Mutant ketoreductase with increased ketoreductase activity, as well as methods and uses related thereto

Abstract

The present invention relates to a mutant ketoreductase having at least one mutation at position 241, a nucleic acid encoding the mutant ketoreductase, a vector comprising the nucleic acid, a method for enzymatic reduction of ketones and formation of chiral alcohols using the mutant ketoreductase, the use of the mutant ketoreductase for ketone reduction and formation of chiral alcohols, and the use of a method for the preparation of pharmaceutically active serine / threonine protein kinase inhibitors.

Description

[Technical Field]

[0001] The present invention relates to a mutant ketoreductase having at least one mutation at position 241, a nucleic acid encoding the mutant ketoreductase, a vector comprising the nucleic acid, a method for enzymatic reduction of ketones and formation of chiral alcohols by the mutant ketoreductase, and the use of the mutant ketoreductase for the preparation of pharmaceutically active serine / threonine protein kinase inhibitors. [Background technology]

[0002] Ketoreductases are a subclass of enzymes belonging to the group of oxidoreductases, that is, enzymes that catalyze redox reactions that enable the transfer of electrons from so-called electron donor molecules to electron acceptor molecules.

[0003] Subclasses of ketoreductase possess the specific ability to catalyze the asymmetric reduction of desired ketones to their corresponding secondary alcohols. During this reduction reaction, electrons are transferred to the ketone group (C=O) by the addition of a hydride to the carbonyl atom of the keto group. Furthermore, a proton is transferred to the oxygen of the carbonyl group. This reaction generally requires a hydride donor as a cofactor, such as NADH or NADPH, which can be regenerated in situ and protonated amino acid residues within the active site of ketoreductase.

[0004] To date, ketoreductase has also been commonly used for the enzymatic reduction of prochiral keto compounds, and therefore for the preparation of intermediates for various pharmaceutical compounds, for example, the formula exemplified in PCT international application WO2008 / 006040A1. It is commonly used, for example, in the preparation of serine / threonine protein kinase inhibitors (TIFF2026509089000001.tif60170). Protein kinase inhibitors are useful, for example, in the treatment of hyperproliferative diseases such as cancer and inflammation in mammals.

[0005] Particularly promising serine / threonine protein kinase inhibitors have formula X and are the clinical AKT inhibitor candidate ipatasertib (CAS registration number 1001264-89-6). TIFF2026509089000002.tif80170

Summary of the Invention

[0006] The object of the present invention is to design improved mutant ketoreductases having increased ketoreductase activity compared to wild-type ketoreductases, particularly the ketoreductase of Sporidiobolus salmonicolor, particularly the ketoreductase of SEQ ID NO: 1. These variants can be used for the production of chiral alcohols, including their production in a scaled-up process, such as the chiral alcohol of formula I.

[0007] Surprisingly, a mutant ketoreductase comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (ketoreductase from Sporidiobolus salmonicolor; designated Q9UUN9 in UniProt), has at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1, and in which the amino acid at the position corresponding to position 241 of SEQ ID NO: 1 is substituted, has been found to exhibit an increase in ketoreductase activity relative to wild-type ketoreductases, particularly the ketoreductase of Sporidiobolus salmonicolor, particularly the ketoreductase of SEQ ID NO: 1.

[0008] As shown in the examples, the substitution introduced at position 241 of the wild-type ketoreductase from Sporidiobolus salmonicolor defined in SEQ ID NO: 1 (designated as Q9UUN9 in UniProt) confers increased activity to the variant compared to the wild-type enzyme. As shown in Table 1, substitution of leucine at position 241 with Met, Asn, Arg, Trp, Ile or Lys increased ketoreductase activity. Furthermore, the increased activity can be demonstrated for further substitutions, especially in combination with substitutions at other positions, namely positions 97, 174, 238, 242, and / or 245 (see Table 2), especially substitutions at positions 97, 241, and 245 (see Table 4). Substitutions at positions 242 and 245 have been shown to be particularly relevant for further increasing enzyme activity (see Tables 4-7), which can be further increased by substituting Phe with Trp at position 97 (see Tables 8 and 9). The inventors have identified further substitutions that can further assist in increasing ketoreductase activity, namely substitutions at positions 134, 174, 224, 228, 234, 238, 246, 316 and / or 342 (see Tables 10-20). It can be confirmed that the increased enzyme activity is present at different times, temperatures and cosubstrate concentrations (see Tables 22-24). The high diastereoselectivity of the wild-type enzyme is maintained in variants with different recycling systems at different scales (see Tables 25-27). For ketoreductases from different fungal species, reductase performance and diastereoselectivity have been confirmed (see Table 21).

[0009] In addition, such ketoreductases have been found to catalyze the enzymatic reduction of ketones and are very active for the formation of chiral alcohols.

[0010] Furthermore, compared to wild-type ketoreductase, the mutant ketoreductase of the present invention has been found to show increased conversion in the enzymatic reduction of ketones.

[0011] In particular, using mutant ketoreductase, Formula I TIFF2026509089000003.tif67170 (in the formula, R 1 is C 1~4 It is alkyl, R 2 is hydrogen or C 1~4 Enzymatic reduction of alkyl (especially methyl or ethyl, especially methyl) ketones yields highly enantiomerized or diastereomerized formula II. TIFF2026509089000004.tif67170 (in the formula, R 1 and R 2 The chiral alcohol is formed as described above. Therefore, the ketoreductase according to the present invention is suitable for the preparation of chiral alcohol key intermediates, particularly the formula exemplified, for example, in PCT international application WO2008 / 006040A1. This is extremely useful for preparing key intermediates in the preparation of serine / threonine protein kinase inhibitors, as described in TIFF2026509089000005.tif60170.

[0012] Therefore, in the first aspect, the present invention relates to a mutant ketoreductase having increased ketoreductase activity compared to wild-type ketoreductase, It contains an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID 1 (ketoreductase from Sporidiobolus salmonicolor; UniProt ID: Q9UUN9); This invention relates to a mutant ketoreductase having at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 241 of SEQ ID NO: 1 is substituted.

[0013] As used herein, the term "ketoreductase" means any protein having the ability to asymmetrically catalyze the reduction of a ketone to its corresponding chiral non-racemic secondary alcohol, particularly the pure enantiomers of each secondary alcohol.

[0014] As used herein, the term "ketone" means a substrate having a prochiral keto functionality that may contain additional chiral centers.

[0015] C 1~4 As used herein, the term "alkyl" for a substituent means a monovalent straight-chain or branched-chain saturated hydrocarbon group of 1 to 4 carbon atoms such as methyl, ethyl, n-propyl, isopropyl, n-butyl, i-butyl, sec-butyl, or t-butyl, preferably t-butyl. 1

[0016] C 1~4 As used herein, the term "alkyl" for a substituent means a monovalent straight-chain saturated hydrocarbon group of 1 to 4 carbon atoms such as methyl, ethyl, n-propyl, or n-butyl, preferably methyl. 2 [[ID=二十二]]

[0017] ​As used herein, the term “wild-type ketoreductase” means any ketoreductase that occurs naturally. As used herein, the term “mutant ketoreductase” means any ketoreductase derived from a corresponding wild-type ketoreductase, in which its amino acid sequence is modified compared to such wild-type ketoreductase. For example, this may include the introduction, deletion, substitution, or post-translational mutation of one or more amino acids at one or more positions. Preferably, the mutant ketoreductase differs from the wild-type ketoreductase by amino acid substitution. Methods for causing mutations in the amino acid sequence, such as amino acid substitutions, are well known to those skilled in the art. For example, such mutations may have already been introduced at the nucleic acid level, resulting in the expression of a desired mutant amino acid sequence. Appropriate methods are well known to those skilled in the art and are also partially described below, for example, in the context of nucleic acids according to a second aspect of the present invention.

[0018] A suitable mutant ketoreductase according to the first embodiment can be derived from the wild-type ketoreductase of any organism. Demonstrated activity has been found using wild-type ketoreductases from Scheffersomyces stipitis, Clavispora lusitaniae, Meyerozyma guilliermondii, Tilletiopsis washingtonensis, Rachicladosporium antarcticum, Lodderomyces elongisporus, Acidomyces richmondensis, or Plicaturopsis crispa. Particularly preferred is the ketoreductase of Sporidiobolos salmonicolor, designated Q9UUN9 in UniProt.

[0019] This application also discloses variants of wild-type ketoreductase from organisms other than Sporidiobolus salmonicolor, and any of the variants or combinations thereof identified for Sporidiobolus salmonicolor and / or defined in this application may be introduced into corresponding sites in other organisms. These organisms may, for example, be Scheffersomyces stipitis, Clavispora lusitaniae, Meyerozyma guilliermondii, Tilletiopsis washingtonensis, Rachicladosporium antarcticum, Lodderomyces elongisporus, Acidomyces richmondensis, or Plicaturopsis crispa. The corresponding sites may be identified, for example, by identifying sequence alignments and homologous regions known to those skilled in the art. Table 21 shows the reductase performance and diastereoselectivity of ketoreductases from different fungal species compared to wild-type ketoreductase from Sporidiobolus salmonicolor (SEQ ID NO: 1; UniProt ID: Q9UUN9).

[0020] According to the present invention, the mutant ketoreductase is active as a ketoreductase. This means that, as detailed above and below, the mutant ketoreductase can convert prochiral ketones to the corresponding secondary alcohols under appropriate conditions. Methods for measuring ketoreductase activity are described herein and given in the examples.

[0021] The mutant ketoreductase according to the first embodiment exhibits increased ketoreductase activity compared to wild-type ketoreductase.

[0022] This activity can be determined in enzyme assays that measure either substrate consumption or product production over time. Numerous different methods exist for measuring substrate and product concentrations, and many enzymes can be assayed in several different ways known to those skilled in the art.

[0023] Methods for determining the enzymatic activity of mutant ketoreductase or wild-type ketoreductase according to the present invention are well known to those skilled in the art. Exemplary methods are also described in the examples. To determine whether the mutant ketoreductase according to the first embodiment exhibits increased ketoreductase activity compared to wild-type ketoreductase, the ketoreductase activity of both ketoreductases is measured using the same method.

[0024] For example, methods for determining the enzymatic activity of ketoreductase may generally be based on fluorescence assays or colorimetric assays. Furthermore, methods for determining the enzymatic activity of ketoreductase generally involve detecting the concentration of the product formed, the concentration of the consumed substrate, or the cofactors required for the reaction to be formed or consumed, such as NAD. + , NADH, NADP + Alternatively, it may include the detection of NADPH concentration.

[0025] A mutant ketoreductase according to the first embodiment, exhibiting increased ketoreductase activity compared to wild-type ketoreductase, for example, shows an increase of more than 1x in ketoreductase activity. Those skilled in the art are familiar with statistical procedures for evaluating whether one value of enzyme activity is increased compared to another, such as Student's t-test or chi-squared test. It is obvious to those skilled in the art that background signals must be subtracted when analyzing the data.

[0026] Furthermore, the mutant ketoreductase according to the first aspect of the present invention contains an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (ketoreductase of Sporidiobolos salmonicolor, referred to as Q9UUN9 in UniProt). Thus, the amino acid sequence of SEQ ID NO: 1 is derived from the ketoreductase of Sporidiobolos salmonicolor, referred to as Q9UUN9 in UniProtKB.

[0027] As used herein, the term "array identity" refers to the percentage of characters that exactly match between two different arrays.

[0028] For example, the term "at least 80% identical to the amino acid sequence of SEQ ID NO: 1" as used herein means that the amino acid sequence of the mutant ketoreductase of the present invention has an amino acid sequence characterized in that, within a stretch of 100 amino acids, at least 80 amino acid residues are identical to the sequence of the corresponding sequence of SEQ ID NO: 1.

[0029] Mutant ketoreductases may also contain amino acid sequences that are at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of Sequence ID No. 1 (ketoreductase from Sporidiobolus salmonicolor, designated Q9UUN9 in UniProt).

[0030] The sequence identity according to the present invention can be determined, for example, by a sequence alignment method that compares sequences. Sequence alignment methods are well known in the art and include various programs and alignment algorithms. Furthermore, the NCBI Basic Local Alignment Search Tool (BLAST) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, and is used in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. For example, the percentage of identity of the variant according to the present invention with respect to the amino acid sequence of SEQ ID NO: 1 is typically characterized using NCBI Blast blastp in a standard configuration. Alternatively, sequence identity can be determined using the software GENEious in a standard configuration. Alignment results can be derived, for example, from the Software CLC Main Workbench (version 21) using a global alignment protocol with free-end gap as the alignment type and Blosum62 as the cost matrix.

[0031] The mutant ketoreductase according to the present invention has at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1. Furthermore, the mutant ketoreductase according to the present invention may have at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1.

[0032] In particular, the mutant ketoreductase according to the present invention has at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 241 of SEQ ID NO: 1 is substituted.

[0033] A method for preparing mutant ketoreductase according to the first aspect of the present invention is well known to those skilled in the art. For example, mutant ketoreductase according to the first aspect can be prepared by any method known to those skilled in the art that is suitable for preparing recombinant enzymes, such as recombinant expression of modified nucleic acids of mutant ketoreductase in a cell culture, followed by protein isolation and purification.

[0034] Preferably, in the mutant ketoreductase of the first embodiment, the amino acid at the position corresponding to position 241 of SEQ ID NO: 1 is substituted with Met (Met241), Asn (Asn241), Arg (Arg241), Trp (Trp241), Ile (241Ile), Lys (Lys241), His (His241), Gln (Gln241), Gly (Gly241), Asp (Asp241), Ser (Ser241), Thr (Thr241), Tyr (Tyr241), Cys (Cys241), Ala (Ala241), Val (Val241), or Phe (Phe241).

[0035] More preferably, in the mutant ketoreductase of the first embodiment, the amino acid at the position corresponding to position 241 of SEQ ID NO: 1 is substituted with Met(Met241), Gln(Gln241), Cys(Cys241), Tyr(Tyr241), Ser(Ser241), Thr(Thr241), Val(Val241), or Ala(Ala241), more preferably Met(Met241).

[0036] If the mutant ketoreductase according to the first embodiment has two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the mutant ketoreductase according to the first embodiment of the present invention preferably includes substitutions at positions corresponding to positions 242 and / or 245 of SEQ ID NO: 1, in addition to the substitution at position 241.

[0037] Therefore, a preferred embodiment of the mutant ketoreductase of the first aspect has at least the following substitutions: -241st and 242nd, or -241st and 245th, or - Ranked 241st, 242nd, and 245th.

[0038] A more preferred embodiment of the mutant ketoleductase of the first embodiment has at least the following substitutions: -The amino acid at position 241 of sequence number 1 is Met(Met241), Asn(Asn241), Arg(Arg241), Trp(Trp241), Ile(241Ile), Lys(Lys241), His(His241), Gln(Gln241), Gly(Gly241), Asp(Asp241), Ser(Ser241), Thr(Thr241), Tyr(Tyr241), Cys(C Substituted with ys241), Ala(Ala241), Val(Val241), or Phe(Phe241), preferably Met(Met241), Gln(Gln241), Cys(Cys241), Tyr(Tyr241), Ser(Ser241), Thr(Thr241), Val(Val241), or Ala(Ala241), more preferably Met(Met241), and / or - The amino acid at position 242 of SEQ ID NO: 1 is substituted with Trp(Trp242), Phe(Phe242), Ile(Ile242), Tyr(Tyr242); Cys(Cys242); Val(Val242), Leu(Leu242), Pro(Pro242), Ala(Ala242), Gln(Gln242) or Ser(Ser242), preferably Trp(Trp242), Phe(Phe242), Ile(Ile242), or Tyr(Tyr242), more preferably Trp(Trp242) or Ile(Ile242), most preferably Trp(Trp242), and / or - The amino acid at position 245 of sequence number 1 is substituted with Ser(Ser245), Thr(Thr245), Asn(Asn245), Met(Met245), Asp(Asp245), Trp(Trp245), Phe(Phe245), Glu(Glu245), Cys(Cys245), or His(His245), preferably Ser(Ser245), Thr(Thr245), or Asn(Asn245), more preferably Ser(Ser245) or Thr(Thr245), most preferably Ser(Ser245).

[0039] More preferably, the mutant ketoreductase of the present invention is -The amino acid at position 241 of sequence number 1 is substituted with Met(Met241), -The amino acid at position 242 of sequence number 1 is substituted with Trp(Trp242), - The amino acid at position 245 of sequence number 1 is substituted with Ser(Ser245). It is characterized by the following:

[0040] Alternatively, or further, if the mutant ketoreductase according to the first embodiment has two or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, for example, three, four, five, six, seven, eight, nine, ten or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the mutant ketoreductase according to the first embodiment of the present invention preferably includes substitutions at positions 97, 134, 135, 174, 224, 228, 234, 238, 242, 245, 246, 316 and / or 342 of SEQ ID NO: 1, in addition to the substitution at position 241, particularly substitutions at positions 97, 134, 224, 238, 242 and / or 245.

[0041] Therefore, in a preferred embodiment of the mutant ketoreductase of the first aspect, - The amino acid at position 97 of sequence number 1 is either substituted with Trp (Trp97) or unsubstituted. - The amino acid at position 134 of SEQ ID NO: 1 is either substituted with Val(Val134), Cys(Cys134), Ala(Ala134), Gln(Gln134), or Met(Met134), or it is unsubstituted. - The amino acid at position 135 of sequence number 1 is either substituted with Cys(Cys135) or Thr(Thr135), or it is unsubstituted. - The amino acid at position 174 of SEQ ID NO: 1 is substituted with or unsubstituted with Thr (Thr174), Val (Val174), Met (Met174), Tyr (Tyr174), Ala (Ala174), Ile (Ile174), Lys (Ly174), Arg (Arg174), Asn (Asn174), Ser (Ser174), or Gln (Gln174), preferably Ala (Ala174), Val (Val174), or Ile (Ile174), and / or - The amino acid at position 224 of sequence number 1 is either substituted with Ala(Ala224) or unsubstituted. - The amino acid at position 228 of sequence number 1 is either substituted with Lys(Lys228), Gln(Gln228), or Arg(Arg228), or it is unsubstituted. - The amino acid at position 234 of sequence number 1 is either substituted with Asp(Asp234) or unsubstituted. - The amino acid at position 238 of Sequence ID No. 1 is either substituted with Lys(Lys238), Arg(Arg238), Leu(Leu238), Gly(Gly238), His(His238), Asn(Asn238), Trp(Trp238), Asp(Asp238), Thr(Thr238), Ser(Ser238), Gln(Gln238), or Tyr(Tyr238), preferably Lys(Lys238), Arg(Arg238), Leu(Leu238), or Gly(Gly238), more preferably Lys(Lys238) or Arg(Arg238), most preferably Lys(Lys238), or unsubstituted. -The amino acid at position 241 of sequence number 1 is Met(Met241), Asn(Asn241), Arg(Arg241), Trp(Trp241), Ile(Ile241), Lys(Lys241), His(His241), Gln(Gln241), Gly(241Gly), Asp(Asp241), Ser(Ser241), Thr(Thr241), Tyr(Tyr241), C It is substituted with ys(Cys241), Ala(Ala241), Val(Val241), or Phe(Phe241), preferably Met(Met241), Gln(Gln241), Cys(Cys241), Tyr(Tyr241), Ser(Ser241), Thr(Thr241), Val(Val241), or Ala(Ala241), more preferably Met(Met241). - The amino acid at position 242 of sequence number 1 is substituted with Trp (Trp242), Phe (Phe242), Ile (Ile242), Tyr (Tyr242); Cys (Cys242); Val (242Val), Leu (Leu242), Pro (Pro242), Ala (Ala242), Gln (Gln242), or Ser (Ser242), preferably Trp (Trp242), Phe (Phe242), Ile (Ile242), or Tyr (Tyr242), more preferably Trp (Trp242) or Ile (Ile242), most preferably Trp (Trp242). - The amino acid at position 245 of Sequence ID No. 1 is substituted with Ser(Ser245), Thr(Thr245), Asn(Asn245), Met(Met245), Asp(Asp245), Trp(Trp245), Phe(Phe245), Cys(Cys245), or His(His245), preferably Ser(Ser245), Thr(Thr245), or Asn(Asn245), more preferably Ser(Ser245) or Thr(Thr245), most preferably Ser(Ser245). - The amino acid at position 246 of Sequence ID No. 1 is either substituted with Gly(Gly246), Lys(Lys246), preferably Gly(Gly246), or unsubstituted. - The amino acid at position 316 of SEQ ID NO: 1 is either substituted with Met(Met316) or unsubstituted, and / or - The amino acid at position 342 of sequence number 1 is either substituted with Met(Met342) or unsubstituted.

[0042] In a more preferred embodiment of the mutant ketoleductase of the first embodiment, - The amino acid at position 97 of sequence number 1 is either substituted with Trp (Trp97) or unsubstituted. - The amino acid at position 134 of sequence number 1 is either substituted with Val(Val134) or unsubstituted. - The amino acid at position 224 of sequence number 1 is either substituted with Ala(Ala224) or unsubstituted. - The amino acid at position 238 of sequence number 1 is either substituted with Lys(Lys238) or unsubstituted. -The amino acid at position 241 of sequence number 1 is substituted with Met(Met241), - The amino acid at position 242 of sequence number 1 is substituted with Trp(Trp242), and / or, - The amino acid at position 245 of sequence number 1 is substituted with Ser(Ser245).

[0043] In a more preferred embodiment of the mutant ketoleductase of the first aspect, -The amino acid at position 241 of sequence number 1 is substituted with Met(Met241), -The amino acid at position 242 of sequence number 1 is substituted with Trp(Trp242), -The amino acid at position 245 of sequence number 1 is substituted with Ser(Ser245). Optionally, - The amino acid at position 97 of sequence number 1 is either substituted with Trp (Trp97) or unsubstituted. - The amino acid at position 134 of sequence number 1 is either substituted with Val(Val134) or unsubstituted. - The amino acid at position 224 of sequence number 1 is either substituted with Ala(Ala224) or unsubstituted. - The amino acid at position 238 of sequence number 1 is either substituted with Lys(Lys238) or unsubstituted. - The amino acid at position 246 of sequence number 1 is either substituted with Gly(Gly246) or unsubstituted. - The amino acid at position 316 of SEQ ID NO: 1 is either substituted with Met(Met316) or unsubstituted, and / or - The amino acid at position 342 of sequence number 1 is either substituted with met (Met342) or unsubstituted.

[0044] Particularly desirable mutant ketoreductases are defined by at least the following mutations: -Having mutations in Trp97, Met241, Trp242 and Ser245, or -Having mutations in Trp97, Met241, Trp242, Ser245, Met316 and Met342, or -Having mutations in Trp97, Lys238, Met241, Trp242, Ser245, Met316 and Met342, or -Having mutations in Trp97, Lys238, Met241, Trp242, Ser245, Gly246, Met316 and Met342, or -Having mutations in Trp97, 224Ala, Lys238, Met241, Trp242, Ser245, Gly246, Met316 and Met342, or -Having mutations in Trp97, Val134, 224Ala, Lys238, Met241, Trp242, Ser245, Gly246, Met316 and Met342, or -Having mutations in Lys238, Met241, Trp242, Ser245, or - It has mutations in Trp97, Lys238, Met241, Trp242, and Ser245.

[0045] Further mutations defined above or below may exist at will.

[0046] A mutant ketoreductase according to a first aspect of the present invention may further include an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and especially 100% identical, to the amino acid sequences of SEQ ID NOs. Sequence identity of the amino acid sequences of two proteins and methods for determining sequence identity are well known to those skilled in the art and are described above.

[0047] In a more preferred embodiment of the mutant ketoreductase of the first embodiment, the mutant ketoreductase consists of or includes an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and particularly 100% identical, to any of the amino acid sequences of SEQ ID NOs: 2 to 9.

[0048] In preferred embodiments of the mutant ketoreductase of the first embodiment, the ketoreductase activity is increased by at least 1.01, 2.0, 5.0, 10, or even 50 times compared to wild-type ketoreductase. Methods for determining the ketoreductase activity of a protein and methods for comparing the ketoreductase activity of two or more proteins are well known to those skilled in the art and are described above.

[0049] The mutant ketoreductase according to the first aspect of the present invention may further exhibit increased conversions compared to wild-type ketoreductase at higher substrate loadings such as 2-10% [w / w] substrate, and at mutant or wild-type ketoreductase loadings of 1-2% [w / w] (s / e5-10) using 2-propanol as a cofactor recycling system, and at 0.1-0.2% [w / w] (s / e50-100) using a glucose / glucose dehydrogenase recycling system.

[0050] As used herein, the term "conversion" means the conversion of any substrate to a product induced by a ketoreductase, such as the mutant ketoreductase or wild-type ketoreductase of the present invention. Such conversions may further depend on various reaction parameters, such as temperature, pressure, or the amount of substrate or ketoreductase enzyme used. Preferred conditions and methods are described in the examples.

[0051] Preferably, the conversion of ketoreductase is determined using 2-propanol as a cofactor recycling system with a 10% [w / w] substrate load and a 2% [w / w] (s / e5) mutant or wild-type ketoreductase load. In this context, the abbreviation "s / e" refers to the "substrate to enzyme" ratio. S / e5 further means that 5 g of substrate is used per 1 g of enzyme, i.e., the substrate to enzyme is used in a 1 / 5 ratio.

[0052] In a further preferred embodiment of the mutant ketoreductase of the first embodiment, the mutant ketoreductase exhibits increased conversion compared to wild-type ketoreductase with 2-10% [w / w] substrate and with 1-2% [w / w] (s / e5-10) mutant or wild-type ketoreductase loading using 2-propanol as a cofactor recycling system, particularly by at least 1.05, 1.10, 1.20, 1.30, 1.40, 1.50, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10-fold conversion compared to wild-type ketoreductase. Alternative cofactor recycling systems such as glucose and glucose dehydrogenase may be applied.

[0053] In preferred embodiments of the mutant ketoreductase of the first embodiment, the mutant ketoreductase can asymmetrically reduce ketones to the corresponding chiral non-racemic secondary alcohols, particularly the pure enantiomers of secondary alcohols, each being a diastereomer.

[0054] In a more preferred embodiment, the ketone has formula I, TIFF2026509089000006.tif67170 Chiral alcohols are given by formula II TIFF2026509089000007.tif73170 (in the formula, R 1 is C 1~4 It is alkyl, R 2 is hydrogen or C 1~4 It has (being alkyl).

[0055] helical bond " "TIFF2026509089000008.tif11170" is " TIFF2026509089000009.tif11170" or " This represents "TIFF2026509089000010.tif11170", and therefore indicates the chirality of the molecule.

[0056] In a more preferred embodiment, R 1 tert.butyl, R 2 It is methyl.

[0057] Mutant ketoreductases can, in principle, asymmetrically catalyze the formation of both the S-enantiomer and R-enantiomer of chiral alcohols, particularly the chiral alcohol of formula II.

[0058] In a preferred embodiment, the mutant ketoreductase is represented by formula IIa TIFF2026509089000011.tif67170 (in the formula, R 1 and R 2 It catalyzes the formation of the R-diastereomer of the chiral alcohol (as described above), more preferably formula IIb It catalyzes the formation of chiral alcohols in TIFF2026509089000012.tif76170.

[0059] A diastereomer excess of at least 95%, 96%, 97%, 98%, or 99% of the R,R-diastereomer of the chiral alcohol can be achieved.

[0060] Furthermore, the mutant ketoreductase according to the first aspect of the present invention can also be combined with further peptides or proteins to form a fusion protein. Therefore, the present invention further relates to a fusion protein comprising the mutant ketoreductase of the present invention.

[0061] Fusion proteins may further contain tags. Tags are attached to proteins for various purposes, such as facilitating purification, assisting proper protein folding, preventing protein precipitation, altering chromatographic properties, modifying proteins, or marking or labeling proteins. In principle, using high-purity enzymes reduces the required enzyme load. Several (affinity) tags or (affinity) markers are currently known. Commonly used tags include Arg tags, His tags, Strep tags, Flag tags, T7 tags, S tags, HAT tags, GST tags, and MBP tags.

[0062] In a second aspect, the present invention relates to a nucleic acid encoding a mutant ketoreductase according to a first aspect of the present invention. Accordingly, the present invention may also relate to a nucleic acid encoding a fusion protein comprising a mutant ketoreductase according to a first aspect of the present invention.

[0063] As used herein, the term “nucleic acid” generally refers to any nucleotide molecule that encodes the mutant ketoreductase of the present invention and may be of variable length. Examples of nucleic acids of the present invention include, but are not limited to, plasmids, vectors, or any type of DNA and / or RNA fragment that can be isolated by standard molecular biology procedures, including, for example, ion-exchange chromatography. Nucleic acids of the present invention may be used for transfection or transduction of certain cells or organisms.

[0064] The nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, such as cDNA and genomic DNA, and may be obtained, for example, by cloning or produced by chemical synthesis techniques or a combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded. Single-stranded DNA may be a coding strand, also known as a sense strand, or a non-coding strand, also called an antisense strand. As used herein, nucleic acid molecules also refer, among other things, to DNA which is a mixture of single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and RNA which is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA which may be single-stranded or more typically double-stranded or triple-stranded, or mixtures of single-stranded and double-stranded regions. Furthermore, as used herein, nucleic acid molecules refer to a triple-stranded region containing RNA or DNA or both RNA and DNA.

[0065] Furthermore, nucleic acids may contain one or more modified bases. Such nucleic acids may also contain modifications, for example, in the ribose-phosphate backbone, to increase the stability and half-life of such molecules in a physiological environment. Thus, DNA or RNA having a modified backbone for stability or other reasons is a “nucleic acid molecule” as intended herein. Furthermore, to give just two examples, DNA or RNA containing unusual bases such as inosine, or modified bases such as tritylated bases, are nucleic acid molecules in the context of the present invention. Naturally, a wide variety of modifications have been made to DNA and RNA to serve many useful purposes known to those skilled in the art. As used herein, the term nucleic acid molecule encompasses such chemically, enzymatically, or metabolically modified forms of nucleic acid molecules, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, such as simple and complex cells, in particular.

[0066] Furthermore, the nucleic acid molecule encoding the mutant ketoreductase of the present invention can be functionally ligated to any desired sequence, such as a regulatory sequence, a leader sequence, a heterologous marker sequence, or a heterologous coding sequence, using standard techniques such as standard cloning techniques, to produce a fusion protein.

[0067] The nucleic acids of the present invention can be formed first in vitro or in cultured cells by generally manipulating nucleic acids with endonucleases and / or exonucleases and / or polymerases and / or ligases and / or recombinases, or by other methods known to those skilled in the art for producing nucleic acids.

[0068] The nucleic acid of the present invention may be contained in an expression vector, and the nucleic acid is operably linked to a promoter sequence that can promote nucleic acid expression in a host cell.

[0069] In preferred embodiments of nucleic acids, the nucleic acid encodes a mutant ketoreductase of the first embodiment, the mutant ketoreductase having or comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and especially 100% identical, to any of the amino acid sequences of SEQ ID NOs. 2 to 9.

[0070] In a third aspect, the present invention relates to a vector comprising a nucleic acid according to a second aspect of the present invention. Accordingly, the present invention may also relate to a vector comprising a nucleic acid encoding a fusion protein comprising a mutant ketoreductase according to a first aspect of the present invention.

[0071] As used herein, the term “vector” generally refers to any type of nucleic acid molecule that can be used to express a protein of interest in a cell (see also the above details relating to the nucleic acids of the present invention). In particular, the vectors of the present invention may be any plasmid or vector known to those skilled in the art that is suitable for expressing proteins in certain host cells, such as mammalian cells, bacterial cells, and yeast cells, but are not limited thereto. The vectors of the present invention may also be nucleic acids that encode the mutant ketoreductase of the present invention and are used for subsequent cloning into the respective vector to ensure expression. Plasmids and vectors for protein expression are well known in the art and can be commercially purchased from a variety of suppliers, including, for example, Promega (Madison, WI, USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, CA, USA), or MoBiTec (Germany). Methods of protein expression are well known to those skilled in the art and are described, for example, in Sambrook et al., 2000, Molecular Cloning: A Laboratory Manual, Third Edition.

[0072] A vector may further include a nucleic acid sequence that enables replication within a host cell, e.g., an origin of replication, one or more therapeutic genes and / or selective marker genes, and other genetic elements known in the art, e.g., regulatory elements that direct the transcription, translation, and / or secretion of the encoded protein. A vector may be used to transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins that are not native to the cell. A vector may optionally include anything that helps achieve entry of nucleic acids into the cell, e.g., viral particles, liposomes, protein coatings, etc. A large number of suitable expression vectors for protein expression are known in the art by standard molecular biology techniques. Such vectors are selected from conventional vector types such as insect, e.g., baculovirus expression, or yeast, fungal, bacterial, or viral expression systems. Many other suitable vectors known in the art can also be used for this purpose. Methods for obtaining such vectors are well known (see, e.g., Sambrook et al., cited above).

[0073] As detailed above, the nucleic acid encoding the mutant ketoreductase of the present invention is operably ligated to a sequence suitable for driving protein expression in a host cell in order to ensure protein expression. However, the vector described in the claims may represent an intermediate product which is then cloned into a suitable vector to ensure protein expression, and this is included in the present invention. The vector of the present invention may further include, but is not limited to, all types of nucleic acid sequences, such as polyadenylation signals, splice donor and splice acceptor signals, intervening sequences, transcriptional enhancer sequences, translational enhancer sequences, and drug resistance genes. Optionally, drug resistance genes may be operably ligated to an internal ribosome entry site (IRES), which may be either cell cycle specific or cell cycle independent.

[0074] As used herein, the term “operatably linked” generally means that gene elements are arranged to function in a coordinated manner for an intended purpose, for example, that transcription is initiated by a promoter and proceeds through the DNA sequence encoding the mutant ketoreductase of the present invention. That is, RNA polymerase transcribes the sequence encoding the mutant ketoreductase into mRNA, which is then spliced ​​into a protein.

[0075] As used in the context of the present invention, the term “promoter sequence” generally refers to any type of regulatory DNA sequence operably ligated to a downstream coding sequence, the promoter being able to bind to RNA polymerase and initiate transcription of an encoded open reading frame within the cell, thereby driving the expression of the downstream coding sequence. The promoter sequence of the present invention may be any type of promoter sequence known to those skilled in the art, including, but not limited to, constitutive promoters, inducible promoters, cell cycle-specific promoters, and cell type-specific promoters.

[0076] Furthermore, the present invention also includes a host cell comprising a mutant ketoreductase or its fusion protein, a nucleic acid according to a second aspect of the present invention, or a vector according to a third aspect of the present invention.

[0077] The “host cell” of the present invention may be any type of organism suitable for application in recombinant DNA technology, but is not limited to any bacterial and yeast strains of any kind suitable for expressing one or more recombinant proteins. Examples of host cells include, for example, Bacillus subtilis strains or E. coli strains. Various E. coli bacterial host cells are known to those skilled in the art and include, but are not limited to, strains such as DH5-alpha, HB101, MV1190, JM109, JM101, or XL-1 blue, which can be commercially purchased from various suppliers, including Stratagene (CA, USA), Promega (WI, USA), or Qiagen (Hilden, Germany). Particularly suitable host cells, namely E. coli BL21(DE3) cells, are also described in the examples. Examples of Bacillus subtilis strains that can be used as host cells include 1012 wild-type:leuA8 metB5 trpC2 hsdRM1 and 168 Marburg:trpC2(Trp-), which are commercially available, for example, from MoBiTec (Germany).

[0078] The cultivation of host cells according to the present invention is a standard procedure known to those skilled in the art. That is, the nucleic acid encoding the mutant ketoreductase of the present invention can be introduced into suitable host cells, and the respective proteins can be produced by recombinant means. These host cells may be any type of suitable cell that can be cultured, preferably bacterial cells such as E. coli. In the first step, this approach may involve cloning the respective gene into a suitable vector, such as a vector according to a second aspect of the present invention. Vectors are widely used for gene cloning and can be readily introduced, i.e., transfected, into bacterial cells that have been temporarily made permeable to DNA. After the protein has been expressed in the respective host cells, the cells can be recovered and serve as starting material for the preparation of cell extracts containing the protein of interest. Cell extracts containing the protein of interest are obtained by cell lysis. Methods for preparing cell extracts by chemical or mechanical cell lysis are well known to those skilled in the art and are not limited to, but include, for example, hypotonic salt treatment, homogenization, or sonication.

[0079] In a fourth aspect, the present invention relates to a method for the enzymatic reduction of ketones and the formation of chiral alcohols in the presence of the mutant ketoreductase of the present invention.

[0080] In a more preferred embodiment, Formula I The ketone in TIFF2026509089000013.tif67170 is reduced, and Equation II A chiral alcohol of TIFF2026509089000014.tif75170, or more preferably formula IIa TIFF2026509089000015.tif69170 (in the formula, R 1 is C 1~4 It is alkyl, R 2 is hydrogen or C 1~4 A chiral alcohol (which is alkyl) is formed.

[0081] More preferably, formula IIb The chiral alcohol of TIFF2026509089000016.tif75170 is of formula Ib It is formed by asymmetrically reducing the ketone in TIFF2026509089000017.tif77170.

[0082] Preferably, the resulting chiral alcohol of formula IIb is a (R,R)-diastereomer.

[0083] Enzymatic reduction by mutant ketoreductase typically occurs in the presence of the cofactor NADH or NADPH, which is regenerated in situ. More preferably, NADP + This is used to regenerate reduced NADPH within the body.

[0084] Oxidative cofactors are, in principle, continuously regenerated with a secondary alcohol as the final reducing agent, a so-called co-substrate or in-situ cofactor recycling system, i.e., glucose dehydrogenase and glucose as the final reducing agent, as is generally known to those skilled in the art of the present invention.

[0085] Typical co-substrates can be selected from 2-propanol, 2-butanol, pentane-1,4-diol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol, hexane-1,5-diol, 2-heptanol, or 2-octanol, preferably 2-propanol. In a more preferred embodiment, the acetone formed when 2-propanol is used as a co-substrate can be continuously removed from the reaction mixture.

[0086] The cofactor loading, i.e., the ratio of substrate (ketone) to cofactor (s / c), can vary between 10 and 250, preferably between 50 and 200, and most preferably between 100.

[0087] In certain embodiments of the present invention, the enzymatic reduction is carried out in an aqueous buffer medium in the presence of a co-substrate, preferably in the presence of 2-propanol. The concentration of the co-substrate is typically in the range of 5% [v / v] to 20% [v / v], and preferably 8% [v / v].

[0088] In certain embodiments of the present invention, enzymatic reduction is carried out in an aqueous buffer medium in the presence of glucose and glucose dehydrogenase. The glucose concentration is typically in the range of 0.2 M to 2 M, at least 1.1 equivalents relative to the target ketone. To neutralize the formed gluconic acid, the addition of a base is required to constantly adjust the pH to the target pH.

[0089] A suitable buffer can be selected from acidic to neutral buffers such as 2-morpholine-4-ethanesulfonic acid, ammonium acetate, acetate, phosphate, and 1,4-piperazinediethanesulfonic acid, which allow the reaction pH to be maintained in the range of pH 6 to pH 10, particularly 6.8 to 7.2, and more specifically, about 7.0 to 7.2.

[0090] The substrate loading, i.e., the ketone loading, may be selected between 1% and 20% [w / w], preferably 10% [w / w], and the substrate-to-enzyme ratio (s / e) depends on the final reducing agent and the applicable cofactor recycling system. When 2-propanol is the final reducing agent, the substrate-to-enzyme ratio (s / e) can be selected between 4 and 50, preferably 4 and 10. When glucose is the final reducing agent, the substrate-to-enzyme ratio (s / e) can be selected between 10 and 200, preferably 50 and 100.

[0091] The reaction temperature is typically maintained in the range of 10°C to 50°C, preferably 20°C to 35°C, and more preferably 23°C to 30°C.

[0092] Once the reaction is complete, the resulting chiral alcohol can be post-treated conventionally by extraction, or preferably by filtration.

[0093] The synthesis of ipatasertib from chiral alcohols formed by enzymatic synthesis according to the present invention can follow the synthesis scheme on page 42 of International Publication No. 2008006040, and corresponding examples applying average techniques in the art.

[0094] In a fifth aspect, the present invention relates to the formula illustrated, for example, in PCT international application WO2008 / 006040A1. The present invention relates to the use of the method (enzymatic reduction of chiral alcohol formation in the presence of ketones and mutant ketoreductase) for the preparation of serine / threonine protein kinase inhibitors, as described in TIFF2026509089000018.tif60170.

[0095] In particular, A, R 1 , R 2 , R 5 and R 10 This may be as defined in claim 1 of PCT international application WO2008 / 006040A1: R 1 This may be H, methyl, ethyl, vinyl, CF3, CHF2, or CH2F. R 2 This may be H or methyl, R 5 This may be H, methyl, ethyl, or CF3. R 10 This may be H or methyl, A is It could also be TIFF2026509089000019.tif47170, In the formula, G is one to four R 9 A 5-6 member heteroaryl group optionally substituted with a phenyl group or optionally substituted with a halogen group; R 6 and R 7 These are independently H, OCH3, (C3~C6 cycloalkyl)-(CH2), (C3~C6 cycloalkyl)-(CH2CH2), V-(CH2) 0~1 (In the formula, V is a 5-6 member heteroaryl), W-(CH2) 1~2(wherein W is phenyl optionally substituted with F, Cl, Br, I, O-methyl, CF3, or methyl), C3-C6 cycloalkyl, hydroxy-(C3-C6 cycloalkyl), fluoro-(C3-C6 cycloalkyl), CH(CH3)CH(OH)phenyl, F, OH, C1-C3 alkyl, cyclopropylmethyl, or C(=O)(C1-C3 alkyl) A 4-6 membered heterocycle optionally substituted with (Kil), or a C1-C6 alkyl group optionally substituted with one or more groups independently selected from OH, oxo, O(C1-C6 alkyl), CN, F, NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, cyclopropyl, phenyl, imidazolyl, piperidinyl, pyrrolidinyl, morpholinyl, tetrahydrofuranyl, oxetanyl, or tetrahydropyranyl, or R 6 and R 7 R is a 4- to 7-membered heterocycle that, along with the nitrogen to which they are bonded, is optionally substituted with one or more groups independently selected from OH, halogen, oxo, CF3, CH2CF3, CH2CH2OH, O(C1-C3 alkyl), C(=O)CH3, NH2, NHMe, N(Me)2, S(O)2CH3, cyclopropylmethyl, and C1-C3 alkyl; a and R b Is H or R a H is H, and R b and R 6 Together with the atoms to which they are bonded, they form a 5-6 member heterocycle having one or two ring nitrogen atoms; R c and R d is either H or Me, or R c and R d Together with the atoms to which they are bonded, they form a cyclopropyl ring; R 8 is either H, Me, F, or OH, or R 8 and R 6 Together with the atoms to which they are bonded, they form a 5-6 member heterocycle having one or two ring nitrogen atoms; each R 9The elements are independently halogen, C1-C6 alkyl, C3-C6 cycloalkyl, O-(C1-C6 alkyl), CF3, OCF3, S(C1-C6 alkyl), CN, OCH2-phenyl, CH2O-phenyl, NH2, NH-(C1-C6 alkyl), N-(C1-C6 alkyl)2, piperidine, pyrrolidine, CH2F, CHF2, OCH2F, OCHF2, OH, SO2(C1-C6 alkyl), C(O)NH2, C(O)NH(C1-C6 alkyl), and C(O)N(C1-C6 alkyl)2, where m, n, and p are independently 0 or 1.

[0096] Enzyme reduction is particularly promising for ipatasertib (CAS registry number 1001264-89-6), a candidate clinical AKT inhibitor, which has formula X. TIFF2026509089000020.tif80170

[0097] With regard to the use of the present invention, terms, examples, and specific embodiments used in the context of other aspects of the present disclosure will be referenced and will also be applicable to these aspects. In particular, the mutant ketoreductase or its fusion protein according to the present invention can be used as detailed with respect to the methods of the present invention.

[0098] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0099] The present invention is not limited to the specific methodologies, protocols, and reagents described herein, for this reason, as these may vary. Any method and materials similar to or equivalent to those described herein may be used in carrying out the present invention, but preferred methods and materials are described herein. Furthermore, the terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present invention.

[0100] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context explicitly indicates otherwise. Similarly, the words “comprise,” “contain,” and “encompass” are interpreted inclusively, not exclusively. Likewise, the word “or” is intended to include “and” unless the context explicitly indicates otherwise. The term “plural” refers to two or more.

[0101] The following figures and examples are intended to illustrate various embodiments of the present invention. Therefore, the specific modifications described should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and it should be understood that such equivalent embodiments are included herein. [Examples]

[0102] Example 1: Enzyme Production Deep-well plate cultivation: E. coli cells expressing wild-type and mutant ketoreductase were cultured in 96-deep-well plate format for screening purposes. Preliminary cultures were initiated by inoculating fresh single transformants or glycerol stocks into 500 μL of Luria-Bertani(LB) medium containing 100-200 mg / L of ampicillin, followed by incubation at 28°C and shaking at 300 rpm for 18 hours (Duetz system, Kuehner shaker, shaking diameter 5 cm). The main culture was initiated by inoculating 8–25 μL of pre-culture into 500 μL of ZYM-5052 autoinducible medium, supplemented with 100–200 mg / L of ampicillin and free of trace elements (10 g / L peptone, 5 g / L yeast extract, 5 g / L glycerol, 0.55 g / L glucose monohydrate, 2.1 g / L lactose monohydrate, 10.6 g / L sodium phosphate dibasic salt, 3.4 g / L potassium phosphate monobasic salt, 2.15 g / L ammonium chloride, 0.59 g / L sodium chloride, 0.663 g / L ammonium sulfate, 2 mM magnesium sulfate). The cultures were incubated in the same shaker at 20°C and 300 rpm for 20 hours. Optical cell density at 600 nm was measured before cell harvesting.

[0103] Cells were disrupted by adding 200 μL of lysis buffer (0.1 M potassium phosphate buffer pH 7, 2 mM MgCl2, 1 mg / mL lysozyme derived from chicken egg white, 0.75 mg / mL polymyxin B sulfate, and 0.2 mg / mL DNase I). The cell suspension was incubated at 30°C and shaken at 300 rpm for 1 hour (Duetz system, Kuehner shaker), followed by centrifugation at 4°C and 3,220 g for 30 minutes. The supernatant of deep-well plate cultured cells was immediately used for UV-based activity assays or 0.2 mL scale biocatalytic reactions with a 10% [w / w] substrate load.

[0104] Culture in shaking flasks: E. coli cells expressing wild-type and mutant ketoreductase were cultured in shaking flasks for biocatalytic reactions on a scale of 1 mL or more. One E. coli transformant was inoculated with 20 mL of LB containing 100 mg / L ampicillin, followed by incubation overnight at 37°C and 180 rpm. Using the pre-culture (5 mL), it was inoculated into a 2 L Erlenmeyer flask containing 500 mL of Terrific Broth (TB) medium containing 100 mg / L ampicillin, followed by incubation at 37°C and 180 rpm until an OD of 0.6–0.8 at 600 nm was reached. After adding 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG), the culture was incubated at 25 or 30°C for 24 hours. Cells were harvested by centrifugation at 4°C and 9,300 g for 45 minutes. The pellets were weighed and resuspended in potassium phosphate buffer pH 7 containing 0.04 mM MgCl2 in a biomass-buffer ratio of 1:2. The cells were sonicated and centrifuged at 30,000 g for 20 minutes at 4°C. The supernatant was collected for lyophilization.

[0105] Lyophilization of cell lysates was carried out overnight using Alpha 2-4LDplus(Christ) set to -85°C and 0.14 mbar. The lyophilized lysates were either immediately used in biocatalytic reactions or stored at -20°C.

[0106] Example 2: Determination of enzyme activity and selectivity UV screening in small-scale reactions Reductase activity was measured in a 96-well Greiner microtiter plate using a spectrophotometer. The reaction was carried out in a total volume of 200 μL containing: (1) 2 mM MgCl2 and 0.01 mg / mL NADP + (1) 178 μL of 0.1 M potassium phosphate buffer pH 7 containing (as sodium salt), (2) 6 μL of clarified lysate (diluted in 0.1 M potassium phosphate buffer pH 7 containing 2 mM MgCl2 for final lysate concentrations of pure or 3%, 1.5%, 1%, 0.5%, or 0.25% [v / v] [v / v]), and (3) 16 μL of stock solution containing 1.25 mg / mL of ketone of formula Ib in 2-propanol [v / v].

[0107] The assay was performed with orbital shaking at 432 rpm and at temperatures fluctuating between 28°C and 32°C (room temperature + 5°C due to shaking). Ketone depletion of formula Ib was tracked at 340 nm and recorded every 5 minutes for 80 minutes. The slope [ΔA / min] within the linear range was used for the parent (FIOP) calculation multiplier increase. The parent could be the wild type (FIOWT) or a different variant. The following table shows the FIOWT or FIOP values ​​for various single and multiple variants compared to the wild type. The diastereomer excess (de) values ​​of the chiral alcohol of formula IIb (R,R-trans alcohol) of the hits were verified by HPLC-UV analysis of UV screening assay samples (see Section: HPLC Analysis of Substrates and Products). For all variants shown in Tables 1-20, the de of the chiral alcohol of formula IIb was ≥99.5%. [Table 1] [Table 2] [Table 3] [Table 4] Table 5 Table 6 Table 7 Table 8 Table 9a Table 9b Table 9c Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19 [Table 20]

[0108] HPLC analysis of substrates and products Achiral method for determining diastereomer excess (de): The reaction mixture was quenched with HPLC-grade methanol in a convenient ratio according to the substrate concentration. After protein precipitation, the sample was centrifuged at 3,300 g for 10 minutes at 4°C. The supernatant was analyzed by HPLC-UV at 260 nm on an Agilent 1290 HPLC system using one of the following methods: i. A Kinetex XB-C18 column (50 mm × 4.6 mm, 2.6 μm) was used, with water and methanol as solvents A and B, respectively. The column was heated to 50°C, the flow rate was set to 0.8 mL / min, and the injection volume was 2 μL. The following gradients were applied: 0-0.1 min, B=50%; 0.1-4 min, B=50-58%; 4-4.1 min, B=58-95%; 4.1-4.6 min, B=95%; 4.6-4.7 min, B=95-50%; 4.7-5 min, B=50%; 5-5.1 min, B=50-95%; 5.1-5.4 min, B=95%; 5.4-5.5 min, B=95-50%; and 5.5-5.9 min, B=50%. ii. An Agilent InfinityLab Poroshell 120 Eclipse EC-C18 column (50 mm × 3.0 mm × 2.7 μm) was used, with water and methanol as solvents A and B, respectively. The column was heated to 50°C, the flow rate was set to 0.8 mL / min, and the injection volume was 2 μL. The following gradients were applied: 0-0.1 min, B=40%; 0.1-6 min, B=40-52.7%; 6-6.2 min, B=52.7-95%; 6.2-7.2 min, B=95%; 7.2-8 min, B=95-40%; 8-9 min, B=40%. iii. A Kinetex EVO-C18 column (50 mm × 4.6 mm, 5 μm) was used, with water and methanol as solvents A and B, respectively. The column was heated to 40°C, the flow rate was set to 2 mL / min, and the injection volume was 1 μL. The following isocratic method was applied: 0 to 7.5 minutes, B = 40%.

[0109] Method i or ii was used after UV assay screening or 0.2 mL scale biocatalytic reactions. Standard substances corresponding to the ketone of formula Ib, the chiral alcohol of formula IIb, and the (R,S)-cis-alcohol product derived from the ketone of formula Ib were treated as samples before HPLC-UV analysis. For rapid verification of selectivity from UV assay samples, the diastereomer excess (de) value was estimated from the relative peak area of ​​the alcohol product (abbreviated as a% area). For 0.2 mL scale biocatalytic reactions, the conversion was calculated using the calibration curve for the chiral alcohol of formula IIb. Method iii was applied to reactions on a scale of 1 mL or more. The conversion and diastereomer excess values ​​were determined from the relative peak area. [Table 21]

[0110] 0.2 mL scale reaction with 10% [w / w] substrate loading using lysates. The reaction was carried out in an Eppendorf tube using 20 mg of ketone Ib, 0.1 M potassium phosphate buffer pH 7.2, 2 mM MgCl2, and 0.1% NADP. + The solution contained a clarified lysate equivalent to disodium salt (s / c=100), 3.5 mg / mL of total protein (determined by BCA assay), and 8%–20% [v / v] 2-propanol. The reaction was carried out in a thermomixer at 25–30°C with shaking at 1,000 rpm. After a given time, the reaction product was quenched with HPLC-grade methanol to achieve a final dilution factor of 100, and then centrifuged. The supernatant was analyzed by HPLC-UV (260 nm). For all mutants shown in Tables 22–24, the diastereomer excess of the product was ≥99.5% for the chiral alcohol of formula IIb. [Table 22] [Table 23] [Table 24]

[0111] 1 mL scale reaction with 10% [w / w] substrate loading using lyophilized lysate. The reaction was carried out in an Eppendorf tube using 0.1 g of ketone Ib, 0.1 M potassium phosphate buffer pH 7.2, 2 mM MgCl2, and 0.1% NADP. + The reaction was carried out at 23–30°C with shaking at 1,500 rpm. The reaction consisted of a disodium salt (s / c=100), 8% [v / v] 2-propanol, and lyophilized lysates derived from selected variants and controls in different substrate-enzyme (s / e) ratios. After a given time, the reaction products were quenched with HPLC-grade methanol and analyzed by HPLC-UV (260 nm). [Table 25]

[0112] Example 3: Preparative-scale biocatalytic reaction 20 mL scale reaction with 10% [w / w] substrate loading using 2-propanol as the final reducing agent. The reaction was carried out in a Scott flask using magnetic stirring, with different substrate-to-enzyme (s / e) ratios, using 2.0 g of formula Ib ketone, 0.1 M potassium phosphate buffer pH 7.2, 2 mM MgCl2, and 0.1% NADP. + The reaction contained disodium salt (s / c=100), 8% [v / v] 2-propanol, and lyophilized lysates derived from selected variants and controls. The stirring reaction was carried out at 23–30°C. After a given time, the reaction sample (0.05 mL) was quenched with HPLC-grade methanol (0.95 mL) and analyzed by HPLC-UV (260 nm). [Table 26]

[0113] 20 mL scale reaction with 10% [w / w] substrate loading using glucose as the final reducing agent. The reaction was carried out in a pH-Stat with overhead stirring, using 2 g of formula Ib ketone, 0.1 M potassium phosphate buffer pH 7.2, 2 mM MgCl2, and 0.1% NADP. + Disodium salt (s / c=100), 8% [v / v] 2-propanol, and lyophilized lysates derived from selected variants and controls were included in different substrate-enzyme (s / e) ratios. The stirring reaction was carried out at 23–30°C, with the pH kept constant by adding 1 M NaOH. After a given time, the reaction sample (0.05 mL) was quenched with HPLC-grade methanol (0.95 mL) and analyzed by HPLC-UV (260 nm). [Table 27]

[0114] A 100 mL scale reaction using 2-propanol as the final reducing agent. The reactants were: ketone of formula Ib (10 g, 0.03 mol, 1 equivalent), water (39 mL), 1 M potassium phosphate buffer pH 7.2 (10 mL), 0.1 M MgCl₂₆H₂O (2 mL), 2-propanol (8 mL), and NADP. +The solution contained (100 mg, 0.004 equivalents, s / c=100, previously dissolved in 1 mL). After stirring for 5 minutes, the reaction was initiated by adding E00185 lyophilized solution (2 g, s / e=5, previously dissolved in 30 mL of water), followed by incubation at 23°C under N2 flow for 30 hours. After complete reduction (ketone of formula Ib less than 1.0%), 2-propanol was depleted by evaporation (40°C, 200-60 mbar), the suspension was cooled to room temperature, the crude product was filtered, washed with water (twice with 25 mL) and heptane (twice with 25 mL), and dried to a constant weight (40°C, less than 10 mbar). The purity of the HPLC product (expressed as area percent, abbreviated as a%) was determined at 254 nm using an Agilent 1290 HPLC system equipped with a Chiralpak IC-3 column (150 mm × 4.6 mm, 3 μm) heated at 30°C, and heptane and ethanol containing 0.1% diethanolamine as solvents A and B, respectively. The flow rate was 0.8 mL / min, and the injection volume was 5 μL. The following formula was used: 0-5 min, B=40%; 5-15 min, B=100%; 15-17 min, B=100%; 17-17.1 min, B=40%.

[0115] 9.5 g (94.2%) of the crude product, a light beige powder, was isolated by HPLC purity of 99.7a% (0.0a% (S,S)-trans-product, <0.02a% cis-product, 0.2a% ketone of formula Ib) for the chiral alcohol of formula IIb.

[0116] A 100 mL scale reaction using glucose as the final reducing agent. The reactants were: ketone of formula Ib (10 g, 0.03 mol, 1 equivalent), water (34 mL), 1 M potassium phosphate buffer pH 7.2 (10 mL), 1 M D(+) glucose monohydrate (7.13 g, 1.2 equivalents, 36 mL), 0.1 M MgCl₂₆H₂O (2 mL), 2-propanol (8 mL), and NADP. +The solution contained (100 mg, 0.004 equivalents, s / c=100) and Codexis glucose dehydrogenase GDH-105 (100 mg, s / e=100). After stirring for 5 minutes, the reaction was initiated by adding E00185 lyophilized solution (0.2 g, s / e=50). The pH was maintained constant by adding 1 M NaOH (Tritisol) (28.9 mL, 0.03 mol, 0.96 equivalents). The reaction suspension was incubated at 23°C for 27 hours to achieve complete reduction (less than 1.0a% of the ketone of formula Ib). Subsequently, 2-propanol was depleted by evaporation (40°C, 200–60 mbar), the suspension was cooled to room temperature, the crude product was filtered, washed with water (twice with 25 mL) and heptane (twice with 25 mL), and dried to a constant weight (40°C, less than 10 mbar). The purity of the HPLC product (expressed as area %) and abbreviated as a% was determined as described in the previous section.

[0117] 9.5 g (94.0%) of the crude product, a light beige powder, was isolated by HPLC purity of 99.5a% (0.0a% (S,S)-trans-product, <0.05a% cis-product, and 0.4a% ketone of formula Ib) for the chiral alcohol of formula IIb.

[0118] array Sequence ID 1 Length: 343 Type: Protein Organism: Sporidiobolus salmonicolor Other information: Wild type MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSFSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPALALMPPQYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA Sequence ID 2 Length: 343 Type: Protein Biological body: artificial Other information: E00128 Mutation: Trp97_Met241_Trp242_Ser245 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPALAMWPPSYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA Sequence ID 3 Length: 343 Type: Protein Biological body: artificial Other information: E00144 Mutations: Trp97_Met241_Trp242_Ser245_Met316_Met342 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPALAMWPPSYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSMEILKSLGRPGWRSIEESIKDLVGSEMA Sequence ID 4 Length: 343 Type: Protein Biological body: artificial Other information: RE1 Mutation: Lys238_Met241_Trp242_Ser245 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSFSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPKLAMWPPSYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA Sequence ID 5 Length: 343 Type: Protein Biological body: artificial Other information: E00178 Mutation: Trp97_Lys238_Met241_Trp242_Ser245 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPKLAMWPPSYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSLEILKSLGRPGWRSIEESIKDLVGSETA Sequence ID 6 Length: 343 Type: Protein Biological body: artificial Other information: E00158 Mutations: Trp97_Lys238_Met241_Trp242_Ser245_Met316_Met342 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPKLAMWPPSYYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSMEILKSLGRPGWRSIEESIKDLVGSEMA Sequence ID 7 Length: 343 Type: Protein Biological body: artificial Other information: E00174 Mutations: Trp97_Lys238_Met241_Trp242_Ser245_Gly246_Met316_Met342 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTSGWMMSLFNGEVSPKLAMWPPSGYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSMEILKSLGRPGWRSIEESIKDLVGSEMA Sequence ID 8 Length: 343 Type: Protein Biological body: artificial Other information: E00184 Mutations: Trp97_Ala224_Lys238_Met241_Trp242_Ser245_Gly246_Met316_Met342 MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSTVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPSDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTAGWMMSLFNGEVSPKLAMWPPSGYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSMEILKSLGRPGWRSIEESIKDLVGSEMA Sequence ID 9 Length: 343 Type: Protein Biological body: artificial Other information: E00185 Mutations: Trp97_Val134_Ala224_Lys238_Met241_Trp242_Ser245_Gly246_Met316_Met342) MAKIDNAVLPEGSLVLVTGANGFVASHVVEQLLEHGYKVRGTARSASKLANLQKRWDAKYPGRFETAVVEDMLKQGAYDEVIKGAAGVAHIASVVSWSNKYDEVVTPAIGGTLNALRAAAATPSVKRFVLTSSVVSALIPKPNVEGIYLDEKSWNLESIDKAKTLPESDPQ KSLWVYAASKTEAELAAWKFMDENKPHFTLNAVLPNYTIGTIFDPETQSGSTAGWMMSLFNGEVSPKLAMWPPSGYVSAVDIGLLHLGCLVLPQIERRRVYGTAGTFDWNTVLATFRKLYPSKTFPADFPDQGQDLSKFDTAPSMEILKSLGRPGWRSIEESIKDLVGSEMA

Claims

1. A mutant ketoreductase having increased ketoreductase activity compared to wild-type ketoreductase, It contains an amino acid sequence that is at least 80% identical to the amino acid sequence of Sequence ID No. 1 (ketoreductase derived from Sporidiobolus salmonicor; UniProt ID: Q9UUN9), A mutant ketoreductase having at least one amino acid substitution to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid at the position corresponding to position 241 of SEQ ID NO: 1 is substituted.

2. The amino acid at the position corresponding to position 241 of Sequence ID No. 1 is Met (Met241), Asn (Asn241), Arg (Arg241), Trp (Trp241), Ile (241Ile), Lys (Lys241), His (His241), Gln (Gln241), Gly (Gly241), Asp (Asp241), Ser (Ser241), Thr (Thr241), Tyr (Tyr241), Cys (Cys241) The mutant ketoreductase according to claim 1, which is substituted with Ala (Ala241), Val (Val241), or Phe (Phe241), preferably Met (Met241), Gln (Gln241), Cys (Cys241), Tyr (Tyr241), Ser (Ser241), Thr (Thr241), Val (Val241), or Ala (Ala241), more preferably Met (Met241).

3. Sequence ID 1 - 241st and 242nd, or - 241st and 245th, or - The amino acids at positions corresponding to positions 241, 242, and 245 are substituted. Preferably, - The amino acid at the position corresponding to position 241 of Sequence ID No. 1 is Met (Met241), Asn (Asn241), Arg (Arg241), Trp (Trp241), Ile (241Ile), Lys (Lys241), His (His241), Gln (Gln241), Gly (Gly241), Asp (Asp241), Ser (Ser241), Thr (Thr241), Tyr (Tyr241), Cys ( Substituted with Cys241), Ala(Ala241), Val(Val241), or Phe(Phe241), preferably Met(Met241), Gln(Gln241), Cys(Cys241), Tyr(Tyr241), Ser(Ser241), Thr(Thr241), Val(Val241), or Ala(Ala241), more preferably Met(Met241), and / or - The amino acid at the position corresponding to position 242 of Sequence ID No. 1 is substituted with Trp (Trp242), Phe (Phe242), Ile (Ile242), Tyr (Tyr242); Cys (Cys242); Val (Val242), Leu (Leu242), Pro (Pro242), Ala (Ala242), Gln (242Gln) or Ser (Ser242), preferably Trp (Trp242), Phe (Phe242), Ile (Ile242), or Tyr (Tyr242), more preferably Trp (Trp242) or Ile (Ile242), most preferably Trp (Trp242), and / or - The mutant ketoreductase according to claim 1 or 2, wherein the amino acid at the position corresponding to position 245 of Sequence ID No. 1 is substituted with Ser (Ser245), Thr (Thr245), Asn (Asn245), Met (Met245), Asp (Asp245), Trp (Trp245), Phe (Phe245), Glu (Glu245), Cys (Cys245), or His (His245), preferably Ser (Ser245), Thr (Thr245), or Asn (Asn245), more preferably Ser (Ser245) or Thr (Thr245), most preferably Ser (Ser245).

4. - The amino acid at the position corresponding to position 241 of sequence number 1 is substituted with Met(Met241), - The amino acid at the position corresponding to position 242 of sequence number 1 is substituted with Trp (Trp242), - The amino acid at the position corresponding to position 245 of SEQ ID NO: 1 is substituted with Ser (Ser245). A mutant ketoreductase according to any one of claims 1 to 3.

5. - The amino acid at position 97 of sequence number 1 is either substituted with Trp (Trp97) or unsubstituted. - The amino acid at position 134 of sequence number 1 is either substituted with Val (Val134), Cys (Cys134), Ala (Ala134), Gln (Gln134), or Met (Met134), or is unsubstituted. - The amino acid at position 174 of Sequence ID No. 1 is substituted with or unsubstituted with Thr (Thr174), Val (Val174), Met (Met174), Tyr (Tyr174), Ala (Ala174), Ile (Ile174), Lys (Lys174), Arg (Arg174), Asn (Asn174), Ser (Ser174), or Gln (Gln174), preferably Ala (Ala174), Val (Val174), or Ile (Ile174), and / or - The amino acid at position 224 of sequence number 1 is either substituted with Ala (Ala224) or unsubstituted. - The amino acid at position 228 of SEQ ID NO: 1 is either substituted with Lys (Lys228), Gln (Gln228), or Arg (Arg228), or it is unsubstituted. - The amino acid at position 234 of sequence number 1 is either substituted with Asp(Asp234) or unsubstituted. - The amino acid at position 238 of Sequence ID No. 1 is either substituted with Lys (Lys238), Arg (Arg238), Leu (238Leu), Gly (Gly238), His (His238), Asn (Asn238), Trp (Trp238), Asp (Asp238), Thr (Thr238), Ser (Ser238), Gln (Gln238), or Tyr (Tyr238), preferably Lys (Lys238), Arg (Arg238), Leu (Leu238), or Gly (Gly238), more preferably Lys (Lys238) or Arg (Arg238), most preferably Lys (Lys238), or unsubstituted. - The amino acid at position 246 of SEQ ID NO: 1 is substituted with Gly (Gly246), Lys (Lys246), Met (met246), or Ser (246Ser), preferably Gly (Gly246), Lys (Lys246), most preferably Gly (Gly246), or is unsubstituted. - The amino acid at position 316 of sequence number 1 is either substituted with Met(Met316) or unsubstituted, and / or - The amino acid at position 342 of sequence number 1 is either substituted with Met(Met342) or unsubstituted. A mutant ketoreductase according to any one of claims 1 to 4.

6. - The amino acid at the position corresponding to position 97 of Sequence ID No. 1 is either substituted with Trp (Trp97) or unsubstituted. - The amino acid at the position corresponding to position 134 of Sequence ID No. 1 is either substituted with Val(Val134) or unsubstituted. - The amino acid at the position corresponding to position 224 of SEQ ID NO: 1 is either substituted with Ala (Ala224) or unsubstituted. - The amino acid at the position corresponding to position 238 of Sequence ID No. 1 is either substituted with Lys(Lys238) or unsubstituted. - The amino acid at the position corresponding to position 241 of sequence number 1 is substituted with Met(Met241), - The amino acid at the position corresponding to position 242 of sequence number 1 is substituted with Trp(Trp242), and / or, - The amino acid at the position corresponding to position 245 of SEQ ID NO: 1 is substituted with Ser (Ser245). A mutant ketoreductase according to any one of claims 1 to 5.

7. - The amino acid at the position corresponding to position 241 of sequence number 1 is substituted with Met(Met241), - The amino acid at the position corresponding to position 242 of sequence number 1 is substituted with Trp (Trp242), - The amino acid at the position corresponding to position 245 of sequence number 1 is substituted with Ser (Ser245), Optionally, - The amino acid at the position corresponding to position 97 of Sequence ID No. 1 is either substituted with Trp (Trp97) or unsubstituted. - The amino acid at the position corresponding to position 134 of Sequence ID No. 1 is either substituted with Val(Val134) or unsubstituted. - The amino acid at the position corresponding to position 224 of SEQ ID NO: 1 is either substituted with Ala (Ala224) or unsubstituted. - The amino acid at the position corresponding to position 238 of Sequence ID No. 1 is either substituted with Lys(Lys238) or unsubstituted. - The amino acid at the position corresponding to position 246 of Sequence ID No. 1 is either substituted with Gly(Gly246) or unsubstituted. - The amino acid at the position corresponding to position 316 of SEQ ID NO: 1 is either substituted with Met(Met316) or unsubstituted, and / or - The amino acid at the position corresponding to position 342 of SEQ ID NO: 1 is either substituted with met(Met342) or unsubstituted. A mutant ketoreductase according to any one of claims 1 to 6.

8. - Having mutations in Trp97, Met241, Trp242 and Ser245, or - Having mutations in Trp97, Met241, Trp242, Ser245, Met316 and Met342, or - Having mutations in Trp97, Lys238, Met241, Trp242, Ser245, Met316 and Met342, or - Having mutations in Trp97, Lys238, Met241, Trp242, Ser245, Gly246, Met316 and Met342, or - Having mutations Trp97, 224Ala, Lys238, Met241, Trp242, Ser245, Gly246, Met316 and Met342, or - Having mutations Trp97, Val134, 224Ala, Lys238, Met241, Trp242, Ser245, Gly246, Met316 and Met342, or - Having mutations Lys238, Met241, Trp242, Ser245, or -Having mutations in Trp97, Lys238, Met241, Trp242, and Ser245, A mutant ketoreductase according to any one of claims 1 to 7.

9. A mutant ketoreductase according to any one of claims 1 to 8, comprising or containing an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99%, particularly 100%, identical to any of the amino acid sequences of SEQ ID NOs: 2 to 9.

10. - The ketoreductase activity is increased by at least 2.0, 5.0, or 10 times compared to the wild-type ketoreductase, and / or - The mutant ketoreductase has increased conversions compared to the wild-type ketoreductase at a substrate load of 2-10% [w / w] and at a mutant or wild-type ketoreductase load of 1-2% [w / w] (s / e 5-10) using a 2-propanol recycling system, and in particular has increased conversions of at least 1.05, 1.10, 1.20, 1.30, 1.40, 1.50, 1.75, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 or 10-fold, and / or - The mutant ketoreductase is capable of converting ketones to chiral alcohols. A mutant ketoreductase according to any one of claims 1 to 9.

11. The aforementioned ketone, Formula I (In the formula, R １ is C １～４ It is alkyl, R ２ is hydrogen or C １～４ (It is alkyl.) The obtained chiral alcohol has formula II (In the formula, R １ and R ２ (As stated above) A mutant ketoreductase according to claim 10, having the characteristics of the mutant ketoreductase according to claim 10.

12. The mutant ketoreductase according to claim 11, having the ability to convert a ketone of formula (II) to the corresponding R-enantiomer or R-diastereomer of a chiral alcohol of formula (I) with an enantiomer or diastereomer excess of at least 95%, 96%, 97%, 98%, or 99%.

13. A nucleic acid encoding a mutant ketoreductase according to any one of claims 1 to 12, which is optionally included in the vector.

14. A method for enzymatic reduction of ketones and formation of chiral alcohols in the presence of mutant ketoreductase according to claims 1 to 12.

15. The aforementioned ketone, Formula I (wherein, R １ is C １～４ alkyl, and R ２ is hydrogen or C １～４ alkyl) The obtained chiral alcohol has formula II (In the formula, R １ and R ２ (As stated above) The method according to claim 14, having the following characteristics.

16. The method according to claim 15, wherein the obtained chiral alcohol is the corresponding R-enantiomer or R-diastereomer.

17. formula (In the formula, A, R １ , R ２ , R ５ and R １０ (As defined in claim 1 of PCT international application WO2008 / 006040A1) Use of the method according to claims 14-16 for the preparation of serine / threonine protein kinase inhibitors.

18. The use according to claim 17 or 18 for the preparation of

18. .

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