Engineering ketoreductase for producing chiral alcohol compounds and method thereof

Abstract

The present disclosure provides an engineered ketoreductase polypeptide capable of producing a chiral alcohol-based compound for catalyzing the production of ethyl (R)-4-chloro-3-hydroxybutyrate from ethyl 4-chloroacetoacetate in particular. The polypeptide has high catalytic activity, high stereoselectivity, good thermal stability, good solvent tolerance, the substrate loading amount in the reaction process reaches 426 g / L, and the conversion rate reaches 99%.

Description

Technical Field

[0001] The present invention belongs to the technical field of biotechnology, and specifically relates to an engineered ketoreductase polypeptide and its use in the production of chiral alcohol-based compounds.

Background Art

[0002] Chiral alcohol-based compounds are a type of widely used compounds. In particular, most of many natural compounds and compounds with biological activities contain the structure of chiral alcohol. L-carnitine, also called vitamin BT, is an amino acid that promotes the conversion of fat into energy. As a food additive, it is widely used in infant foods, sports foods, pharmaceuticals, nutritional supplements for middle-aged and elderly people, nutritional supplements for vegetarians, and animal feed additives. Among them, ethyl (R)-4-chloro-3-hydroxybutyrate, which is a chiral alcohol-based compound, is an important intermediate for synthesizing L-carnitine. Therefore, researching the efficient production of ethyl (R)-4-chloro-3-hydroxybutyrate has high important application value and can be expected for the future of the market.

[0003] In industrial production, chemical methods and enzyme-catalyzed methods are the main methods for producing chiral alcohols. In the enzyme-catalyzed method, usually, using a carbonyl compound as a substrate, a carbonyl substrate is asymmetrically reduced by keto reductase (abbreviated as KRED) or alcohol dehydrogenase (abbreviated as ADH) to obtain a chiral alcohol. The enzyme method is popular in the industry due to advantages such as high selectivity, high conversion rate, mild reaction conditions, low cost, and low pollution. As for the reaction mechanism, keto reductase or alcohol dehydrogenase catalyzes the reduction of a carbonyl (or ketone group) compound to an alcohol compound by the reducing power H from the cofactor NADH or NADPH, and the cofactor NADH or NADPH is converted to NAD+ or NADP+ respectively (Figure 1). However, because the price of the cofactor NADH or NADPH is high, the production cost becomes very high. Currently, in the industry, usually, a cofactor recycling system with lower cost is adopted. That is, during the progress of this reaction process, NAD+ or NADP+ is converted to NADH or NADPH respectively by another enzyme reaction, that is, the regeneration cycle of NADH or NADPH is achieved. There are usually three methods to convert NAD+ or NADP+ to NADH or NADPH respectively. The first one is to add glucose dehydrogenase (GDH) and glucose to the reaction in Figure 1 simultaneously. During the process where glucose dehydrogenase catalyzes the conversion of glucose to gluconic acid, NAD+ or NADP+ is converted to NADH or NADPH respectively. The second one is to add formate dehydrogenase (FDH) and formate to the reaction in Figure 1 simultaneously. During the process where formate dehydrogenase catalyzes the conversion of formate to carbon dioxide, NAD+ or NADP+ is converted to NADH or NADPH respectively.The third is to add isopropanol to the reaction in Fig. 1, and by the action of the same ketoreductase or alcohol dehydrogenase, another alcohol compound (e.g., isopropanol) is converted to a carbonyl compound (e.g., acetone, etc.), and NAD+ or NADP+ is reduced to NADH or NADPH respectively. The reaction is shown in Fig. 2.

[0004] Among these, since both the GDH cycle system and the FDH cycle system use additional enzymes, the production cost of either the first cycle system or the second cycle system is clearly higher than that of the third system which uses only one enzyme. Furthermore, solid waste is generated by the application of the GDH cycle system. Therefore, in the present invention, from the perspective of avoiding high costs and the large generation of solid waste, an isopropanol-acetone cycle system is adopted.

[0005] Ethyl (R)-4-chloro-3-hydroxybutyrate is an important chiral alcohol compound and an important intermediate of L-carnitine. In the chemical synthesis method, usually, ethyl 4-chloroacetoacetate is reduced using a metal complex as a catalyst to obtain L-carnitine. However, the chemical method has a high cost due to harsh reaction conditions and the use of metal catalysts, and the subsequent treatment process also becomes complicated.

[0006] Compared with chemical methods, the enzymatic method is more economical and environmentally friendly in the process, and the reaction conditions are also mild. Although some progress has been made in the biocatalytic synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate, the reaction control is not easy. For example, problems such as "exhaust gas, wastewater, and solid waste" still exist. For example, the use of a circulation system leads to the generation of solid waste. Therefore, in order to avoid the generation of a large amount of solid waste, the present invention adopts an isopropanol-acetone circulation system. At the same time, the isopropanol-acetone circulation system uses the same enzyme as the enzyme that catalyzes the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate, and the production cost is also lower than that of the GDH circulation system. However, there are few reports on the prior art using an isopropanol-acetone circulation system to synthesize ethyl (R)-4-chloro-3-hydroxybutyrate. In fact, there are some problems with the use of an isopropanol-acetone circulation system in the synthesis reaction. Usually, protein molecules are more stable in an aqueous phase system. Since most enzymes, especially wild-type enzymes existing in nature, cannot adapt to a harsh organic phase environment, enzymatic reactions are carried out in an aqueous phase system. However, ethyl 4-chloroacetoacetate, the substrate used in the present invention, is unstable in an aqueous system and is prone to hydrolysis. Moreover, the presence of isopropanol further promotes the hydrolysis of the substrate. In order to avoid spontaneous hydrolysis reactions, the reaction system in this application adopts a water-toluene two-phase system. When a water-toluene two-phase system is adopted, the enzymes disclosed in the prior art cannot effectively catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate and maintain sufficient activity and stability to regenerate NADH by converting isopropanol to acetone. Therefore, in this application, by implementing the directed evolution of wild-type ketoreductase, a series of engineered ketoreductase catalysts with high activity and stability in a two-phase system and capable of withstanding higher substrate concentrations were obtained.Based on this, the present application can obtain an enzyme catalyst and a reaction process for producing ethyl (R)-4-chloro-3-hydroxybutyrate with a high conversion rate while achieving low costs.

[0007] The two-phase system used in the present invention can not only avoid the spontaneous hydrolysis reaction of the substrate and the generation of emulsification, but also facilitate the pH control of the aqueous phase during the reaction process, which is beneficial for mass production. In addition, the organic solvent can be recycled and the wastewater can be reduced, which is more environmentally friendly. In the all-aqueous phase system commonly used in the prior art, more industrial wastewater is generated, and organic solvent extraction is inevitably used in the subsequent purified products. On the other hand, the two-phase reaction of the present invention can reduce the amount of water used, thereby significantly reducing the generation of wastewater and reducing the additional organic reagent extraction step, which is more in line with the requirements of an environmentally friendly manufacturing process and sustainable development.

[0008] In addition, since the existing ketoreductase that can catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate is unstable in the two-phase system, the use of the two-phase system will reduce the conversion rate or overall yield of the enzyme reaction. The enzyme modified by the directed evolution disclosed in the present application can maintain high activity and stability even in the two-phase system, achieve a reaction process with a high substrate concentration and a high conversion rate (>99%), and the ee value also reaches 99.8% or more. Furthermore, the enzyme modified in the present application has high thermal stability, further meeting the needs of industrial applications.

Summary of the Invention

[0009] 1 Summary The present invention provides an engineered ketoreductase polypeptide that is highly stereoselective, has high catalytic activity, good stability, and high substrate tolerance, and is useful for the production of chiral alcohol compounds, particularly the asymmetric synthesis of ethyl (R)-4-chloro-3-hydroxybutyrate. Further provided are a gene for the engineered ketoreductase polypeptide, a recombinant expression vector containing this gene, an engineered strain and an efficient production method thereof, and a reaction process for producing chiral alcohol compounds using the engineered ketoreductase polypeptide.

[0010] In some embodiments, the engineered ketoreductase polypeptide disclosed in the present application can catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate with higher activity than SEQ ID No:2. The improved engineered ketoreductase polypeptide according to the present invention has higher activity, stability, and resistance to solvents and substrates compared to the wild-type ketoreductase corresponding to SEQ ID NO:2, and can more effectively catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate. Alternatively, even when the substrate concentration is high, the improved engineered ketoreductase polypeptide according to the present invention can catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate without inhibition. These improved ketoreductase polypeptides may include an amino acid sequence having one or more residue differences from the SEQ ID NO:2 sequence at residue positions corresponding to X18, X22, X42, X46, X47, X48, X53, X67, X79, X80, X94, X96, X111, X115, X149, X204, X211, X229, X238, X239, X262, X282, X283, X284, X286, X303, X327. The improved ketoreductase polypeptide includes an amino acid sequence containing at least one of the characteristics of V18I, A22K, I42L, D46G, D46F, D46V, M47I, M47S, P48R, P48H, I53Q, I53G, I53P, I53T, I53S, I53H, I53N, I53R, I53K, T67V, T67I, E79K, T80V, A94R, A94Y, A94C, A94H, H96R, E111H, T115R, T115C, T115S, T115H, V149A, L204I, L211F, D229A, E238R, E238K, G239S, I262V, F282Q, F282G, M283N, M283D, I284M, I284L, I284V, F286V, F286H, F286R, D303E, T327L, T327E, or, in addition to these differences, includes one or more amino acid residue insertions or deletions.

[0011] More specifically, in some embodiments, the engineered ketoreductase polypeptide improved based on SEQ ID NO:2 comprises a polypeptide consisting of the amino acid sequences shown in SEQ ID No:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128.

[0012] In some embodiments, the improved engineered ketoreductase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference sequences of SEQ ID No:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128. In some embodiments, the ketoreductase polypeptide catalyzes the formation of ethyl (R)-4-chloro-3-hydroxybutyrate from ethyl 4-chloroacetoacetate, the polypeptide has at least 90% sequence identity with the reference sequence SEQ ID NO:2, and comprises an amino acid sequence having at least two residue differences at residue positions X204 and X211 compared to SEQ ID NO:2. The amino acid residue at residue position X204 is selected from I, and the amino acid residue at residue position X211 is selected from F. The polypeptide has high activity, stability and resistance to solvents and substrates, and the ee value of the product is at least 99%.

[0013] The identity between two amino acid sequences or two nucleotide sequences can be obtained by algorithms commonly used in the art, and may be calculated according to the default parameters using NCBI Blastp and Blastn software, or the Clustal W algorithm (Nucleic Acid Research, 22(22):4673-4680, 1994) may be employed.

[0014] In another aspect, the present invention provides a polynucleotide sequence encoding an engineered ketoreductase polypeptide. In some embodiments, the polynucleotide may be part of an expression vector having one or more control sequences for expressing the engineered ketoreductase polypeptide. In some embodiments, the polynucleotide may comprise a polynucleotide sequence corresponding to the sequence shown in SEQ ID No: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127.

[0015] As is known to those skilled in the art, due to the degeneracy of nucleotide codons, the polynucleotide sequences encoding the amino acid sequences of SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 are not limited to SEQ ID No: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127. The nucleic acid sequence of the ketoreductase gene of the present invention may be any other nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 in the sequence listing.

[0016] In another aspect, the present disclosure provides a polynucleotide comprising a codon-optimized ketoreductase, or an expression vector and a host cell capable of expressing the engineered ketoreductase. In some embodiments, the host cell may be a bacterial host cell such as Escherichia coli. The host cell is used to express and isolate the engineered ketoreductase described herein, or may be used directly in a reaction to convert a substrate into a product.

[0017] In some embodiments, the engineered ketoreductase in the form of a whole cell, a crude extract, a separated polypeptide, or a purified polypeptide may be used alone or in an immobilized form (e.g., immobilized on a resin).

[0018] In another aspect, the improved engineered ketoreductase polypeptide described herein can convert a carbonyl compound into a chiral alcohol compound in the presence of the cofactor NADH. The present disclosure also provides a method for producing a wide range of compounds (I) or structural analogs thereof using the engineered ketoreductase polypeptide disclosed herein. In some embodiments, the engineered ketoreductase polypeptide can be used in a method for producing a compound of structural formula (I). JPEG2025518973000002.jpg35170

[0019] The alcohol product of the structural formula (I) has a chiral center represented by * with the indicated stereochemical configuration, and the alcohol product of the indicated structure (I) is in excess compared to the corresponding enantiomer.

[0020] Here, R 1 is optionally substituted aryl or heteroaryl, or optionally substituted C1-C8 hydrocarbyl, which may also be cyclo hydrocarbyl or heterocyclyl, R 2is an optionally substituted C1-C6 hydrocarbyl, halogen (e.g., -F, -Cl, -Br, and -I), alkenyl, alkynyl, aryl, heteroaryl, -NO2, -NO, -SO2R' or -SOR', -SR', -NR'R', -OR', -CO2R', or -COR', -C(O)NR', -SO2NH 2、 or -SONH2, -CN, -CF3, and R' is each independently -H, (C1-C4) hydrocarbyl, halogen, C1-C8 hydrocarbyl, C2-C 12 alkenyl, C2-C 12 alkynyl, cyclo hydrocarbyl, aryl, or heterocyclyl.

[0021] The method comprises contacting a carbonyl substrate of structural formula (II) with a ketoreductase polypeptide under reaction conditions suitable for the conversion of the carbonyl substrate to an alcohol product JPEG2025518973000003.jpg30170

[0022] and the ketoreductase polypeptide comprises a polypeptide consisting of an amino acid sequence shown by SEQ ID No: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128 described herein.

[0023] 2 Details 2.1 Definitions For the present disclosure, unless otherwise expressly defined, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0024] "Protein", "polypeptide", and "peptide" are used interchangeably herein and refer to polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids.

[0025] "Engineered ketoreductase", "engineered ketoreductase polypeptide", "improved ketoreductase polypeptide", and "engineered polypeptide" are used interchangeably herein.

[0026] Ketoreductase or alcohol dehydrogenase are used interchangeably herein.

[0027] "Polynucleotide" and "nucleic acid" are used interchangeably herein.

[0028] As used herein, "cofactor" refers to a non-protein compound that acts in concert with an enzyme in a catalytic reaction. As used herein, "cofactor" includes NADH (nicotinamide adenine dinucleotide) or NADPH (nicotinamide adenine dinucleotide phosphate) and their oxidized forms NAD+ or NADP+, which may also be referred to as coenzymes.

[0029] "Coding sequence" refers to the nucleic acid portion (e.g., gene) that encodes the amino acid sequence of a protein.

[0030] "Naturally occurring" or "wild-type" 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, is isolatable from a natural source, and has not been intentionally modified by human manipulation.

[0031] "Recombinant", "engineered", or "non-naturally occurring", when used to refer to, for example, a cell, nucleic acid, or polypeptide, refers to a material that has been modified in a way that does not occur in nature, or equivalently, a material that is produced or obtained from synthetic materials and / or by manipulation using recombinant techniques, or a material corresponding to the natural or native form of the material or materials.

[0032] "Sequence identity" and "homology" are used interchangeably herein to refer to a comparison between polynucleotides or polypeptides (where "sequence identity" and "homology" are typically expressed as a percentage) and are determined by comparing the two most favorably aligned sequences in a comparison window. Here, a portion of the polynucleotide or polypeptide sequence within the comparison window may include additions or deletions (i.e., gaps) for optimal alignment of the two sequences compared to a reference sequence. This percentage can be calculated as follows. Determine the number of positions in which the same nucleotide base or amino acid residue exists in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions within the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Optionally, this percentage can be calculated as follows. Determine the number of positions in which the same nucleotide base or amino acid residue exists in both sequences, or the number of positions in which the nucleotide base or amino acid residue is aligned with a gap, to obtain the number of matching positions, divide the number of matching positions by the total number of positions within the comparison window, and multiply the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will recognize that there are numerous established algorithms that can be used to align two sequences.Optimal alignment of arrays for comparison can utilize, for example, the local homology algorithm described in Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the homology alignment algorithm described in Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the similarity search method described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, the computer implementations of these algorithms (GAP, BESTFIT, FASTA, or TFASTA in the GCG Wisconsin software package) or visual inspection (generally see Current Protocols in Molecular Biology, F.M. Ausubel et al. eds., Current Protocols, a joint venture of Greene Publishing Associates Inc. and John Wiley & Sons, Inc., (1995 supplement) (Ausubel)). Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. The software used to perform BLAST analysis is publicly available on the website of the National Center for Biotechnology Information. This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits function as seeds to initiate a search to find longer HSPs that contain them.Subsequently, word matches are extended in both directions along each sequence until the cumulative alignment score can no longer increase. In the case of nucleotide sequences, the cumulative score uses parameters M (reward score for matching residue pairs, always >0) and N (penalty score for mismatched residues, always >0). In the case of amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word match string in each direction ends when: the cumulative alignment score drops by an amount X from its maximum achieved value; the cumulative score becomes zero or reaches either end of either sequence due to the accumulation of one or more negatively scored residue alignments. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands as default values. For amino acid sequences, the BLASTP program uses, as default values, a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary determinations of sequence alignment and percent sequence identity can be made using the BESTFIT or GAP programs of the GCG Wisconsin software package (Accelrys, Madison, Wisconsin) using the default parameters provided.

[0033] "Reference sequence" refers to a defined sequence used as a basis for sequence comparison. The reference sequence may be a subset of a larger sequence, such as a fragment of a full-length gene or polypeptide sequence. Generally, the reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, or the full length of a nucleic acid or polypeptide. Two polynucleotides or polypeptides may each (1) contain sequences that are similar between the two sequences (i.e., part of the complete sequence), and (2) may further contain sequences that differ between the two sequences. The comparison of sequences between two (or more) polynucleotides or polypeptides is usually performed by comparing the sequences of the two polynucleotides or polypeptides within a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, the "reference sequence" is not intended to be limited to a wild-type sequence and may include engineered or modified sequences. For example, a "reference sequence based on SEQ ID NO:2 having a proline at the residue corresponding to X53" means having a reference sequence in which the corresponding residue (isoleucine) of X53 in SEQ ID NO:2 is changed to proline.

[0034] "Comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues that can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acid residues, where a portion of the sequence within the comparison window may contain no more than 20% addition or deletion (i.e., gap) compared to the reference sequence (without addition or deletion) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues and optionally includes windows of 30, 40, 50, 100, or more.

[0035] When used in reference to the numbering of a specified amino acid sequence or polynucleotide sequence, "corresponding to", "referring to", or "relative to" refers to the number of the specified reference sequence residue when the specified amino acid sequence or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given sequence is not the actual numerical position of the residue within the given amino acid or polynucleotide sequence, but is assigned based on the reference sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered ketoreductase, can be aligned with a reference sequence, which is done by introducing gaps to optimize residue matches between the two sequences. In these cases, gaps are present, but the residue numbers of the given amino acid or polynucleotide sequence are determined relative to the reference sequence to which it is aligned.

[0036] "Difference in amino acid" or "residue difference" refers to the difference in an amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. A position where the amino acid is different is generally referred to herein as "Xn", where n refers to the corresponding position within the reference sequence that underlies the residue difference. For example, "residue difference at X53 compared to SEQ ID NO:2" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 53 of SEQ ID NO:2. Thus, if the reference polypeptide of SEQ ID NO:2 has isoleucine at position 53, "residue difference at X53 compared to SEQ ID NO:2" refers to any amino acid substitution other than isoleucine at the polypeptide position corresponding to position 53 of SEQ ID NO:2. In most examples herein, the difference in a particular amino acid residue at a position is denoted "XnY", where "Xn" refers to the corresponding position as described above and "Y" is the one-letter identifier of the amino acid found within the engineered polypeptide (i.e., a residue different from the residue of the reference polypeptide). In some examples (e.g., Table 1), the present disclosure also provides for differences in particular amino acids represented by the conventional notation "AnB". Here, A is the one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position within the reference sequence, and B is the one-letter identifier of the residue substitution within the sequence of the engineered polypeptide. In some examples, the polypeptides of the present disclosure may include differences in one or more amino acid residues relative to a reference sequence, represented by a list of specific positions having residue differences relative to the reference sequence.

[0037] "Deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions may include the removal of one or more, two or more, five or more, ten or more, fifteen or more, or twenty or more amino acids, while retaining the enzymatic activity of the engineered ketoreductase and / or retaining the improved properties of the engineered ketoreductase, and include up to 10% of the total number of amino acids constituting the reference enzyme, or up to 20% of the total number of amino acids constituting the reference enzyme. Deletions may include the internal portion and / or the terminal portion of the polypeptide. In various embodiments, deletions may include contiguous segments or may be discontinuous.

[0038] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered ketoreductase includes one or more amino acid insertions into a naturally occurring ketoreductase polypeptide and one or more amino acid insertions into another improved ketoreductase polypeptide. Insertions can be made into the internal portion of the polypeptide or at the carboxyl or amino terminus. As used herein, insertions include fusion proteins known in the art. Insertions may be contiguous segments of amino acids or may be separated by one or more amino acids within a naturally occurring polypeptide.

[0039] As used herein, "fragment" refers to a polypeptide having deletions at the amino and / or carboxyl termini, but having the remaining amino acid sequence identical to the corresponding positions within the sequence. Fragments may be at least 10 amino acids in length, at least 20 amino acids in length, at least 50 amino acids in length, and may be up to 70%, 80%, 90%, 95%, 98%, and 99% of the full-length ketoreductase polypeptide.

[0040] "Isolated polypeptide" refers to a polypeptide substantially isolated from other substances that are naturally associated, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cell or in vitro synthesis). The improved ketoreductase polypeptide can be present intracellularly, in cell culture medium, or can be produced in various forms such as lysates or isolated products. Thus, in some embodiments, the improved ketoreductase polypeptide may be an isolated polypeptide.

[0041] "Chiral center" refers to a carbon atom to which four different groups are attached.

[0042] "Stereoselectivity" refers to the preferential formation of one stereoisomer over another or others in a chemical or enzymatic reaction. Stereoselectivity may be partial, where one stereoisomer is formed preferentially over another, or complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is called enantioselectivity, and the excess of one enantiomer over the other in a mixture of two enantiomers (usually reported as a percentage) is usually optionally reported as the "enantiomeric excess" (abbreviated ee). When the stereoisomers are diastereoisomers, the stereoselectivity is called diastereoselectivity, i.e., the excess of one diastereoisomer over the other in a mixture of two diastereoisomers (usually reported as a percentage), and is usually optionally reported as the "diastereoisomeric excess" (abbreviated de). This ratio (typically a percentage) is often alternatively reported in the art as the enantiomeric excess (ee) calculated from the formula {major enantiomer concentration - minor enantiomer concentration} / {major enantiomer concentration + minor enantiomer concentration}.

[0043] The terms "stereoisomer", "stereoisomeric form" and similar expressions are used interchangeably herein and refer to all isomers of a molecule that differ only in the orientation of the atoms in space. This includes enantiomers and isomers of compounds with multiple chiral centers that are not mirror images of each other (i.e., "diastereomers").

[0044] "Improved enzyme properties" refers to a ketoreductase polypeptide that exhibits some improved enzyme properties as compared to a reference ketoreductase such as a wild-type ketoreductase or another engineered ketoreductase. Enzyme properties that may be desirable to improve include, but are not limited to, enzyme activity (which can be expressed as the percentage of substrate conversion), thermal stability, solvent stability, pH-activity profile, cofactor requirements, resistance to inhibitors (e.g., inhibitors of the substrate or product), stereospecificity, and stereoselectivity.

[0045] "Conversion" refers to the enzymatic conversion of a substrate to the corresponding product. "Percentage of conversion" or "conversion rate" refers to the percentage of substrate within the reaction system that is converted to product under specified reaction conditions and within a specified reaction time. Thus, the "enzyme activity" or "activity" of a ketoreductase polypeptide can be expressed as the "percentage of conversion" from substrate to product. The conversion rate is generally calculated by taking a sample and measuring the product concentration and substrate concentration within the reaction system as {product molar concentration} / {substrate molar concentration + product molar concentration}.

[0046] "Thermal stability" means that the ketoreductase polypeptide maintains similar activity compared to the wild-type enzyme after being exposed to high temperature (e.g., 68°C or higher) for a certain period (e.g., 2.5 hours or more).

[0047] "Solvent stability" or "solvent tolerance" means that the ketoreductase polypeptide maintains similar activity compared to the wild-type enzyme after being exposed to solvents (such as methanol, ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) at different concentrations (e.g., 5 - 99%) for a certain period (e.g., 0.5 - 24 hours).

[0048] "Suitable reaction conditions" refers to the conditions in the biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, co-solvent, etc.), and also includes reactive species that regenerate cofactors under conditions where the ketoreductase polypeptide of the present disclosure can convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in the present disclosure and illustrated by the examples.

[0049] "Hydrocarbyl" refers to a straight-chain or branched-chain hydrocarbyl group. A number with a subscript following the symbol "C" designates the number of carbon atoms that may be included in a particular group. For example, "C1-C8" refers to a straight-chain or branched-chain hydrocarbyl group having 1 to 8 carbon atoms. The hydrocarbyl group may be optionally substituted by one or more substituents. "Aryl" refers to a monovalent aromatic hydrocarbyl having 6 to about 20 carbon atoms. "Heteroaryl" and "heteroaromatic" refer to an aryl group in which one or more carbon atoms of the parent aromatic ring system are substituted with heteroatoms (O, N, or S). When used to modify a particular group or radical, "substituted" means that one or more hydrogen atoms of the particular group or radical are each independent of one another. As used herein, "compound" refers to any compound encompassed by the structural formula and / or chemical name associated with the compounds disclosed herein. The compound may be indicated by its chemical structure and / or chemical name. If the chemical structure conflicts with the chemical name, the compound is determined by the chemical structure. Unless otherwise specified, the chemical structures described herein include all possible isomeric forms of the described compounds.

[0050] It is replaced by the same or different substituents. "Substituted hydrocarbyl, aryl or heteroaryl" refers to a hydrocarbyl, aryl or heteroaryl group in which one or more hydrogen atoms are replaced by another substituent. "Optional" or "optionally" means that the described event or situation may or may not occur. For example, "optionally substituted aryl" refers to an aryl group that may or may not be substituted. This description includes both substituted and unsubstituted aryl groups.

[0051] As used herein, "compound" refers to any compound encompassed by the structural formula and / or chemical name associated with the compounds disclosed herein. A compound may be indicated by its chemical structure and / or chemical name. If the chemical structure conflicts with the chemical name, the compound is determined by the chemical structure. Unless otherwise specified, the chemical structures described herein include all possible isomeric forms of the described compounds.

[0052] 2.2 Improved Engineering Ketoreductase Table 1 below lists and describes the engineering ketoreductase polypeptides developed according to the present invention. Each row shows the nucleotide sequence number and amino acid sequence number of a specific engineering ketoreductase polypeptide, as well as the difference in residues compared to SEQ ID NO:2. The catalytic performance of each exemplified engineering ketoreductase peptide under different reaction conditions.

[0053] JPEG2025518973000004.jpg217170JPEG2025518973000005.jpg230170JPEG2025518973000006.jpg75170Note: The multiple of catalytic activity described in Table 1 refers to the ratio of the catalytic activity of the engineered ketoreductase that has undergone directed evolution to SEQ ID No:2. Table 1 shows that ethyl (R)-4-chloro-3-hydroxybutyrate catalyzed by the engineered ketoreductase had an ee value higher than 99%. Reaction condition 1: Ethyl 4-chloroacetoacetate 180 g / L, wet cells 10 g / L, NAD + 0.5 g / L, IPA 25% (V / V), 0.1 M PB, 30 °C, pH 7.0, 24 h Reaction condition 2: Ethyl 4-chloroacetoacetate 250 g / L, wet cells 10 g / L, NAD + 0.5 g / L, IPA / toluene / water: 25% / 40% / 35% (V / V / V), 0.1 M PB, 30 °C, pH 7.0, 24 h Reaction condition 3: Ethyl 4-chloroacetoacetate 250 g / L, wet cells 10 g / L after heat treatment at [68 °C, 4 h], NAD + 0.5 g / L, IPA / toluene / water: 25% / 40% / 35% (V / V / V), 0.1 M PB, 30 °C, pH 7.0, 24 h Reaction condition 4: Ethyl 4-chloroacetoacetate 400 g / L, wet cells 30 g / L, NAD + 0.5 g / L, IPA / toluene / water: 25% / 40% / 35% (V / V / V), 0.1 M PB, 30 °C, pH 7.0, 24 h For the manufacturing process of the wet cells described in Reaction conditions 1, 2, and 4, refer to Example 2. The wet cells after heat treatment described in Reaction condition 3 are the wet cells heat-treated at [68 °C, 4 h] based on the above-mentioned wet cells.

[0054] Under reaction condition 1, the catalytic activity of the engineered ketoreductase disclosed in the present application is clearly higher than that of the wild-type enzyme, reaching up to 8.5 times the maximum catalytic activity of the wild-type enzyme. Under reaction condition 2, in the presence of the organic solvent toluene, the engineered ketoreductase disclosed in the present application is more suitable for the two-phase system, has excellent solvent tolerance, and the catalytic activity of the engineered ketoreductase is about 7 to 12 times that of the wild-type enzyme. Under reaction condition 3, due to the difference in the thermal stability of the ketoreductase polypeptide corresponding to the amino acid sequence in Table 1, different ketoreductase polypeptides show different degrees of inactivation after heat treatment, and as a result, the catalytic performance changes. The catalytic activity of the engineered ketoreductase disclosed in the present application is about 15 to 26 times that of the wild-type enzyme. From the above, it was found that the engineered ketoreductase disclosed in the present application has high thermal stability. Under reaction condition 4, when the concentration of the substrate ethyl 4-chloroacetoacetate was increased to 400 g / L, due to the inhibitory effect of the substrate on the wild-type enzyme, the conversion rate of SEQ ID NO:2 decreased. On the other hand, the engineered enzyme substrate has high tolerance. Even when the substrate concentration is 400 g / L, the engineered ketoreductase disclosed in the present application can achieve a conversion rate of 94% or more, up to 99.97%.

[0055] 2.3 Polynucleotides, control sequences, expression vectors and host cells that can be used for the production of engineered ketoreductase polypeptides In another aspect, the present disclosure provides a polynucleotide encoding an engineered polypeptide having the ketoreductase activity described herein. The polynucleotide can be operably linked to one or more heterologous regulatory sequences that control gene expression to generate a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered ketoreductase can be introduced into a suitable host cell to express the corresponding engineered ketoreductase polypeptide.

[0056] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provide a description of all polynucleotides that can encode a protein sequence of interest. Due to the degeneracy of the genetic code, where the same amino acid is encoded by alternative or synonymous codons, a very large number of nucleic acids can be generated, all of which encode the improved ketoreductase polypeptides disclosed herein. Thus, after determining a particular amino acid sequence, one skilled in the art can generate any number of different nucleic acids by modifying the sequence of only one or more codons in a way that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates any possible variation of the polynucleotides that can be produced by selecting combinations based on possible codon usage, and for any polypeptide disclosed herein, the amino acid sequences of the exemplary engineered polypeptides provided in Table 1 are included.

[0057] In various embodiments, the codons are preferably selected to be compatible with the host cell in which the protein is to be produced. For example, preferred codons used in bacteria are those used for gene expression in bacteria. Preferred codons used in yeast are those used for gene expression in yeast, and preferred codons used in mammals are those used for gene expression in mammalian cells.

[0058] In some embodiments, the polynucleotide encodes a ketoreductase polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an even-numbered reference sequence selected from SEQ ID NOs: 4 - 128, the polypeptide has ketoreductase activity, and has the ability to convert a substrate to a product with increased activity compared to, for example, the polypeptide of SEQ ID NO: 2, having one or more of the improved properties described herein.

[0059] In some embodiments, the polynucleotide encoding the engineered ketoreductase polypeptide comprises a sequence selected from the odd numbered SEQ ID NOs: 3 to 127.

[0060] In some embodiments, the polynucleotide encodes the polypeptide described herein and has at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered ketoreductase. In some embodiments, the reference polynucleotide sequence is selected from the sequences having odd numbered SEQ ID NOs: 3 to 127.

[0061] The isolated polynucleotide encoding the engineered ketoreductase polypeptide can be manipulated in a variety of ways to provide for expression of the polypeptide. Such methods include further alteration of the sequence to improve expression by codon optimization, insertion into appropriate expression elements with or without additional control sequences, and transformation into a host cell suitable for expression and production of the polypeptide.

[0062] Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide before insertion into the vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel, F. ed., Greene Pub. Associates, 1998, updated 2010.

[0063] In another aspect, the present disclosure also relates to a recombinant expression vector comprising a polynucleotide encoding an engineered ketoreductase polypeptide or a variant thereof, and one or more expression regulatory regions, such as a promoter, a terminator, an origin of replication, etc., according to the type of host to be introduced. Optionally, the nucleic acid sequence of the present disclosure can be expressed by inserting the nucleic acid sequence or a nucleic acid construct containing the sequence into an appropriate expression vector. When constructing an expression vector, the coding sequence is placed within the vector such that the coding sequence is operably linked to an appropriate control sequence for expression.

[0064] The recombinant expression vector can be conveniently used in DNA recombination steps and can be any vector (e.g., a plasmid or a virus) that can bring about the expression of a polynucleotide sequence. The choice of vector usually depends on the compatibility between the vector and the host cell into which the vector is introduced. The vector may be a linear plasmid or a circular plasmid. The expression vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, and its replication is independent of the replication of chromosomes such as plasmids, extrachromosomal elements, mini-chromosomes, or artificial chromosomes. The vector may contain any means for ensuring self-replication. Optionally, the vector may be integrated into the genome when introduced into the host cell and replicate together with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain all the DNA introduced into the host cell genome can be used.

[0065] Many expression vectors useful in the embodiments of the present disclosure are commercially available. An exemplary expression vector can be produced by operably linking a polynucleotide encoding an improved ketoreductase polypeptide to the plasmid pACYC-Duet-1 (Novagen).

[0066] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding an engineered ketoreductase polypeptide, wherein the polynucleotide is operably linked to one or more control sequences for expressing ketoreductase in the host cell. Host cells for expressing the polypeptide encoded by the expression vector of the present disclosure are well known in the art and include bacterial cells such as Escherichia coli, Arthrobacter sp. KNK168, Streptomyces spp., Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells such as Drosophila S2 cells and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells, but are not limited thereto. An exemplary host cell is Escherichia coli BL21(DE3). The above host cells may be wild-type or engineered cells that have undergone genome editing such as knocking out the wild-type ketoreductase gene contained in the host cell genome. Media and growth conditions suitable for the above host cells are well known in the art.

[0067] The polynucleotide for expressing ketoreductase can be introduced into cells by various methods known in the art. Techniques include electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, protoplast fusion, etc. Various methods for introducing polynucleotides into cells will be apparent to those skilled in the art.

[0068] 2.4 Method for Producing Engineered Ketoreductase Polypeptide When the sequence of an engineering polypeptide is known, the polynucleotide encoding the polypeptide can be produced by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then ligated (e.g., by enzymatic or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides of the present disclosure can be produced through chemical synthesis, for example, by the classical phosphoramidite method described in Beaucage et al., 1981, Tet Lett 22:1859-69, or the method described in Matthes et al., 1984, EMBO J. 3:801-05, which are typically carried out in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized, purified, annealed, ligated, and cloned into an appropriate vector, for example, using an automated DNA synthesizer. Also, essentially any nucleic acid can be obtained from any of a variety of commercial sources.

[0069] In some embodiments, the present disclosure also provides a method for producing or generating an engineered ketoreductase polypeptide, the method including culturing a host cell capable of expressing a polynucleotide encoding the engineered polypeptide under culture conditions suitable for the expression of the polypeptide. In some embodiments, the method for producing the polypeptide further includes isolating the polypeptide. The engineered polypeptide is expressed in a suitable cell and can be isolated (or recovered) from the host cell and / or the medium using any one or more of the techniques well known for protein purification. The protein purification techniques include, but are not limited to, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, chromatography, and the like.

[0070] 2.5 Methods Using Engineered Ketoreductase and Compounds Produced Thereby In another aspect, the engineered ketoreductase polypeptide described herein can convert a carbonyl compound to a chiral alcohol-based compound in the presence of the cofactor NADH. The present disclosure also provides a method for producing a wide range of compounds (I) or structural analogs thereof using the engineered ketoreductase polypeptides disclosed herein. In some embodiments, the engineered ketoreductase polypeptide can be used in a method for producing a compound of structural formula (I). JPEG2025518973000007.jpg36170

[0071] The alcohol product of the structural formula (I) has a chiral center represented by * with the indicated stereochemical configuration, and the alcohol product of the indicated structure (I) is in excess compared to the corresponding enantiomer.

[0072] R 1 is optionally substituted aryl or heteroaryl, or optionally substituted C1-C8 hydrocarbyl, which may be cyclo hydrocarbyl or heterocyclyl, R 2 is optionally substituted C1-C6 hydrocarbyl, halogen (e.g., -F, -Cl, -Br, and -I), alkenyl, alkynyl, aryl, heteroaryl, -NO2, -NO, -SO2R' or -SOR', -SR', -NR'R', -OR', -CO2R' or -COR', -C(O)NR', -SO2NH2, or -SONH2, -CN, -CF3, and R' is each independently, -H, (C1-C4) hydrocarbyl, halogen, C1-C8 hydrocarbyl, C2-C 12 alkenyl, C2-C 12 alkynyl, cyclo hydrocarbyl, aryl or heterocyclyl.

[0073] The method is a carbonyl substrate of structural formula (II) under reaction conditions suitable for the conversion of a carbonyl substrate to an alcohol product Contacting JPEG2025518973000008.jpg29170 with a keto-reductase polypeptide, wherein the keto-reductase polypeptide is an engineered keto-reductase polypeptide as described herein.

[0074] In some embodiments, the improved ketoreductase polypeptide according to the present disclosure reduces ethyl 4-chloroacetoacetate with higher activity to produce ethyl (R)-4-chloro-3-hydroxybutyrate. The improved engineered ketoreductase polypeptide according to the present invention has higher activity, stability, and resistance to solvents and substrates compared to the wild-type ketoreductase corresponding to SEQ ID NO:2, and can more effectively catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate. Alternatively, even when the substrate concentration is low, the improved engineered ketoreductase polypeptide according to the present invention can catalyze the reduction of ethyl 4-chloroacetoacetate to produce ethyl (R)-4-chloro-3-hydroxybutyrate without inhibition. These improved ketoreductase polypeptides may include an amino acid sequence having one or more residue differences from the SEQ ID NO:2 sequence at residue positions corresponding to X18, X22, X42, X46, X47, X48, X53, X67, X79, X80, X94, X96, X111, X115, X149, X204, X211, X229, X238, X239, X262, X282, X283, X284, X286, X303, X327. The improved ketoreductase polypeptide includes an amino acid sequence comprising at least one of the characteristics of V18I, A22K, I42L, D46G, D46F, D46V, M47I, M47S, P48R, P48H, I53Q, I53G, I53P, I53T, I53S, I53H, I53N, I53R, I53K, T67V, T67I, E79K, T80V, A94R, A94Y, A94C, A94H, H96R, E111H, T115R, T115C, T115S, T115H, V149A, L204I, L211F, D229A, E238R, E238K, G239S, I262V, F282Q, F282G, M283N, M283D, I284M, I284L, I284V, F286V, F286H, F286R, D303E, T327L, T327E, or includes one or more amino acid residue insertions or deletions in addition to these differences.

[0075] More specifically, in some embodiments, the engineered ketoreductase polypeptide improved based on SEQ ID NO:2 comprises a polypeptide consisting of an amino acid sequence shown in SEQ ID No:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128.

[0076] In some embodiments, the improved engineered ketoreductase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the reference sequences of SEQ ID No:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128.

[0077] In some embodiments of this method, the chiral alcohol product of structural formula (I) is ethyl (R)-(+)-4-chloro-3-hydroxybutyrate, The carbonyl substrate of structural formula (II) in JPEG2025518973000009.jpg28170 is ethyl 4-chloroacetoacetate. JPEG2025518973000010.jpg33170

[0078] In some embodiments of this method, the chiral alcohol product of structural formula (I) is methyl (S)-(-)-3-hydroxybutyrate, The carbonyl substrate of structural formula (II) in JPEG2025518973000011.jpg25170 is methyl acetoacetate. JPEG2025518973000012.jpg29170

[0079] In some embodiments of this method, the chiral alcohol product of structural formula (I) is ethyl (S)-(-)-3-hydroxybutyrate, The carbonyl substrate of structural formula (II) in JPEG2025518973000013.jpg33170 is ethyl acetoacetate. JPEG2025518973000014.jpg30170

[0080] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol, The carbonyl substrate of structural formula (II) in JPEG2025518973000015.jpg49170 is 3,5-bis(trifluoromethyl)acetophenone. JPEG2025518973000016.jpg45170

[0081] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (S)-1,3-butanediol, The carbonyl substrate of structural formula (II) in JPEG2025518973000017.jpg27170 is 4-hydroxy-2-butanone. JPEG2025518973000018.jpg30170

[0082] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (2S,5S)-2,5-hexanediol, JPEG2025518973000019.jpg31170 The carbonyl substrate of structural formula (II) is 2,5 - hexanedione. JPEG2025518973000020.jpg35170

[0083] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (S)-1 - tert - butoxycarbonyl - 3 - hydroxypiperidine, JPEG2025518973000021.jpg40170 The carbonyl substrate of structural formula (II) is N - tert - butoxycarbonyl - 3 - piperidone. JPEG2025518973000022.jpg39170

[0084] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (S)-(-)-3 - chloro - 1 - phenyl - 1 - propanol, JPEG2025518973000023.jpg34170 The carbonyl substrate of structural formula (II) is 3 - chloropropiophenone. JPEG2025518973000024.jpg40170

[0085] In some embodiments of this method, the chiral alcohol product of structural formula (I) is methyl (R)-(+)-4 - chloro - 3 - hydroxybutyrate, JPEG2025518973000025.jpg28170 The carbonyl substrate of structural formula (II) is methyl 4 - chloroacetoacetate. JPEG2025518973000026.jpg29170

[0086] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (2S,3S)-2,3 - butanediol, JPEG2025518973000027.jpg36170 The carbonyl substrate of structural formula (II) is 2,3 - butanedione. JPEG2025518973000028.jpg37170

[0087] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (2S,4S)-2,4-pentanediol, and The carbonyl substrate of structural formula (II) in JPEG2025518973000029.jpg28170 is acetylacetone. JPEG2025518973000030.jpg29170

[0088] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (2S,5S)-2,5-hexanediol, and The carbonyl substrate of structural formula (II) in JPEG2025518973000031.jpg36170 is 2,5-hexanedione. JPEG2025518973000032.jpg39170

[0089] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (3S,5S)-3,5-heptanediol, and The carbonyl substrate of structural formula (II) in JPEG2025518973000033.jpg21170 is 3,5-heptanedione. JPEG2025518973000034.jpg26170

[0090] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (3S,6S)-3,6-octanediol, and The carbonyl substrate of structural formula (II) in JPEG2025518973000035.jpg33170 is 3,6-octanedione. JPEG2025518973000036.jpg32170

[0091] In some embodiments of this method, the chiral alcohol product of structural formula (I) is (3S,6S)-2,7-dimethyl-3,6-octanediol, JPEG2025518973000037.jpg The carbonyl substrate of Structural Formula (II) is 2,7-dimethyloctane-3,6-dione. JPEG2025518973000038.jpg35170

Brief Description of the Drawings

[0092]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0093] The following examples are used to further illustrate the present invention, but the present invention is not limited thereto. In the following examples, experimental methods without specifying specific conditions usually follow conventional conditions or conditions recommended by the manufacturer.

[0094] Example 1: Gene Cloning and Construction of Expression Vector The wild-type ketoreductase derived from Rhodococcus with the amino acid sequence shown in SEQ ID NO:2 was obtained by searching NCBI. Next, the corresponding nucleic acid was synthesized by a technique commonly used in the art and cloned into the expression vector pACYC-Duet-1. Under the transformation conditions of heat shock at 42°C for 90 seconds, the recombinant expression plasmid was transformed into competent cells of E.coil BL21(DE3). The transformation solution was spread on an LB plate containing chloramphenicol and cultured overnight at 37°C with inversion to obtain recombinant transformants.

[0095] Example 2: Expression of Keto Reductase Polypeptide and Production of Wet Bacterial Cells or Enzyme Solution Containing Keto Reductase Polypeptide The production steps of the enzyme solution of the present invention are as follows. The recombinant E. coli BL21(DE3) obtained in Example 1 was inoculated into a 250 mL Erlenmeyer flask containing 500 mL of LB medium (peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, pH 7.0 ± 0.2, 25 °C) containing chloramphenicol, and placed on a shaker at 30 °C and 250 rpm for overnight shaking culture. When the OD600 of the culture solution reached 2, it was inoculated into a 1000 mL Erlenmeyer flask containing 250 mL of TB medium (tryptone 12 g / L, yeast extract powder 24 g / L, disodium hydrogen phosphate 9.4 g / L, dipotassium hydrogen phosphate 2.2 g / L, lactose 6 g / L, pH 7.0 ± 0.2, 30 °C) at an inoculation amount of 5% (v / v). Here, ZnCl2 with a final concentration of 1 mM was also added to this medium. It was placed on a shaker at 30 °C and 250 rpm for shaking culture. During the shaking culture, lactose induced the expression of the keto reductase polypeptide. After 20 h, the culture solution was centrifuged (8000 rpm, 10 minutes). After centrifugation, the supernatant was discarded, and the cells were collected to obtain wet bacterial cells containing the keto reductase polypeptide. The wet bacterial cells can be directly used for the reaction or stored frozen at -20 °C until use.

[0096] It is also possible to directly use the wet bacterial cells to produce an enzyme solution. The obtained wet bacterial cells were suspended in 30 mL of phosphate buffer (PB, pH 7.0), sonicated in an ice bath, and centrifuged (4000 rpm, 15 minutes), and the supernatant was collected to obtain an enzyme solution containing the keto reductase polypeptide.

[0097] According to the shaking flask production method, the culture solution was obtained by scaling down to a 96-well plate for culture at the same ratio, and the culture supernatant was removed by centrifugation to obtain wet bacterial cells. The enzyme solution can be obtained by a chemical disruption method well known in the art.

[0098] Example 3: Heat Treatment Using the production method described in Example 2, 25 mL of an enzyme solution of the ketoreductase polypeptide corresponding to SEQ ID No: 38 was produced. It was placed in a 50 mL centrifuge tube, put into a water bath at 68 °C and stirred to uniformly heat the enzyme solution. After 4 hours, the enzyme solution was centrifuged (4000 rpm, 30 minutes), and the supernatant was collected to obtain the enzyme solution after heat treatment. A sample of the enzyme solution after heat treatment was subjected to SDS-PAGE electrophoresis analysis, and the electrophoresis results are shown in Figure 3. Sample 1 is the enzyme solution corresponding to SEQ ID No: 38, and Sample 2 is the enzyme solution after heat treatment corresponding to SEQ ID No: 38. The results in Figure 3 show that after heat treatment at [68 °C, 4 h], a large amount of background proteins can be removed, indicating that SEQ ID No: 38 has very good thermal stability.

[0099] Example 4: Construction of a ketoreductase mutant library All reagents used here are commercially available, preferably the Quikchange kit (supplier: Agilent). The sequence design of the mutation primers was carried out according to the kit instructions. Here, the construction of a saturation mutation library at a single residue position was used as an example for explanation. The PCR system was 10 μL of 5x Buffer, 1 μL of 10 mM dNTP, 1 μL of plasmid DNA template (50 ng / uL), 0.75 μL of upstream and downstream primers (10 uM), 0.5 μL of high-fidelity enzyme, and 36 μL of ddH2O. The codon of the PCR primer at the mutation position was NNK.

[0100] The PCR amplification steps were as follows. (1) Pre-denaturation at 98 °C for 3 minutes, (2) Denaturation at 98 °C for 10 seconds, (3) Annealing and extension at 72 °C for 3 minutes. Steps (2)-(3) were repeated 25 times. (5) Extension was continued at 72 °C for 10 minutes and cooled to 4 °C. 2 μL of DpnI was added to the PCR product and digested overnight at 37 °C to remove the plasmid template. The digested PCR product was transformed into E. coli BL21(DE3) competent cells by electroporation and plated on an LB plate containing chloramphenicol to obtain a saturation mutation library at the target residue position.

[0101] Example 5: High-throughput screening of a ketoreductase mutant library For the expression of the ketoreductase mutant library, culturing and expression induction were carried out using a 96-well plate according to the method described in Example 2 to obtain wet bacterial cells.

[0102] 200 μL / well of PB buffer (0.1 M phosphate buffer, pH 7.0) was added to the deep-well plate containing the wet bacterial cells, sealed with a film, then placed on a plate shaker and shaken at 700 rpm for 1 h. Thereafter, 40 μL of the bacterial solution per well was sampled into a 96-well plate that had been pre-filled with a reaction mother liquor (160 μL / well). Next, the 96-well plate was heat-sealed with an aluminum film, placed on a shaker at 30 °C and 200 rpm to initiate the reaction. After reacting for 24 h, 1 mL of ethyl acetate was added to this well plate to stop the reaction, placed on a flat shaker and shaken for 30 min (800 rpm), then centrifuged (4000 rpm, 10 min), and the supernatant after centrifugation was subjected to GC analysis to detect the conversion rate.

[0103] The reaction mother liquor was prepared as follows. a) Ethyl 4-chloroacetoacetate was dissolved in isopropanol. b) NAD⁺ was dissolved in PB buffer, and a) and b) were mixed to obtain a reaction mother liquor with a final concentration of [ethyl 4-chloroacetoacetate 304.5 g / L, isopropanol 31.25% (v / v), NAD⁺ 0.625 g / L, PB 0.1 M, pH 7.0].

[0104] Example 6: Analysis and detection method for ethyl (R)-(+)-4-chloro-3-hydroxybutyrate GC analysis method The chromatographic column was Agilent DB-WAX 15m * 0.25mm * 0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 140°C, the split ratio was 10:1, the detector temperature was 300°C, the injection volume was 2μL, and the column temperature was 100°C. The temperature was increased to 140°C at 15°C / min and maintained for 3.9 minutes. The retention time of ethyl 4-chloroacetoacetate was 3.8 minutes, and the retention time of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate was 5.4 minutes.

[0105] HPLC chiral analysis method The chromatographic column was Daicel CHIRALCEL AD-H 250mm * 4.6mm * 5μm, the mobile phase was 95% n-hexane + 5% isopropanol, the column temperature was 30°C, the injection volume was 20μL, the flow rate was 1ml / min, and the wavelength was 210nm. The retention time of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate was 11.5 minutes, the retention time of ethyl (S)-(-)-4-chloro-3-hydroxybutyrate was 11.4 minutes, and the retention time of ethyl 4-chloroacetoacetate was 4.1 minutes.

[0106] Example 7: Fermentation expression A single microbial colony of Escherichia coli BL21(DE3) containing the target engineering ketoreductase polypeptide expression plasmid was inoculated into 50 mL of LB medium (5.0 g / L yeast extract, 10 g / L tryptone, 10 g / L sodium chloride) containing 30 μg / mL chloramphenicol. The culture was incubated at 30°C and 250 rpm on a shaker for 16 h. When the OD600 of the culture broth reached 3.5 - 5.0, the medium was taken out from the shaker.

[0107] A 1.0 L fermentation tank containing 0.6 L of the fermentation basal medium was sterilized in a high-pressure steam sterilizer at 121 °C for 30 minutes. ZnCl2 with a final concentration of 1.0 mM was added to the bottom of the fermentation tank, mixed uniformly, and then inoculated with the above shaking flask culture solution. The fermentation temperature was controlled to be maintained at 37 °C, the speed of the stirring paddle was controlled within the range of 200 - 1000 rpm, air was supplied to the fermentation vessel at 0.4 - 0.8 L / min to maintain the dissolved oxygen level above 35%, 25 - 28% v / v ammonium hydroxide was added to maintain the pH of the culture solution at 7.0. The growth of the bacteria was maintained by supplying a feed solution containing 500 g / L of edible glucose dextrose monohydrate, 12 g / L of ammonium chloride, and 5 g / L of magnesium sulfate heptahydrate. After culturing for 8 h, when the OD600 of the culture solution reached 35 ± 5, the temperature of the culture solution was lowered and maintained at 30 °C. Next, α-lactose monohydrate was added to a final concentration of 15 g / L to induce the expression of ketoreductase. Fermentation was continued for 16 h, and the fermentation broth was collected from the tank. Using a Thermo Multifuge X3R centrifuge, the fermentation broth was centrifuged at 4 °C and 8000 rpm for 10 minutes to collect the wet bacterial cells. The collected wet bacterial cells can be directly used in the post-treatment process or stored frozen at -20 °C until use.

[0108] Example 8: Reaction process for catalyzing the formation of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineered ketoreductase The following is a typical reaction flow and post-treatment process with a volume of 100 mL. 1.0 g of wet bacterial cells (containing the enzyme with the SEQ ID No: 34 sequence), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD+, 25 mL of isopropanol, and 40 mL of toluene were added to a 250 mL three-neck reaction flask. Finally, 24.4 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction was stirred at a reaction temperature of 30 °C and 250 rpm. After reacting for 24 h, during which the pH was controlled in real time, when reacting for 16 h, a water pump was used to vacuum aspirate once at 0.07 MPa to remove the acetone generated by the reaction. After 24 h, a sample was taken for detection, and the conversion rate was 99.5%.

[0109] After the reaction was completed, the mixture was allowed to stand, and 18 mL of wastewater was separated as the lower layer, and the toluene layer was separated as the upper layer. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone, and toluene. After changing to an oil pump and raising the temperature, 22.4 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 90.7%, a purity of 99%, and an ee ≥ 99.8%.

[0110] Example 9: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a typical reaction flow and post-treatment process with a volume of 100 mL. 1.0 g of wet cells (containing the enzyme with the sequence of SEQ ID No: 6), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD+, 25 mL of isopropanol, and 35 mL of toluene were added to a 250 mL three-neck reaction flask. Finally, 30.5 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction was stirred at a reaction temperature of 30 °C and 250 rpm. After reacting for 24 h, during which the pH was controlled in real time, when reacting for 16 h, the water pump was used to aspirate under vacuum at 0.07 MPa once to remove the acetone generated by the reaction. After 24 h, a sample was taken for detection, and the conversion rate was 99.5%.

[0111] After the reaction was completed, the mixture was allowed to stand, and 18 mL of wastewater was separated as the lower layer, and the toluene layer was separated as the upper layer. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone, and toluene. After changing to an oil pump and raising the temperature, 28.1 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91%, a purity of 99%, and an ee ≥ 99.8%.

[0112] Example 10: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a representative reaction flow and post-treatment process with a volume of 100 mL. Add 1.0 g of wet cells (containing the enzyme of SEQ ID No: 56), 10 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD⁺, 25 mL of isopropanol, and 45 mL of toluene to a 250 mL three-necked reaction flask. Finally, add 24.4 g of ethyl 4-chloroacetoacetate and start stirring. Stir at 250 rpm at a reaction temperature of 30 °C. React for 24 h, during which the pH is controlled in real time. When reacting for 16 h, aspirate the reaction flask to vacuum once with a water pump at 0.07 MPa to remove the acetone generated by the reaction. After 24 h, samples were taken for detection, and the conversion rate was 99.5%.

[0113] After the reaction was completed, the mixture was allowed to stand, and 12 mL of waste water was separated as the lower layer, and the toluene layer was separated as the upper layer. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone, and toluene. After replacing with an oil pump and raising the temperature, 22.7 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91.9%, a purity of 99%, and an ee ≥ 99.8%.

[0114] Example 11: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a representative reaction flow and post-treatment process with a volume of 100 mL. Add 1.0 g of wet cells (containing the enzyme of SEQ ID No: 70), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD⁺, 25 mL of isopropanol, and 27 mL of toluene to a 250 mL three-necked reaction flask. Finally, add 40 g of ethyl 4-chloroacetoacetate and start stirring. Stir at 250 rpm at a reaction temperature of 30 °C. React for 24 h, during which the pH is controlled in real time. When reacting for 16 h, aspirate the reaction flask to vacuum once with a water pump at 0.07 MPa to remove the acetone generated by the reaction. After 24 h, samples were taken for detection, and the conversion rate was 99.5%.

[0115] After the reaction was completed, the mixture was allowed to stand, and 18 mL of wastewater was separated into the lower layer and the toluene layer was separated into the upper layer by liquid separation. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone and toluene. After changing to an oil pump and raising the temperature, 37 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91.4%, a purity of 99%, and an ee of ≥99.8%.

[0116] Example 12: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a typical reaction flow and post-treatment process with a volume of 100 mL. 1.0 g of wet cells (containing the enzyme of SEQ ID No: 92), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD+, 25 mL of isopropanol and 35 mL of toluene were added to a 250 mL three-necked reaction flask. Finally, 30.5 g of ethyl 4-chloroacetoacetate was added and stirring was started. The reaction was stirred at a reaction temperature of 30 °C and 250 rpm. After reacting for 24 h, during which the pH was controlled in real time at 6.5 - 7.5, when reacting for 16 h, the water pump was used to vacuum aspirate once at 0.07 MPa to remove the acetone produced by the reaction. After 24 h, a sample was taken for detection, and the conversion rate was 99.5%.

[0117] After the reaction was completed, the mixture was allowed to stand, and 18 mL of wastewater was separated into the lower layer and the toluene layer was separated into the upper layer by liquid separation. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone and toluene. After changing to an oil pump and raising the temperature, 28.1 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91%, a purity of 99%, and an ee of ≥99.8%.

[0118] Example 13: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a typical reaction flow and post-treatment process for a 100 mL volume. Into a 250 mL three-neck reaction flask, 1.0 g of wet cells (containing the enzyme of SEQ ID No: 92), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD+, 25 mL of isopropanol, and 35 mL of toluene were added. Finally, 30.6 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction was carried out at a reaction temperature of 30 °C with stirring at 250 rpm. The reaction was allowed to proceed for 24 h, during which the pH was controlled in real-time at 5.5 - 6.5. When the reaction had proceeded for 16 h, a water pump was used to perform a vacuum suction once at 0.07 MPa to remove the acetone produced by the reaction. After 24 h, a sample was taken for detection, and the conversion rate was found to be 99.5%.

[0119] After the reaction was completed, it was allowed to stand, and 18 mL of waste water was separated as the lower layer, and the toluene layer was separated as the upper layer. The toluene layer was subjected to vacuum distillation using a water pump to remove isopropanol, acetone, and toluene. The oil pump was replaced and the temperature was raised, and 28.5 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 92%, a purity of 99%, and an ee ≥ 99.8%.

[0120] Example 14: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a pure aqueous phase reaction process with a volume of 100 mL. Into a 250 mL three-neck reaction flask, 2.0 g of wet cells (containing the enzyme of SEQ ID No: 56), 55 mL of 0.1 M phosphate buffer with pH = 7.6, 50 mg of coenzyme NAD+, and 25 mL of isopropanol were added. Finally, 24.4 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction was carried out at a reaction temperature of 30 °C with stirring at 250 rpm, and the pH was controlled. The reaction was allowed to proceed for 24 h. During the reaction, the pH decreased rapidly, and it was difficult to control the pH throughout the process. When an alkali was added, the reaction solution changed from light red to black. After 24 h, a sample was taken for detection, and the conversion rate was 87.5%, and the yield of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate was 75%.

[0121] Example 15: Comparison between engineered ketoreductase SEQ ID No: 102 and wild-type ketoreductase SEQ ID NO: 2 when catalyzing the reaction for producing ethyl (R)-(+)-4-chloro-3-hydroxybutyrate According to the method described in Example 2, cells of a) SEQ ID NO: 2 and b) SEQ ID No: 102 were prepared. a) and b) were weighed and redissolved in 0.1 M PB at pH 7.0 to obtain c) a bacterial solution of 100 g / L of SEQ ID NO: 2 and d) a bacterial solution of 100 g / L of SEQ ID No: 102, respectively. A portion of the bacterial solutions c) and d) was taken out and heat-treated at 68 °C for 4 h to obtain e) and f), respectively.

[0122] Four reaction flasks with a volume of 30 mL were prepared. 1.218 g of ethyl 4-chloroacetoacetate and 1.25 mL of isopropanol were added to the reaction flasks. 0.5 mL of different bacterial solutions of c), d), e), and f) were added to the four reaction flasks. Then, 0.25 mL of an NAD+ cofactor stock solution (2 g / L) and 2.25 mL of toluene were added to the reaction flasks so that the final concentration of the substrate in the reaction flask was 240 g / L, the final concentration of isopropanol was 25% v / v, the final concentration of NAD+ was 0.5 g / L, and the final concentration of toluene was 45% v / v. A magnetic stirrer was added to the reactants, the temperature in the stirrer was controlled at 30 °C, and the stirring speed was set at 400 rpm. After reacting for 24 h, a 50 μL reaction solution sample was taken from the reaction flask. 950 μL of ethyl acetate was added to quench the sample, and it was detected according to the analysis method of Example 6, and the conversion rate was calculated. The results are shown in Table 2. JPEG2025518973000039.jpg41170

[0123] Example 16: Reaction process for catalyzing the formation of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineered ketoreductase The following is a representative reaction flow and post-treatment process with a volume of 100 mL. Into a 250 mL three-necked reaction flask, 1.0 g of wet cells (containing the enzyme of SEQ ID No: 118), 10 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD, 25 mL of isopropanol, and 45 mL of toluene were added. Finally, 24.6 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction was carried out at a temperature of 40 °C with stirring at 250 rpm. After reacting for 24 h, during which the pH was controlled at 6.5 - 7.5 in real time, when reacting for 16 h, a water pump was used to perform a vacuum suction once at 0.07 MPa to remove the acetone generated by the reaction. After 24 h, a sample was taken for detection, and the conversion rate was 99.6%.

[0124] After the reaction was completed, it was allowed to stand, and 12 mL of waste water was separated as the lower layer, and the toluene layer was separated as the upper layer. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone, and toluene. After switching to an oil pump and raising the temperature, 22.8 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91.5%, a purity of 99%, and an ee ≥ 99.8%.

[0125] Example 17: Reaction process for catalyzing the formation of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a representative reaction flow and post-treatment process with a volume of 100 mL. Into a 250 mL three-necked reaction flask, 1.0 g of wet cells (containing the enzyme of SEQ ID No: 120), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 10 mg of coenzyme NAD, 25 mL of isopropanol, and 35 mL of toluene were added. Finally, 30.6 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction was carried out at a temperature of 30 °C with stirring at 250 rpm. After reacting for 24 h, during which the pH was controlled at 6.5 - 7.5 in real time, when reacting for 16 h, 5 mL of isopropanol was replenished. After 24 h, a sample was taken for detection, and the conversion rate was 99.0%.

[0126] After the reaction was completed, the mixture was allowed to stand, and 20 mL of waste water was separated into the lower layer and the toluene layer was separated into the upper layer by liquid separation. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone and toluene. The oil pump was replaced and the temperature was raised, and 28.2 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91%, a purity of 98.5% and an ee ≥ 99.8%.

[0127] Example 18: Reaction process for catalyzing the production of ethyl (R)-(+)-4-chloro-3-hydroxybutyrate by engineering ketoreductase The following is a typical reaction flow and post-treatment process with a volume of 100 mL. 2.0 g of wet cells (containing the enzyme of SEQ ID No: 120), 15 mL of 0.1 M phosphate buffer with pH = 7.6, 20 mg of coenzyme NAD, 30 mL of isopropanol and 20 mL of toluene were added to a 250 mL three-necked reaction flask, and finally, 42.6 g of ethyl 4-chloroacetoacetate was added, and stirring was started. The reaction temperature was 30 °C and stirring was carried out at 250 rpm. The reaction was carried out for 24 h, during which the pH was controlled at 6.5 - 7.5 in real time. When the reaction was carried out for 16 h, the water pump was used to vacuum aspirate once at 0.07 MPa to remove the acetone produced by the reaction. After 24 h, a sample was taken for detection, and the conversion rate was 96.0%.

[0128] After the reaction was completed, the mixture was allowed to stand, and 17 mL of waste water separated by liquid separation was separated into the lower layer and the toluene layer was separated into the upper layer by liquid separation. The toluene layer was vacuum distilled with a water pump to remove isopropanol, acetone and toluene. The oil pump was replaced and the temperature was raised, and 39.3 g of the product ethyl R-4-chloro-3-hydroxybutyrate was collected with a yield of 91.5%, a purity of 95% and an ee ≥ 99.8%.

[0129] Example 19: Catalysis of the production of (S)-(+)-1,3-butanediol by engineering ketoreductase Prepare one reaction flask with a volume of 30 mL, add 0.25 g of the bacterial cells of SEQ ID No:4 as the bacterial cells to the reaction flask. Next, add 0.25 g of 4-hydroxy-2-butanone, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor of the NAD+ cofactor (10 g / L) to the reaction flask, and supplement the final reaction volume in the reaction flask to 5.0 mL with 0.01 M PB at pH 7. Thus, the final concentration of the substrate (4-hydroxy-2-butanone) in the reaction flask was 50 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. Place the reaction flask on an IKA magnetic stirrer at 30 °C, set the stirring speed to 400 rpm, and start the reaction. After reacting for 24 h, collect samples from the reaction flask, process and detect them, calculate the conversion rate and ee value, and show the obtained results in Table 3. JPEG2025518973000040.jpg20170

[0130] Analysis method Conversion rate analysis method The chromatography column was Agilent DB-WAX 15m*0.25mm*0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250 °C, the split ratio was 28:1, the detector temperature was 300 °C, the injection volume was 1 μL, and the column temperature was 130 °C. The temperature was raised to 150 °C at 10 °C / min, and then raised to 160 °C at 20 °C / min. Here, the retention time of 4-hydroxy-2-butanone was 1.5 minutes, and the retention time of (S)-(+)-1,3-butanediol was 2.3 minutes.

[0131] Chiral analysis method Sample pretreatment method: 200 μL of the sample was put into 50 μL of MSTFA and 30 μL of anhydrous pyridine, uniformly mixed in a 1.5 mL centrifuge tube, and subjected to a shaking reaction for 30 minutes. The chromatography column was Agilent CP-Chirasil Dex CB (CP7502) 25 m * 0.25 mm * 0.25 μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250 °C, the split ratio was 28:1, the detector temperature was 300 °C, the injection volume was 2 μL, the column temperature was 80 °C, the stop time was 35 minutes, the retention time of (R)-(-)-1,3-butanediol was 20.7 minutes, and the retention time of (S)-(+)-1,3-butanediol was 23.0 minutes.

[0132] Example 20: Catalysis of the production of methyl (S)-(-)-3-hydroxybutyrate by engineering ketoreductase Prepare one 30 mL reaction flask, add 0.1 g of the cells of SEQ ID No: 18 as the cells to the reaction flask, and then add 0.25 g of methyl acetoacetate, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor (10 g / L) of the NAD+ cofactor to the reaction flask. Supplement the final reaction volume in the reaction flask to 5.0 mL with 0.1 M PB at pH 7. Thus, the final concentration of the substrate (methyl acetoacetate) in the reaction flask was 50 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. Place the reaction flask on an IKA magnetic stirrer at 30 °C, set the stirring speed to 400 rpm, and start the reaction. After reacting for 24 h, samples were taken from the reaction flask, processed and detected, the conversion rate and ee value were calculated, and the obtained results are shown in Table 4. JPEG2025518973000041.jpg23170

[0133] Analysis method Conversion rate analysis method The chromatography column was Agilent HP-5 30m*0.32mm*0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250°C, the split ratio was 50:1, the detector temperature was 300°C, the injection volume was 1μL, the column temperature was 50°C and it was held for 5 minutes. Next, the temperature was raised to 100°C at 5°C / min and then to 225°C at 50°C / min and held for 5.5 minutes. Here, the retention time of methyl acetoacetate was 9.5 minutes and the retention time of methyl (R)-(-)-3-hydroxybutyrate was 8.9 minutes.

[0134] Chiral analysis method The chromatography column was Agilent CP-Chirasil Dex CB(CP7502) 25m*0.25mm*0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250°C, the split ratio was 50:1, the detector temperature was 250°C, the injection volume was 1μL, the column temperature was 100°C, the stop time was 15 minutes, the retention time of methyl (R)-(-)-3-hydroxybutyrate was 6.6 minutes, and the retention time of methyl (S)-(+)-3-hydroxycibutyrate was 7.0 minutes.

[0135] Example 21: Catalysis of the production of ethyl (S)-(-)-3-hydroxybutyrate by engineering ketoreductase One 30 mL reaction flask was prepared, 0.1 g of the cells of SEQ ID No:70 were added to the reaction flask as the cells, then 0.25 g of ethyl acetoacetate, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor of the NAD+ cofactor (10 g / L) were added to the reaction flask, and the final reaction volume in the reaction flask was supplemented to 5.0 mL with 0.1M PB at pH 7. Thus, the final concentration of the substrate (ethyl acetoacetate) in the reaction flask was 50 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. The reaction flask was placed on an IKA magnetic stirrer at 30°C, the stirring speed was set to 400 rpm, and the reaction was started. After reacting for 24 h, samples were taken from the reaction flask, processed, detected, the conversion rate and the ee value were calculated, and the obtained results are shown in Table 5. JPEG2025518973000042.jpg22170

[0136] Analysis method Conversion rate analysis method The chromatographic column was Agilent DB-WAX 15m * 0.25mm * 0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250 ° C, the split ratio was 50:1, the detector temperature was 300 ° C, the injection volume was 1 μL, and the column temperature was 120 ° C and held for 4.5 minutes. Here, the retention time of ethyl acetoacetate was 2.4 minutes and the retention time of ethyl 3-hydroxybutyrate was 2.8 minutes.

[0137] Chiral analysis method The chromatographic column was Agilent CP-Chirasil Dex CB (CP7502) 25m * 0.25mm * 0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250 ° C, the split ratio was 50:1, the detector temperature was 250 ° C, the injection volume was 1 μL, the column temperature was 110 ° C, the stop time was 11 minutes, the retention time of (R)-(-)-ethyl 3-hydroxybutyrate was 9.4 minutes, and the retention time of (S)-(+)-ethyl 3-hydroxybutyrate was 9.7 minutes.

[0138] Example 22: Catalyst for the production of (2S,5S)-2,5-hexanediol by engineering ketoreductase Prepare one reaction flask with a volume of 30 mL, add 0.1 g of the bacterial cells of SEQ ID No: 128 as the bacterial cells to the reaction flask. Next, add 0.25 g of 2,5 - hexanedione, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor of NAD+ cofactor (10 g / L) to the reaction flask, and supplement the final reaction volume in the reaction flask to 5.0 mL with 0.1 M PB at pH 7. Thus, the final concentration of the substrate (2,5 - hexanedione) in the reaction flask was 50 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. Place the reaction flask on an IKA magnetic stirrer at 30 °C, set the stirring speed to 400 rpm, and start the reaction. After reacting for 24 h, collect samples from the reaction flask, process and detect them, calculate the conversion rate and ee value, and show the obtained results in Table 6. JPEG2025518973000043.jpg22170

[0139] Analysis method Conversion rate analysis method The chromatography column was Agilent DB - WAX 30m * 0.25mm * 0.25μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250 °C, the split ratio was 20:1, the detector temperature was 300 °C, the injection volume was 3 μL, the column temperature was 100 °C and was held for 3 minutes. Next, the temperature was raised to 150 °C at a rate of 5 °C / min and held for 3 minutes. Here, the retention time of 2,5 - hexanedione was 7.1 minutes and the retention time of 2,5 - hexanediol was 14.1 minutes.

[0140] Chiral analysis method 200 μL of the reaction sample dissolved in chloroform was added to 200 μL of trifluoroacetic anhydride, sealed with a breathable membrane, and then the solvent was removed by evaporation over 20 minutes in a constant temperature water bath at 60 °C. After that, 200 μL of chloroform was added for redissolution and injection. The chromatography column was Agilent CP-Chirasil Dex CB (CP7502) 25 m * 0.25 mm * 0.25 μm, the carrier gas was N2, the detector was FID, the inlet temperature was 270 °C, the split ratio was 20:1, the detector temperature was 300 °C, the injection volume was 1 μL, the column temperature was 70 °C, the stop time was 30 minutes, the retention time of (2S,5S)-2,5-hexanediol was 15.0 minutes, and the retention time of (2R,5R)-2,5-hexanediol was 16.7 minutes.

[0141] Example 23: Catalysis of the formation of (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol by engineering ketoreductase One 30 mL reaction flask was prepared, 0.1 g of the cells of SEQ ID No:56 were added to the reaction flask as the cells, and then 0.25 g of 3,5-bis(trifluoromethyl)acetophenone, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor of the NAD+ cofactor (10 g / L) were added to the reaction flask. The final reaction volume in the reaction flask was made up to 5.0 mL with 0.1 M PB at pH 7. Thus, the final concentration of the substrate (3,5-bis(trifluoromethyl)acetophenone) in the reaction flask was 50 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. The reaction flask was placed on an IKA magnetic stirrer at 30 °C, the stirring speed was set at 400 rpm, and the reaction was started. After reacting for 24 h, samples were taken from the reaction flask, processed and detected, the conversion rate and ee value were calculated, and the results obtained are shown in Table 7. JPEG2025518973000044.jpg21170

[0142] Analysis method Conversion rate analysis method The chromatographic column is ZORBAX SB-C18 (150 m * 4.6 mm * 5 μm), the mobile phase is 60% acetonitrile + 40% water, the column temperature is 30 °C, the injection volume is 10 μL, the flow rate is 1.0 ml / min, and the wavelength is 218 nm. Here, the retention time of (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol is 5.0 minutes, and that of 3,5-bistrifluoromethylacetophenone is 7.0 minutes.

[0143] Chiral analysis method The chromatographic column is Agilent CYCLODEX-B 30 m * 0.320 mm, 0.25 μm, the carrier gas is N2, the detector is FID, the inlet temperature is 200 °C, the split ratio is 30:1, the detector temperature is 250 °C, the injection volume is 1 μL, the column temperature is 115 °C, the stop time is 20 minutes, the retention time of (S)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol is 8.3 minutes, and the retention time of (R)-1-[3,5-bis(trifluoromethyl)phenyl]ethanol is 8.8 minutes.

[0144] Example 24: Catalytic production of (S)-1-tert-butoxycarbonyl-3-hydroxypiperidine by engineering ketoreductase Prepare one 30 mL reaction flask, add 0.1 g of the bacterial cells of SEQ ID No: 102 as the bacterial cells to the reaction flask. Next, add 0.25 g of N-tert-butoxycarbonyl-3-piperidone, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor of the NAD+ cofactor (10 g / L) to the reaction flask, and supplement the final reaction volume in the reaction flask to 5.0 mL with 0.1 M PB at pH 7. Thus, the final concentration of the substrate (N-tert-butoxycarbonyl-3-piperidone) in the reaction flask was 50 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. Place the reaction flask on an IKA magnetic stirrer at 30 °C, set the stirring speed to 400 rpm, and start the reaction. After reacting for 24 h, samples were taken from the reaction flask, processed, detected, the conversion rate and ee value were calculated, and the obtained results are shown in Table 8. JPEG2025518973000045.jpg23170

[0145] Analysis method Conversion rate analysis method The chromatographic column is Agilent DB-WAX 30m * 0.250mm, 0.25μm, the carrier gas is N2, the detector is FID, the inlet temperature is 250°C, the split ratio is 20:1, the detector temperature is 250°C, the injection volume is 3μL, the column flow rate is 1.0mL / min, and the initial column temperature is 30°C. The temperature was raised to 150°C at 20°C / min and held for 5 minutes, then raised to 230°C at 5°C / min and held for 5 minutes. The stop time was 32 minutes, the retention time of N-tert-butoxycarbonyl-3-piperidone was 21.3 minutes, and that of (S)-1-tert-butoxycarbonyl-3-hydroxypiperidine was 24.3 minutes.

[0146] Example 25: Catalysis of the formation of (S)-(-)-3-chloro-1-phenyl-1-propanol by engineering ketoreductase Prepare one 30 mL reaction flask, add 0.1 g of the bacterial cells of SEQ ID No: 34 as the bacterial cells to the reaction flask, then add 0.01 g of 3-chloropropiophenone, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor (10 g / L) of the NAD+ cofactor to the reaction flask, and supplement the final reaction volume in the reaction flask to 5.0 mL with pure water. Thus, the final concentration of the substrate (3-chloropropiophenone) in the reaction flask is 2 g / L, the final concentration of isopropanol is 10% v / v, and the final concentration of NAD+ is 0.5 g / L. Place the reaction flask on an IKA magnetic stirrer at 30°C, set the stirring speed to 400 rpm, and start the reaction. After reacting for 24 h, samples were taken from the reaction flask, processed, detected, the conversion rate and ee value were calculated, and the obtained results are shown in Table 9. JPEG2025518973000046.jpg28170

[0147] Analysis method Conversion rate analysis method The chromatographic column is ZORBAX SB-C18 (150m * 4.6mm * 5μm), the mobile phase is 50% acetonitrile + 50% HClO4, the column temperature is 45°C, the injection volume is 10 μL, the flow rate is 2.0 ml / min, and the wavelength is 215 nm. Here, the retention time of (S)-(-)-3-chloro-1-phenyl-1-propanol is 1.9 minutes, and the retention time of 3-chloropropiophenone is 2.7 minutes.

[0148] Chiral analysis method Sample pretreatment method: 200 μL of the sample was placed in 50 μL of MSTFA and 30 μL of anhydrous pyridine, mixed uniformly in a 1.5 mL centrifuge tube, and subjected to a shaking reaction for 30 minutes. The chromatographic column is Agilent CP-Chirasil Dex CB (CP7502) 25m * 0.25mm * 0.25μm, the carrier gas is N2, the detector is FID, the inlet temperature is 250°C, the split ratio is 10:1, the detector temperature is 260°C, the column flow rate is 1.1 mL / min, the injection volume is 1 μL, the initial column temperature is 100°C and was maintained for 20 minutes. The temperature was raised to 120°C at 5°C / min and held for 20 minutes, the stop time was 44 minutes, the retention time of (S)-(-)-3-chloro-1-phenyl-1-propanol was 35.6 minutes, and the retention time of (R)-(+)-3-chloro-1-phenyl-1-propanol was 38.8 minutes.

[0149] Example 26: Catalyst for the production of 2,7-dimethyl-3,6-octanediol by engineering ketoreductase Prepare one reaction flask with a volume of 30 mL, add 0.125 g of the bacterial cells of SEQ ID No: 60 as the bacterial cells to the reaction flask. Next, add 0.05 g of 2,7-dimethyl-3,6-octanedione, 0.5 mL of isopropanol, and 0.25 mL of the mother liquor of the NAD+ cofactor (10 g / L) to the reaction flask, and supplement the final reaction volume in the reaction flask to 5.0 mL with 0.1 M PB at pH 7. Thus, the final concentration of the substrate (2,7-dimethyl-3,6-octanedione) in the reaction flask was 10 g / L, the final concentration of isopropanol was 10% v / v, and the final concentration of NAD+ was 0.5 g / L. Place the reaction flask on an IKA magnetic stirrer at 30 °C, set the stirring speed to 400 rpm, and start the reaction. After reacting for 18 h, samples were taken from the reaction flask, processed, detected, the conversion rate was calculated, and the obtained results are shown in Table 10. In addition, ketoreductase can also catalyze the formation of (2S,3S)-2,3-butanediol from 2,3-butanedione, the formation of (2S,4S)-2,4-pentanediol from acetylacetone, the formation of (3S,6S)-3,6-octanediol from 3,6-octanedione, and the formation of (3S,5S)-3,5-heptanediol from 3,5-heptanedione. JPEG2025518973000047.jpg25170

[0150] Analysis method Conversion rate analysis method The chromatography column was Agilent CP-Chirasil-Dex CB (CP7502) 25 m * 0.25 mm * 0.25 μm, the carrier gas was N2, the detector was FID, the inlet temperature was 250 °C, the split ratio was 20:1, the detector temperature was 300 °C, the injection volume was 1 μL, the column temperature was 40 °C, and it was held for 0.5 min. Next, the temperature was raised to 77 °C at 10 °C / min and held for 0.5 min, and then further raised to 190 °C at 40 °C / min and held for 5.0 min. Here, the retention time of 2,7-dimethyl-3,6-octanedione was 8.6 min.

[0151] After reading the above content of the present invention, those skilled in the art should understand that various changes or modifications can be made to the present invention. These equivalent forms are similarly included within the scope limited by the claims attached to this application.

Claims

1. It is used to catalyze the production of ethyl (R)-4-chloro-3-hydroxybutyrate from ethyl 4-chloroacetoacetate, with an ee value of at least 99%. The polypeptide has at least 90% sequence identity with the reference sequence SEQ ID NO: 2 and contains an amino acid sequence having differences in at least two residues at residue positions X204 and X211 compared to SEQ ID NO:

2. The amino acid residue at residue position X204 is selected from I, and the amino acid residue at residue position X211 is selected from F. An engineered ketoreductase polypeptide.

2. It can reduce ethyl 4-chloroacetoacetate with catalytic activity and / or stability superior to that of SEQ ID NO: 2 to produce ethyl (R)-4-chloro-3-hydroxybutyrate. The amino acid sequence is a sequence selected from the group consisting of the amino acid sequences represented by SEQ ID Nos: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128. An engineered ketoreductase polypeptide.

3. The suitable reaction conditions include an ethyl 4-chloroacetoacetate loading of about 5 g / L to 426 g / L, a pH of about 5.0 to 8.0, and a temperature of about 10 to 80 °C. The ketoreductase polypeptide according to claims 1 to 2.

4. A polypeptide immobilized on a solid material by chemical bonding or physical adsorption, selected from the ketoreductase polypeptides according to any one of claims 1 to 3.

5. A polynucleotide encoding the polypeptide according to any one of claims 1 to 4.

6. The polynucleotide sequence is the sequence corresponding to SEQ ID No: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, the polynucleotide according to claim 2.

7. An expression vector comprising the polynucleotide according to claims 5 to 6.

8. The expression vector according to claim 6, comprising a plasmid, cosmid, phage, or viral vector.

9. A host cell comprising the expression vector according to any one of claims 7 to 8, wherein the host cell is preferably Escherichia coli (E. coli).

10. A method for producing a ketoreductase polypeptide, comprising culturing the host cell according to claim 9 and obtaining a ketoreductase polypeptide from the culture.

11. Produced by the method according to claim 10, obtaining a host cell or culture solution containing a ketoreductase polypeptide from the culture, or processing a product using the same, The product refers to an extract obtained from transformed cells, and is a separated product obtained by separating or purifying ketoreductase in the extract, or is an immobilized product obtained by immobilizing transformed cells, their extract, or a separated product of the extract, a ketoreductase catalyst.

12. A method for producing a compound represented by structural formula (I), comprising contacting a carbonyl substrate of structural formula (II) with the engineering polypeptide according to any one of claims 1 to 4. (R １ is optionally substituted aryl or heteroaryl, or optionally substituted C １ ~C ８ hydrocarbyl, which may be cyclo hydrocarbyl or heterocyclyl, R ２ is optionally substituted C １ ~C ６ hydrocarbyl, halogen (e.g., -F, -Cl, -Br, and -I), alkenyl, alkynyl, aryl, heteroaryl, -NO ２ , -NO, -SO ２ R', or -SOR', -SR', -NR'R', -OR', -CO ２ R', or -COR', -C(O)NR', -SO ２ NH ２ , or -SONH ２ , -CN, -CF ３ wherein R' is each independently -H, (C １ -C ４ ) hydrocarbyl, halogen, C １ ~C ８ hydrocarbyl, C ２ ~C １２ alkenyl, C ２ ~C １２ alkynyl, cyclo hydrocarbyl, aryl, or heterocyclyl. )

13. The product of structural formula (I) is The carbonyl substrate of structural formula (II) is The method according to claim 12.

14. Ethyl 4-chloroacetoacetate as the carbonyl substrate A method for producing ethyl (R)-(+)-4-chloro-3-hydroxybutyrate of the following compound, comprising the step of contacting ethyl 4-chloroacetoacetate with the engineering ketoreductase polypeptide according to any one of claims 1 to 4.

15. The reaction according to any one of claims 12 to 14, wherein water, toluene, isopropanol, and acetone are included as solvents, the temperature is 10°C to 80°C, and the pH is 5.0 to 8.0.

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