, nitro or alkyl; R3 is selected from hydrogen, halogen, C1-C3 alkyl.

[0124] Table 4

[0125]

[0126] 4.2 Optimal mutant enzyme catalysis

[0127] Using recombinant 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 to the substrate ethyl 4-chloroacetoacetate within the temperature range of 25℃-35℃ are as follows: Figure 1 The conversion of the substrate ethyl 4-chloroacetoacetate and the product ee value of the mutant V14E+I42L+A97H+G170R+A242L+I262A (SEQ ID NO:6) at different temperatures were investigated. The catalytic results are as follows: Figure 1 , Figure 2 , Figure 3 and Figure 4 Compared with the recombinant E. coli Rosetta (pET-28a-RR), the catalytic time of the mutant V14E+I42L+A97H+G170R+A242L+I262A was shortened from 17 h to 1 h, and the catalytic conversion rate was greater than 99%. Within the temperature range of 25℃-35℃, the ee value of the recombinant E. coli Rosetta (pET-28a-RR) decreased significantly with increasing temperature, while for the mutant V14E+I42L+A97H+G170R+A242L+I262A, increasing temperature did not significantly change the ee value.

[0128] 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%, and a space-time conversion rate of 1440 g / L / d, exhibiting extremely high catalytic efficiency. Second, the catalytic system operates under mild conditions, primarily at room temperature, in a neutral, aqueous environment, with minimal use of organic solvents. The reaction process is less hazardous, environmentally friendly, meets current production requirements, and possesses significant commercial potential.

[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. SEQUENCE LISTING <110> Anhui Puli Pharmaceutical Co., Ltd. <120> A method for producing chiral alcohols <130> hnpoly001 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 346 <212> PRT <213> artificial sequence <220> <223> The ketone reductase 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> forward 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> Includes the V14E+I42L+A97H+G170R+A242L+I262A mutation. <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 method for producing a chiral alcohol, the method comprising using a ketone reductase mutant to catalyze the reduction reaction of a prochiral ketone compound to produce the chiral alcohol. The amino acid sequence of the ketone reductase mutant is shown in SEQ ID NO:6; The structure of the prochiral ketone compound is shown below:

2. The method according to claim 1, characterized in that, The prochiral ketone compound is or .

3. The method according to claim 1 or 2, characterized in that, The reaction system contains a coenzyme, a coenzyme regeneration system, and a buffer solution. The concentration of the prochiral ketone compound in the reaction system is 1 g / L to 200 g / L; The pH value of the reaction system is 5.0 to 9.0, and the reaction temperature is 4 to 60°C. The coenzyme is NADH or NADPH. The buffer solution is a phosphate buffer, Tris-HCl buffer, sodium barbital-HCl buffer, or citrate-sodium citrate buffer.

4. The method according to claim 3, wherein the coenzyme regeneration system is any one of the following systems: (1) a system composed of isopropanol and coenzyme NADH or NAD+; (2) a system composed of glucose, glucose dehydrogenase and coenzyme NADH or NAD+; (3) a system composed of formate compound, formate dehydrogenase and coenzyme NADH or NAD+.

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