CN110713992A

CN110713992A - Ketoreductase mutant and method for producing chiral alcohol

Info

Publication number: CN110713992A
Application number: CN201911083349.5A
Authority: CN
Inventors: 洪浩; 詹姆斯·盖吉; 肖毅; 张娜; 焦学成; 张克俭; 杨益明; 王翔; 张燕青
Original assignee: Kaileying Pharmaceutical Chemistry Fuxin Technology Co Ltd
Current assignee: Kaileying Pharmaceutical Chemistry Fuxin Technology Co Ltd; Asymchem Laboratories Fuxin Co Ltd
Priority date: 2019-11-07
Filing date: 2019-11-07
Publication date: 2020-01-21
Anticipated expiration: 2039-11-07
Also published as: CN110713992B

Abstract

The invention discloses a ketoreductase mutant and a method for producing chiral alcohol. Wherein, the ketoreductase mutant has a sequence with amino acid mutation of the sequence shown in SEQ ID NO. 1, and the mutation site comprises K200H. The mutant obtained by mutation can efficiently produce chiral alcohol by using a ketone compound as a raw material through stereoselective reduction, has greatly improved stability, and is suitable for popularization and application in industrial production of chiral alcohol.

Description

Ketoreductase mutant and method for producing chiral alcohol

Technical Field

The invention relates to the technical field of compound synthesis, in particular to a ketoreductase mutant and a method for producing chiral alcohol.

Background

Chiral alcohols are widely found in nature, are structural units of many important bioactive molecules, and are important intermediates for synthesizing natural products and chiral drugs. Many chiral drugs contain one or more chiral centers, and different chiral drugs differ significantly in pharmacological activity, metabolic processes, metabolic rates, and toxicity, usually with one enantiomer being effective and the other enantiomer being ineffective or ineffective, and even toxic. Therefore, how to efficiently and stereoselectively construct compounds containing chiral centers has important significance in medicine research and development.

Ketoreductases (KREDases), also known as carbonyl reductases (carbonyl-reductases), are commonly used to reduce latent chiral aldehydes or ketones to produce chiral alcohols, with the enzyme class number EC 1.1.1.184. KRED can not only convert aldehyde or ketone substrate into corresponding alcohol product, but also can catalyze its reverse reaction, namely catalytic oxidation alcohol substrate to obtain corresponding aldehyde or ketone. In the ketoreductase-catalyzed reaction, the participation of cofactors is required, including reduced Nicotinamide Adenine Dinucleotide (NADH), reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH), oxidized Nicotinamide Adenine Dinucleotide (NAD)⁺) Or oxidized form of Nicotinamide Adenine Dinucleotide Phosphate (NADP)⁺)。

In general, the reduced cofactor NADH or NADPH is involved in the reduction of an aldehyde or ketone, and the oxidized cofactor NAD is added in the actual reaction⁺Or NADP⁺Then regenerated to reduced NADH or by a suitable cofactor regeneration systemNADPH. Commonly used cofactor regeneration systems include glucose and glucose dehydrogenases, formate and formate dehydrogenases, secondary and secondary alcohol dehydrogenases, phosphite and phosphite dehydrogenases, and other similar systems. Generally, replacement of the coenzyme regeneration system does not materially affect the function of the ketoreductase.

Although many KREDs have been used in commercial production, KREDs generally suffer from low stability in application, particularly in thermal stability and tolerance to organic solvents. The enzyme is modified by means of directed evolution, so that the stability of the enzyme can be improved, and the enzyme can be better applied to production.

Disclosure of Invention

The invention aims to provide a ketoreductase mutant and a method for producing chiral alcohol so as to improve the stability of ketoreductase.

In order to achieve the above object, according to one aspect of the present invention, there is provided a ketoreductase mutant. The ketoreductase mutant has a sequence with amino acid mutation of a sequence shown in SEQ ID NO. 1, and the mutation site comprises K200H.

Further, the mutated site also includes at least one of the following sites: a15, K28, G36, K39, G43, Q44, a46, V47, F59, K61, T65, K71, a94V, a144, M146, Y152, N156, I86, K208, and K237; or a ketoreductase mutant having an amino acid sequence which has a mutation site in the mutated amino acid sequence and which has 95% or more identity to the mutated amino acid sequence.

Further, the site of mutation also includes at least one of the following mutations: A15C, K28A/E/M/Q/R/S, G36C, K39I/V, G43C/M, Q44R, A46C, V47C, F59C, K61E/H, T65A, K71R, A94V, A144T, M146I, Y152F, N156S, I86V, K208R and K237E.

Further, the amino acid mutation includes any one of the following site combination mutations: Q44R + N156S + K200H, Q44R + N156S + K200H + G201D, Q44R + N156S + K200H + G201D + M146I, Q44R + N156S + K200H + G201D + M146I + K61H, Q44H + N156H + K200H + G201H + M146H + K61H + I86H, Q44H + N156H + K H + N H + a H + N H + H.

Further, the occurrence of the amino acid mutation includes any one of the following site combination mutations: i86 + M146 + K200, M146 + N156 + K200, M146 + K200, K200 + G201, Q44 + M146 + K200, K61 + K200 + K237, I86 + M146 + K200, M146 + K200 + G201, N156 + K200 + G201, K200 + G201 + K237, K28 + M146 + K200 + G201, K28 + N156 + K200 + G201, Q44 + M146 + K200 + G201, Q44 + N156 + K200 + G201, I86 + M146 + K61, I86 + M200 + K208, I86 + M146 + K200 + G201, I86 + M146 + K201, I86 + M146 + K200G 201, Q44 + K156 + K200 + K156 + K61 + K200 + K61 + K146 + K44 + K146 + K146 + K201, Q44 + K + M146 + K + 201, Q44 + M146 + K + M146 + K + 200 + K + 201, Q44 + K + 200 + K + M + K + M + 201, Q44 + K + M + K + M + 200 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + I86 + A94, Q44 + N156 + K200 + G201 + M146 + K61 + I86 + K208, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K39, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + K94 + K39, Q44 + N156 + K200 + G146 + K61 + K208 + A94 + T65, Q44 + N156 + K2 + K43 + K17 + K + 61 + K + 2 + K + 200, Q44 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A43 + K208 + K43 + A94 + K43 + K + A43 + K43 + K + A43 + K43 + K + A46 + K43 + K17 + K17 + K,

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+I39V、Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+K71R、

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+A144T、

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+Y152F、

Q44R + N156S + K200H + G201D + M146I + K61H + K208R + A94V + K39I + A15C + A46C + G43M + K28E, and

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+G36C。

according to another aspect of the invention, a DNA molecule is provided. The DNA molecule encodes the ketoreductase mutant described above.

According to still another aspect of the present invention, there is provided a recombinant plasmid. The recombinant plasmid is connected with the DNA molecule.

Further, the recombinant plasmid is 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-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), pET-32a (+), and pET-35b (+), or, pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19.

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

Further, host cells include prokaryotic or eukaryotic cells; preferably the prokaryotic cell is E.coli.

According to another aspect of the present invention, there is provided a method of producing a chiral alcohol. The method comprises the step of producing chiral alcohol by adopting ketoreductase to catalyze the prochiral ketone compound to carry out reduction reaction, wherein the ketoreductase is any one of the ketoreductase mutants.

Further, the chiral ketone compound has the following structural formulaWherein R is₁And R₂Each independently is alkyl, cycloalkyl, aryl or heteroaryl, or R₁And R₂Taken together with the carbon on the carbonyl group to form a heterocyclic, carbocyclic, or heteroaryl group, each heteroatom in the heterocyclic and heteroaryl groups being independently selected from at least one of nitrogen, oxygen, and sulfur, each aryl group in the aryl group, heteroaryl group in the heteroaryl group, carbocyclic group in the carbocyclic group, or heterocyclic group in the heterocyclic group being independently unsubstituted or substituted with at least one halogen, alkoxy, or alkyl group;

R₁and R₂Each independently is C₁～C₈Alkyl radical, C₅～C₁₀Cycloalkyl radical, C₅～C₁₀Aryl or C₅～C₁₀Heteroaryl, or R₁And R₂Together with carbon on carbonyl to form C₅～C₁₀Heterocyclic group, C₅～C₁₀Carbocyclic radical or C₅～C₁₀Heteroaryl group, C₅～C₁₀Heterocyclyl and C₅～C₁₀Each heteroatom in the heteroaryl group is independently selected from at least one of nitrogen, oxygen and sulfur, C₅～C₁₀Aryl of aryl, C₅～C₁₀Heteroaryl of heteroaryl, C₅～C₁₀Carbocyclic group or C of carbocyclic groups₅～C₁₀Each of the heterocyclic groups in the heterocyclic group is independently unsubstituted or substituted with at least one group selected from halogen, alkoxy and alkyl;

preferably, the ketone compound has the structureWherein R is₃Is H, F, Cl, Br or CH₃，R₄Is H, F, Cl, Br or CH₃，R₅Is H, F, Cl, Br, CH₃，OCH₃Or CH₂CH₃；

More preferably, the ketone compound is

Furthermore, the reaction system for producing chiral alcohol by using ketoreductase to carry out reduction reaction on ketone compounds also comprises coenzyme, a coenzyme regeneration system and buffer solution.

Furthermore, the concentration of the ketone compound in the reaction system is 1 g/L-200 g/L.

Further, the pH value of the reaction system is 5-9, and the reaction temperature of the reaction system is 4-60 ℃.

Further, the coenzyme is NADH.

Further, the coenzyme regeneration system comprises: isopropanol, coenzyme NAD⁺And ketoreductases.

Further, the buffer solution is phosphate buffer solution, Tris-hydrochloric acid buffer solution, barbital sodium-hydrochloric acid buffer solution or citric acid-sodium citrate buffer solution.

The mutant obtained by mutation can efficiently produce chiral alcohol by using a ketone compound as a raw material through stereoselective reduction, has greatly improved stability, and is suitable for popularization and application in industrial production of chiral alcohol.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

Name interpretation:

"ketoreductase" and "KRED" are used interchangeably herein and refer to a polypeptide that is capable of reducing a keto group to its corresponding alcohol. In particular, the ketoreductase polypeptides of the present application are capable of stereoselectively reducing a ketone compound to the corresponding alcohol product. The polypeptide typically utilizes the cofactor reduced Nicotinamide Adenine Dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide smoke as a reducing agent. In the present application, ketoreductases include naturally occurring (wild-type) ketoreductases as well as non-naturally occurring ketoreductase mutants produced by artificial processing.

"naturally occurring" or "wild type" as opposed to "mutant" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism, which can be isolated from a source in nature, and which has not been intentionally modified or altered by man.

In this application, reference to, for example, a cell, nucleic acid, or polypeptide being "recombinant" refers to a cell, nucleic acid, or polypeptide that has been modified in a manner not found in nature, or that is the same as the form found in nature, but has been made or derived from synthetic materials and/or by processing using recombinant techniques, or that corresponds to a native or native form. Non-limiting examples include, among others, recombinant cells that express genes other than the native (non-recombinant) form in the cell or express native genes at different levels.

"percent of sequence identity" refers to the alignment between polynucleotides and is determined by comparing two optimally aligned sequences across a comparison window, wherein the portion of the polynucleotide sequence in the comparison bed may include additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentages can be calculated as follows: the percentage of sequence identity is determined by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100. Alternatively, the percentage may be calculated as follows: the percentage of sequence identity is determined by determining the number of positions in the two sequences at which the identical nucleobase or amino acid residue occurs or is aligned with a gap to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Wherein "reference sequence" refers to a designated sequence that is used as a basis for sequence comparison. The reference sequence may be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence.

Site-directed mutagenesis: it is intended to introduce a desired change (usually, a change indicating a favorable direction) including addition, deletion, point mutation or the like of a base into a DNA fragment of interest (which may be a genome or a plasmid) by a method such as Polymerase Chain Reaction (PCR). The site-directed mutation can rapidly and efficiently improve the character and the characterization of target protein expressed by DNA, and is a very useful means in gene research work.

Ketoreductase KRED mutant E144A + L152Y + L198Q + E201G + G6S + L146M +147V + D42E + T199V (as the template of the invention, having the amino acid sequence of SEQ ID NO: 1) derived from Acetobacter pasteianus 386B (in the invention, taking "E144A" as an example, the invention shows that "original amino acid + site + mutated amino acid", namely, E at position 144 is changed into A), can catalyze a target substrate to obtain a product, but the stability of the product is required to be further improved. The present invention seeks to improve the stability of KRED by directed evolution methods.

SEQ ID NO：1：

MARVASKVAIVSGAANGIGKATAQLLAKEGAKVVIGDLKEEEGQKAVAEIKAAGGEAAFVKLNVTDEAAWKAAIGQTLKLYGRLDIAVNNAGIAYSGSVESTSLEDWRRVQSINLDGVFLGTQVAIEAMKKSGGGSIVNLSSIAGMVGDPMYAAYNASKGGVRLFTKSAALHCAKSGYKIRVNSVHPGYIWTPMVAGQVKGDAAARQKLVDLHPIGHLGEPNDIAYGILYLASDESKFVTGSELVIDGGYTAQ

In the application, firstly, a mutation site is introduced on KRED in a site-specific mutation mode, the activity of the mutant is detected, and the mutant with improved activity is selected. Wherein the mutant E144A + L152Y + L198Q + E201G + G6S + L146M +147V + D42E + T199V + K200H has about 3-fold improved stability compared with the initial template. Subsequently, E144A + L152Y + L198Q + E201G + G6S + L146M +147V + D42E + T199V + K200H are used as templates to carry out continuous mutation, so that a mutant with more remarkable stability improvement is obtained.

The introduction of site-directed mutagenesis by whole plasmid PCR is simple and effective, and currently, a lot of means are used. The principle is as follows: a pair of primers (forward and reverse) containing mutation sites and a polymerase are used for ' cycle extension ' after template plasmid annealing (the cycle extension refers to that the polymerase extends the primer according to the template, returns to the 5 ' end of the primer after one cycle to terminate, and then is subjected to a cycle of repeated heating annealing extension, the reaction is different from rolling circle amplification, and a plurality of tandem copies are not formed, the extension products of the forward and reverse primers are annealed and then matched into an open-loop plasmid with a nick, Dpn I enzyme-cut extension product, because the original template plasmid is from conventional escherichia coli, is modified by dam methylation and is sensitive to Dpn I and cut up, and the plasmid with a mutation sequence synthesized in vitro is not cut up because of no methylation, the subsequent transformation can be successfully carried out, and the clone of the mutation plasmid can be obtained, the mutation plasmid is transformed into a host cell, and the target protein is induced and expressed, then the crude enzyme is obtained by a method of disrupting cells by ultrasonication. Optimal conditions for ketoreductase-induced expression: induction was carried out at 25 ℃ with 0.1mM IPTG for 16 h.

In an exemplary embodiment of the invention, a ketoreductase mutant is provided. The ketoreductase mutant has a sequence with amino acid mutation of a sequence shown in SEQ ID NO. 1, and the mutation site comprises K200H. The mutant obtained by mutation can efficiently produce chiral alcohol by using a ketone compound as a raw material through stereoselective reduction, has greatly improved stability, and is suitable for popularization and application in industrial production of chiral alcohol.

Preferably, the site of mutation further includes at least one of the following sites: a15, K28, G36, K39, G43, Q44, a46, V47, F59, K61, T65, K71, a94V, a144, M146, Y152, N156, I86, K208, and K237; or a ketoreductase mutant having an amino acid sequence which has a mutation site in the mutated amino acid sequence and which has 95% or more identity to the mutated amino acid sequence. Mutation of the above-mentioned site can further improve the stability of the enzyme. More preferably, the site of mutation further comprises at least one of the following mutations: A15C, K28A/E/M/Q/R/S, G36C, K39I/V, G43C/M, Q44R, A46C, V47C, F59C, K61E/H, T65A, K71R, A94V, A144T, M146I, Y152F, N156S, I86V, K208R and K237E. Where "/" represents "or".

In a typical embodiment of the present invention, the amino acid mutation comprises any one of the following site combination mutations: Q44R + N156S + K200H + G201D, Q44R + N156R + K200R + G201R + M146R + K61R + K208R + a 94R + K39R, Q44R + N156R + K200R, Q44R + N156R + K200R + G201R + Q44R + N156R + K200R + G R + M146R, Q44R + N156R + N R + N R + N R + N R +.

More preferably, the amino acid mutation includes any one of the following site combination mutations: i86 + M146 + K200, M146 + N156 + K200, M146 + K200, K200 + G201, Q44 + M146 + K200, K61 + K200 + K237, I86 + M146 + K200, M146 + K200 + G201, N156 + K200 + G201, K200 + G201 + K237, K28 + M146 + K200 + G201, K28 + N156 + K200 + G201, Q44 + M146 + K200 + G201, Q44 + N156 + K200 + G201, I86 + M146 + K61, I86 + M200 + K208, I86 + M146 + K200 + G201, I86 + M146 + K201, I86 + M146 + K200G 201, Q44 + K156 + K200 + K156 + K61 + K200 + K61 + K146 + K44 + K146 + K146 + K201, Q44 + K + M146 + K + 201, Q44 + M146 + K + M146 + K + 200 + K + 201, Q44 + K + 200 + K + M + K + M + 201, Q44 + K + M + K + M + 200 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + I86 + A94, Q44 + N156 + K200 + G201 + M146 + K61 + I86 + K208, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K39, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + K94 + K39, Q44 + N156 + K200 + G146 + K61 + K208 + A94 + T65, Q44 + N156 + K2 + K43 + K17 + K + 61 + K + 2 + K + 200, Q44 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A43 + K208 + K43 + A94 + K43 + K + A43 + K43 + K + A43 + K43 + K + A46 + K43 + K17 + K17 + K,

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+A144T、

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+Y152F、

Q44R+N156S+K200H+G201D+M146I+K61H+K208R+A94V+K39I+A15C+A46C+G43M+G36C。

according to an exemplary embodiment of the present invention, a DNA molecule is provided. The DNA molecule encodes the ketoreductase mutant described above. The ketoreductase coded by the DNA molecule has good activity.

The above-described DNA molecules of the invention may also be present in the form of "expression cassettes". An "expression cassette" refers to a nucleic acid molecule, linear or circular, encompassing DNA and RNA sequences capable of directing the expression of a particular nucleotide sequence in an appropriate host cell. Generally, a promoter is included that is operably linked to a nucleotide of interest, optionally operably linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but also encodes a functional RNA of interest in the sense or antisense orientation, e.g., an antisense RNA or an untranslated RNA. An expression cassette comprising a polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component. The expression cassette may also be naturally occurring but obtained with efficient recombinant formation for heterologous expression.

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

Although the term "comprising" is used in the present invention when defining the above DNA molecule, it does not mean that other sequences unrelated to their functions may be arbitrarily added to both ends of the DNA sequence. Those skilled in the art know that in order to satisfy the requirements of recombinant operation, it is necessary to add suitable restriction sites for restriction enzymes at both ends of a DNA sequence, or additionally add initiation codons, termination codons, etc., and thus, if defined by closed expressions, these cases cannot be truly covered.

The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage or Agrobacterium binary nucleic acid molecule, preferably a recombinant expression plasmid, either prokaryotic or eukaryotic, but preferably prokaryotic, selected from the group consisting of 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-23b (+), pET-24a (+), and, 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-43b (+), pET-44a (+), pET-49b (+), pQE2, QEP 9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pR A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6-p-1, pGEX-6-P-2-pGEX-2 b (+), pET-39b (+), pET-40b (+) pBV220, pBV221, pBV222, pTrc99A, pTwin1, pEZZ18, pKK232-18, pUC-18 or pUC-19. More preferably, the above recombinant plasmid is pET-22b (+).

According to a typical embodiment of the present invention, there is provided a host cell containing any one of the above recombinant plasmids. Host cells suitable for use in the present invention include, but are not limited to, prokaryotic cells, yeast, or eukaryotic cells. Preferably the prokaryotic cell is a eubacterium, such as a gram-negative or gram-positive bacterium. More preferably, the prokaryotic cell is an E.coli BL21 cell or an E.coli DH5 alpha competent cell.

According to an exemplary embodiment of the present invention, a method for producing a chiral alcohol is provided. The method comprises the step of carrying out reduction reaction on chiral ketone compounds by adopting ketoreductase to produce chiral alcohol, wherein the ketoreductase is any one of ketoreductase mutants. Because the ketoreductase mutant of the invention has good activity characteristics, the chiral alcohol prepared by the ketoreductase mutant of the invention can improve the reaction rate, improve the substrate concentration, reduce the enzyme dosage and reduce the difficulty of post-treatment.

In the present application, chiral ketones include, but are not limited to, compounds having the formula

Wherein R is₁And R₂Each independently is an alkaneAryl, cycloalkyl, aryl or heteroaryl, or R₁And R₂Taken together with the carbon on the carbonyl group to form a heterocyclic, carbocyclic, or heteroaryl group, each heteroatom in the heterocyclic and heteroaryl groups being independently selected from at least one of nitrogen, oxygen, and sulfur, each aryl, heteroaryl, carbocyclyl, or heterocyclic group in the aryl group being independently unsubstituted or substituted with at least one halogen, alkoxy, or alkyl group; preferably, R₁And R₂Each independently is C₁～C₈Alkyl radical, C₅～C₁₀Cycloalkyl radical, C₅～C₁₀Aryl or C₅～C₁₀Heteroaryl, or R₁And R₂Together with carbon on carbonyl to form C₅～C₁₀Heterocyclic group, C₅～C₁₀Carbocyclic radical or C₅～C₁₀Heteroaryl group, C₅～C₁₀Heterocyclyl and C₅～C₁₀Each heteroatom in the heteroaryl group is independently selected from at least one of nitrogen, oxygen and sulfur, C₅～C₁₀Aryl of aryl, C₅～C₁₀Heteroaryl of heteroaryl, C₅～C₁₀Carbocyclic group or C of carbocyclic groups₅～C₁₀Each of the heterocyclic groups in the heterocyclic group is independently unsubstituted or substituted with at least one group selected from halogen, alkoxy and alkyl;

preferably, the ketone compound has the structure

Wherein R is₃Is H, F, Cl, Br or CH₃，R₄Is H, F, Cl, Br or CH₃，R₅Is H, F, Cl, Br, CH₃，OCH₃Or CH₂CH₃；

More preferably, the ketone compound is

The host cells described earlier herein can be used for expression and isolation of the ketoreductase enzyme, or alternatively, they can be used directly to convert the ketone substrate to the chiral alcohol product. Preferably, the prokaryotic cell is E.coli.

The reduction reactions set forth above generally require a cofactor, which is usually NADH or NADPH, and can include systems for regenerating the cofactor, such as D-glucose, coenzyme NAD⁺And glucose dehydrogenase GDH; formate compound, coenzyme NAD⁺And formate dehydrogenase FDH; or isopropanol, coenzyme NAD⁺And alcohol dehydrogenase ADH. In some embodiments using purified ketoreductase enzymes, such cofactors, and optionally such cofactor regeneration systems, will generally be added to the reaction medium along with the substrate and ketoreductase enzyme. Similar to the ketoreductase enzyme, any enzyme comprising a cofactor regeneration system may be in the form of an extract or lysate of such cells, or added to the reaction mixture as a purified enzyme. In embodiments where a cellular extract or cell lysate is used, the cells used to produce the extract or lysate may be cells expressing an enzyme containing either only the cofactor regeneration system or both the cofactor regeneration system and the ketoreductase enzyme. In embodiments using whole cells, the cells can be allowed to express an enzyme containing a cofactor regeneration system and a ketoreductase enzyme.

Whether whole cells, cell extracts, or purified ketoreductases are used, a single ketoreductase can be used, or alternatively, a mixture of two or more ketoreductases can be used.

The reaction system for producing chiral alcohol by using ketone reductase to perform reduction reaction on chiral ketone compounds also comprises coenzyme, a coenzyme regeneration system and buffer solution.

The ketoreductase mutant of the invention has higher catalytic activity, can increase the concentration of a substrate and improve the production efficiency, and the concentration of the chiral ketone compound in a reaction system is 1 g/L-200 g/L.

The pH value of the reaction system is 5-9, and the reaction temperature of the reaction system is 4-60 ℃; the buffer solution is phosphate buffer solution, Tris-hydrochloric acid buffer solution, barbital sodium-hydrochloric acid buffer solution or citric acid-sodium citrate buffer solution.

The following examples are provided to further illustrate the advantageous effects of the present invention.

In the present application, the enzyme activity detection method is as follows:

1. preparing a reagent:

substrate (R) -1- (2, 4-dichloroacetophenone) mother liquor 60 mM: weighing 56.7mg of the suspension, dissolving in 5mL of 0.1M phosphate buffer () PB), pH 7.0buffer, and dissolving in a water bath kettle at 50 ℃;

10mM NADH mother liquor: 33.17mg of NADH was weighed out and dissolved in 5mL of 0.1M PB pH 7.0 buffer.

2. The enzyme activity system is as follows:

firstly adding enzyme, then adding a mixture of a substrate (R) -1- (2, 4-dichloroacetophenone), NADH and Buffer, putting the mixture into an enzyme-labeling instrument, and detecting the enzyme activity at 30 ℃ and 340nm wavelength.

The test system is formulated as shown in Table 1.

TABLE 1

System of	Amount of addition	Final concentration
			Enzyme mutants	20μL	N/A
Substrate	50μL	10mM
			NADH	10μL	0.33mM
0.1M PB pH 7.0buffer	220μL	N/A

Stability is expressed as the residual activity, i.e. the percentage of activity after treatment to activity before treatment.

The ketoreductase KRED mutant E144A + L152Y + L198Q + E201G + G6S + L146M +147V + D42E + T199V is simply called as a "template" in the invention, and the listed mutation sites are mutations performed on the basis of the "template".

Example 1

The "template" and mutant were incubated at 45 ℃ and 53 ℃ for 1h, respectively, and their activity was then determined, compared to the non-incubated one, with stability expressed as the percentage of their residual activity compared to the initial activity. The thermostability of all mutants was determined at 53 ℃ and the results are shown in Table 2.

TABLE 2

+ represents stability, + residual activity 0-5%, + + residual activity 5-10%, + + + + residual activity 10-50%, and ++++ residual activity 50-95%.

Example 2

Combining saturation mutation can obtain several mutants with synergistic effect between mutation sites, and can optimize the combination of amino acid composition. Mutation point combination was performed using Q44R + N156S + K200H + G201D as a template, at 65 ℃ for 1h in enzyme solution treatment conditions, and then the activity was measured and compared to that of the non-incubated samples, the stability was expressed as the percentage of the residual activity compared to the initial activity.

The preparation method of the enzyme solution in the high-throughput screening comprises the following steps: the supernatant medium was removed by centrifugation in a 96-well plate, 200. mu.L of an enzymatic solution (lysozyme 2mg/mL, polymyxin 0.5mg/mL, pH 7.0) was added to each well, and the mixture was disrupted at 37 ℃ for 3 hours. The enzyme activity detection method comprises the following steps: the enzyme was added first, then the mixture of substrate (R) -1- (2, 4-dichloroacetophenone), NADH and Buffer was added, and the enzyme activity was measured in an enzyme-linked microplate reader at 30 ℃ and 340nm wavelength, the results are shown in Table 3.

TABLE 3

Example 3

The mutation was continued using Q44R + N156S + K200H + G201D + M146I + K61H + K208R + A94V + K39I as a template. The enzyme solution was then treated at 71 ℃ for 1h and the activity was measured and compared to the non-incubated solution, the stability being expressed as the percentage of the residual activity compared to the initial activity.

The preparation method of the enzyme solution in the high-throughput screening comprises the following steps: the supernatant medium was removed by centrifugation in a 96-well plate, 200. mu.L of an enzymatic solution (lysozyme 2mg/mL, polymyxin 0.5mg/mL, pH 7.0) was added to each well, and the mixture was disrupted at 37 ℃ for 3 hours. The enzyme activity detection method comprises the following steps: the enzyme was added first, then the mixture of substrate (R) -1- (2, 4-dichloroacetophenone), NADH and Buffer was added, and the enzyme activity was measured in an enzyme-linked microplate reader at 30 ℃ and 340nm wavelength, the results are shown in Table 4.

TABLE 4

Example 4

In the process of improving the stability of the enzyme, the rigidity of the whole enzyme is improved, so that the mutant with improved stability simultaneously shows improved tolerance to chemical substances such as glutaraldehyde and organic solvents such as methanol, and the like, and the mutant is actually an external expression of the improvement of the rigidity of the whole mutant.

The mutants described hereinbefore were tested for glutaraldehyde and methanol tolerance. The glutaraldehyde resistance was held at 1% glutaraldehyde for 1h and then the residual activity was determined, with the aldehyde resistance being expressed as a percentage of the activity without and with the temperature. Methanol tolerance was measured after 1h incubation in 60% methanol solution, as a percentage of the activity without and with incubation, and the results are shown in table 5.

TABLE 5

Represents aldehyde tolerance, 0-5% residual activity, 5-10% residual activity, 10-50% residual activity, 50-95% residual activity.

# represents methanol tolerance, residual activity of # 0-5%, # 5-10%, # 10-50%, and # 50-95%.

Example 5

Different substrate reaction validation was performed using mutant Q44R + N156S + K200H + G201D + M146I + K61H + K208R + a94V + K39I + a15C + a46C + G43M + G36C, with the results shown in table 6.

1) Adding 2g of substrate 2-chloroacetophenone into a 25mL reaction bottle, adding 0.1M PB 7.0, 2g of isopropanol, 20mg of NAD + and 0.05g of ketone reductase mutant, uniformly mixing, wherein the total volume is 10mL, and reacting for 16 hours at 50 ℃ by using a 200-turn table;

2) adding 2g of substrate 3-fluoro acetophenone, 0.1M PB 7.0, 2g of isopropanol, 20mg of NAD +, 0.05g of ketone reductase mutant into a 25mL reaction bottle, uniformly mixing, wherein the total volume is 10mL, and reacting for 16 hours at 50 ℃ by using a 200-turn table;

3) adding 2g of substrate 4-methoxyacetophenone, 0.1M of PB 7.0, 2g of isopropanol and 20mg of NAD + into a 25mL reaction flask, uniformly mixing, wherein the total volume is 10mL, and reacting for 16 hours at 50 ℃ in a 200-turn table;

4) a25 mL reaction flask was charged with 2g of ethyl acetoacetate as a substrate, 0.1M PB 7.0, 2g of isopropanol, 20mg of NAD +, and 0.05g of ketoreductase mutant, mixed well in a total volume of 10mL, and reacted at 50 ℃ for 16 hours on a 200-turn shaker.

TABLE 6

Serial number	Substrate	Conversion (%)	ee
				1	2-Chloroacetophenone	99	>99％
2	3-fluoro acetophenone	99	>99％
				3	4-methoxy acetophenone	99	>99％
4	Acetoacetic acid ethyl ester	99	>99％

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Kailai pharmaceutical chemistry (Fuxin) technology, Inc

<120> ketoreductase mutant and method for producing chiral alcohol

<130>PN116848KLY

<160>1

<170>SIPOSequenceListing 1.0

<210>1

<211>253

<212>PRT

<213>Acetobacter pasteurianus 386B

<400>1

Met Ala Arg Val Ala Ser Lys Val Ala Ile Val Ser Gly Ala Ala Asn

1 5 10 15

Gly Ile Gly Lys Ala Thr Ala Gln Leu Leu Ala Lys Glu Gly Ala Lys

20 25 30

Val Val Ile Gly Asp Leu Lys Glu Glu Glu Gly Gln Lys Ala Val Ala

35 40 45

Glu Ile Lys Ala Ala Gly Gly Glu Ala Ala Phe Val Lys Leu Asn Val

50 55 60

Thr Asp Glu Ala Ala Trp Lys Ala Ala Ile Gly Gln Thr Leu Lys Leu

65 70 75 80

Tyr Gly Arg Leu Asp Ile Ala Val Asn Asn Ala Gly Ile Ala Tyr Ser

8590 95

Gly Ser Val Glu Ser Thr Ser Leu Glu Asp Trp Arg Arg Val Gln Ser

100 105 110

Ile Asn Leu Asp Gly Val Phe Leu Gly Thr Gln Val Ala Ile Glu Ala

115 120 125

Met Lys Lys Ser Gly Gly Gly Ser Ile Val Asn Leu Ser Ser Ile Ala

130 135 140

Gly Met Val Gly Asp Pro Met Tyr Ala Ala Tyr Asn Ala Ser Lys Gly

145 150 155 160

Gly Val Arg Leu Phe Thr Lys Ser Ala Ala Leu His Cys Ala Lys Ser

165 170 175

Gly Tyr Lys Ile Arg Val Asn Ser Val His Pro Gly Tyr Ile Trp Thr

180 185 190

Pro Met Val Ala Gly Gln Val Lys Gly Asp Ala Ala Ala Arg Gln Lys

195 200 205

Leu Val Asp Leu His Pro Ile Gly His Leu Gly Glu Pro Asn Asp Ile

210 215 220

Ala Tyr Gly Ile Leu Tyr Leu Ala Ser Asp Glu Ser Lys Phe Val Thr

225 230 235 240

Gly Ser Glu Leu Val Ile Asp Gly Gly Tyr Thr Ala Gln

245 250

Claims

1. A ketoreductase mutant, which is characterized in that the ketoreductase mutant has a sequence with amino acid mutation of the sequence shown in SEQ ID NO. 1, and the site of the mutation comprises K200H.

2. The ketoreductase mutant according to claim 1, wherein the mutated sites further comprise at least one of the following sites: a15, K28, G36, K39, G43, Q44, a46, V47, F59, K61, T65, K71, a94V, a144, M146, Y152, N156, I86, K208, and K237; or the ketoreductase mutant has an amino acid sequence which has a mutation site in the mutated amino acid sequence and has 95% or more identity with the mutated amino acid sequence.

3. The ketoreductase mutant according to claim 2, wherein the site of the mutation further comprises at least one of the following mutations: A15C, K28A/E/M/Q/R/S, G36C, K39I/V, G43C/M, Q44R, A46C, V47C, F59C, K61E/H, T65A, K71R, A94V, A144T, M146I, Y152F, N156S, I86V, K208R and K237E.

4. The ketoreductase mutant according to claim 3, wherein the amino acid mutation comprises any one of the following site combination mutations: Q44R + N156S + K200H, Q44R + N156S + K200H + G201D, Q44R + N156S + K200H + G201D + M146I, Q44R + N156S + K200H + G201D + M146I + K61H, Q44H + N156H + K200H + G201H + M146H + K61H + I86H, Q44H + N156H + K H + N H + a H + N H + H.

5. The ketoreductase mutant according to claim 1, wherein the occurrence of the amino acid mutation comprises any one of the following site combination mutations: i86 + M146 + K200, M146 + N156 + K200, M146 + K200, K200 + G201, Q44 + M146 + K200, K61 + K200 + K237, I86 + M146 + K200, M146 + K200 + G201, N156 + K200 + G201, K200 + G201 + K237, K28 + M146 + K200 + G201, K28 + N156 + K200 + G201, Q44 + M146 + K200 + G201, Q44 + N156 + K200 + G201, I86 + M146 + K61, I86 + M200 + K208, I86 + M146 + K200 + G201, I86 + M146 + K201, I86 + M146 + K200G 201, Q44 + K156 + K200 + K156 + K61 + K200 + K61 + K146 + K44 + K146 + K146 + K201, Q44 + K + M146 + K + 201, Q44 + M146 + K + M146 + K + 200 + K + 201, Q44 + K + 200 + K + M + K + M + 201, Q44 + K + M + K + M + 200 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + I86 + A94, Q44 + N156 + K200 + G201 + M146 + K61 + I86 + K208, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K39, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + K94 + K39, Q44 + N156 + K200 + G146 + K61 + K208 + A94 + T65, Q44 + N156 + K2 + K43 + K17 + K + 61 + K + 2 + K + 200, Q44 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + K28, Q44 + N156 + K200 + G201 + M146 + K61 + K28 + A146 + K61 + K43 + K + A43 + K43 + K43 + K + A43 + K + 61 + K17 + K + 61 + K + 32 + K + 61 + K + 17 + K + 32 + K + 61 + K + 17 + K +, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + G43 + A144, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + G43 + Y152, Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + G43 + K28, and Q44 + N156 + K200 + G201 + M146 + K61 + K208 + A94 + K39 + A15 + A46 + G43 + G36.

6. A DNA molecule encoding the ketoreductase mutant of any one of claims 1 to 5.

7. A recombinant plasmid having the DNA molecule of claim 6 attached thereto.

8. The recombinant plasmid of claim 7, wherein the recombinant plasmid is 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-23b (+), pET-24a (+), pET-25b (+), pET-26b (+), pET-27b (+), pET-28a (+), pET-29a (+), pET-30a (+), pET-31b (+), or, pET-32a (+), pET-35b (+), pET-38b (+), pET-39b (+), pET-40b (+), pET-41a (+), pET-41b (+), pET-42a (+), pET-43b (+), pET-44a (+), pET-49b (+), pQE2, pQE9, pQE30, pQE31, pQE32, pQE40, pQE70, pQE80, pRSET-A, pRSET-B, pRSET-C, pGEX-5X-1, pGEX-6p-2, pBV220, pBV221, pBV222, pTrc 53999, pTwin1, pZZ 685 18, pK 232-18, pK-18-19 or pK-19.

9. A host cell comprising the recombinant plasmid of claim 7 or 8.

10. The host cell of claim 9, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is E.coli.

11. A method of producing a chiral alcohol comprising the step of catalyzing a reduction reaction of a prochiral ketonic compound with a ketoreductase to produce a chiral alcohol, wherein the ketoreductase is a ketoreductase mutant according to any one of claims 1 to 5.

12. The method of claim 11, wherein the chiral ketone compound has the formula

Wherein R is₁And R₂Each independently is alkyl, cycloalkyl, aryl or heteroaryl, or R₁And R₂Taken together with the carbon on the carbonyl group to form a heterocyclic, carbocyclic, or heteroaryl group, each heteroatom in the heterocyclic and heteroaryl groups being independently selected from at least one of nitrogen, oxygen, and sulfur, each aryl, heteroaryl, carbocyclyl, or heterocyclic group in the aryl group being independently unsubstituted or substituted with at least one halogen, alkoxy, or alkyl group;

R₁and R₂Each independently is C₁～C₈Alkyl radical, C₅～C₁₀Cycloalkyl radical, C₅～C₁₀Aryl or C₅～C₁₀Heteroaryl, or R₁And R₂Together with carbon on carbonyl to form C₅～C₁₀Heterocyclic group, C₅～C₁₀Carbocyclic radical or C₅～C₁₀Heteroaryl of said C₅～C₁₀Heterocyclyl and C₅～C₁₀Each heteroatom in the heteroaryl group is independently selected from at least one of nitrogen, oxygen and sulfur, and C₅～C₁₀Aryl of aryl, C₅～C₁₀Heteroaryl of heteroaryl, C₅～C₁₀Carbocyclic group or C of carbocyclic groups₅～C₁₀Each of the heterocyclic groups in the heterocyclic group is independently unsubstituted or substituted with at least one group selected from halogen, alkoxy and alkyl;

preferably, the ketone compound has the structure

More preferably, the ketone compound is

13. The method of claim 11, wherein the reaction system for producing the chiral alcohol by the reduction reaction of the ketone compound with the ketoreductase further comprises a coenzyme, a coenzyme regeneration system, and a buffer solution.

14. The method according to claim 13, wherein the concentration of the ketone compound in the reaction system is 1g/L to 200 g/L.

15. The method according to claim 13, wherein the pH value of the reaction system is 5-9, and the reaction temperature of the reaction system is 4-60 ℃.

16. The method of claim 13, wherein the coenzyme is NADH.

17. The method of claim 16, wherein the coenzyme regeneration system comprises: isopropanol, coenzyme NAD⁺And ketoreductases.

18. The method of claim 13, wherein the buffer is a phosphate buffer, a Tris-hydrochloric acid buffer, a barbiturate-hydrochloric acid buffer, or a citric acid-sodium citrate buffer.