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

Abstract

The present invention relates to a mutant ketoreductase, a nucleic acid encoding the mutant ketoreductase, a vector containing the nucleic acid, a method for the enzymatic reduction of prochiral ketones and the formation of chiral alcohols using the mutant ketoreductase, the use of the mutant ketoreductase for the preparation of chiral alcohols, and the use of a method for the preparation of a pharmaceutically active morpholine compound.

Description

Technical Field

[0001] The present invention relates to a mutant ketoreductase, a nucleic acid encoding the mutant ketoreductase, a vector containing the nucleic acid, a method for the enzymatic reduction of a prochiral ketone and the formation of chiral alcohols using the mutant ketoreductase, the use of the mutant ketoreductase for the preparation of chiral alcohols, and the use of a method for the preparation of a pharmaceutically active morpholine compound.

Background Art

[0002] Ketoreductase is a subclass of enzymes belonging to the group of oxidoreductases, i.e., an enzyme that catalyzes a redox reaction that enables the transfer of electrons from a so-called electron donor molecule to an electron acceptor molecule.

[0003] Subclasses of ketoreductase have the specific ability to catalyze the enantioselective conversion of a desired prochiral ketone to its corresponding secondary alcohol. During this reduction reaction, electrons are transferred to the ketone group (C=O), whereby a first hydrogen is added to the carbon of the ketone group and a second hydrogen is added to the oxygen. This reaction generally requires an electron donor as a cofactor such as NADH or NADPH, which can be regenerated in situ.

[0004] To date, ketoreductases have also been commonly used for the enzymatic reduction of prochiral ketocompounds and are thus used for the preparation of intermediates for various pharmaceutical compounds, for example, the preparation of compounds having good affinity for trace amine-associated receptors (TAAR).

[0005] TAAR belongs to the group of G protein-coupled receptors and functions as a receptor for various endogenous and exogenous compounds. Six functional human TAARs are known. One of them is TAAR1, which has been identified as a receptor for metabolic derivatives, such as metabolic derivatives of the amino acids phenylalanine, tyrosine, and tryptophan. Furthermore, TAAR1 acts as a receptor for exogenous compounds such as ephedrine or synthetic psychostimulants, for example, amphetamine and methamphetamine.

[0006] The chiral alcohol of formula I TIFF2025523053000001.tif27170(wherein R X represents hydrogen, C 1~4 alkyl or a halogen atom) is an important intermediate for preparing compounds having good affinity for TAAR, particularly for TAAR1, as outlined, for example, in WO 2012 / 016879, WO 2012 / 126922, and WO 2017 / 157873. Such compounds can be used, for example, in the treatment of mental disorders such as schizophrenia and mood disorders.

[0007] A particular promising TAAR1 clinical candidate is ralmitaront having formula X. TIFF2025523053000002.tif38170

[0008] The preparation of the chiral alcohol of formula I is described, for example, in WO 2015 / 086495. SUMMARY OF THE INVENTION

[0009] 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 Lactobacillus brevis, particularly the ketoreductase of SEQ ID NO: 1. These variants can be used in the production of chiral alcohols, including their production in a scaled-up process, such as the chiral alcohol of formula I.

[0010] 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 (Lactobacillus brevis ATCC 14869 ketoreductase, called Q84EX5 in UniProtKB), wherein the mutant ketoreductase has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, and the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu, Ala, Cys, Met or Thr (Leu145, Ala145, Cys145, Met145 or Thr145, respectively), and the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Cys, Glu, Ile, Leu or Thr (Cys202, Glu202, Ile202, Leu202 or Thr202, respectively). It has been found that the mutant ketoreductase exhibits increased ketoreductase activity compared to the wild-type ketoreductase, particularly the ketoreductase of Lactobacillus brevis, particularly the ketoreductase of SEQ ID NO: 1.

[0011] As shown in the examples, various single and double mutations introduced into the ketoreductase of Lactobacillus brevis, particularly the ketoreductase of SEQ ID NO: 1, increased the ketoreductase activity compared to the wild type, as can be seen from the value of Fold Improvement Over the Parent (FIOP) (see Tables 1 and 2). The combination of mutations at positions 145 and 202 has proven to be particularly appropriate. Substitutions with Leu, Ala, Cys, Met, or Thr at position 145 (Leu145, Ala145, Cys145, Met145, or Thr145, respectively) significantly increased the ketoreductase activity (more than 8-fold as shown in Table 2). Further mutations at position 202, namely substitutions with Cys, Glu, Ile, Leu, or Thr (Cys202, Glu202, Ile202, Leu202, or Thr202, respectively), further increased the activity (more than 11-fold as shown in Table 2). Additional mutations such as substitutions at positions 141, 144, or 199 may be added (see Tables 3 to 7). Furthermore, mutations at positions 16 and 43 also showed beneficial effects (see Tables 1 and 2).

[0012] Furthermore, such ketoreductases have been found to be very active for catalyzing the enzymatic reduction of prochiral ketones and for the formation of chiral alcohols that can serve as intermediates in the preparation of compounds having good affinity for TAARs such as TAAR1.

[0013] Furthermore, the mutant ketoreductases of the present invention have been found to catalyze the enzymatic reduction of prochiral ketones with increased selectivity and increased conversion compared to the wild-type ketoreductase.

[0014] In particular, the enzymatic reduction of a ketone of the formula TIFF2025523053000003.tif27170(wherein R x is hydrogen, C 1~4 alkyl or a halogen atom) using the mutant ketoreductase gives a very high enantiomeric excess of the formula TIFF2025523053000004.tif27170(wherein R x is as described above) has been found to form a chiral alcohol. Thus, the ketoreductase according to the present invention is important for the preparation of important intermediates, particularly for the preparation of compounds having good affinity for TAAR such as TAARI, more specifically for the preparation of TAARI clinical candidates of formula X TIFF2025523053000005.tif36170 is extremely useful.

[0015] Thus, in a first aspect, the present invention relates to a mutant ketoreductase having increased ketoreductase activity compared to wild-type ketoreductase, wherein the mutant ketoreductase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (Lactobacillus brevis ATCC 14869 ketoreductase), the mutant ketoreductase has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu, Ala, Cys, Met or Thr (Leu145, Ala145, Cys145, Met145 or Thr145, respectively), - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Cys, Glu, Ile, Leu or Thr (Cys202, Glu202, Ile202, Leu202 or Thr202, respectively), relates to the mutant ketoreductase.

[0016] As used herein, the term "ketoreductase" means any protein having the ability to catalyze the enantioselective conversion of a prochiral ketone to the corresponding secondary alcohol.

[0017] As used herein, the term "wild-type ketoreductase" means any ketoreductase that occurs naturally per se. As used herein, the term "mutant ketoreductase" means any ketoreductase derived from the corresponding wild-type ketoreductase and having its amino acid sequence modified as compared to such wild-type ketoreductase. For example, this may include the introduction, deletion, substitution or post-translational modification 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 producing mutations, such as amino acid substitutions, in the amino acid sequence are well known to those skilled in the art. For example, such mutations may already be introduced at the nucleic acid level that results in the expression of the desired mutant amino acid sequence. Accordingly, suitable methods are well known to those skilled in the art and are also described in part below, for example, in the context of the nucleic acids according to the second aspect of the present invention.

[0018] Suitable mutant ketoreductases according to the first aspect may be derived from the wild-type ketoreductase of any organism. A preferred source is Lactobacillus brevis. Particularly preferred is the Lactobacillus brevis ATCC 14869 ketoreductase called Q84EX5.

[0019] 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 a prochiral ketone to the corresponding secondary alcohol under appropriate conditions. Methods for measuring ketoreductase activity are described herein and given in the examples.

[0020] The mutant ketoreductase according to the first aspect exhibits increased ketoreductase activity as compared to the wild-type ketoreductase.

[0021] This activity can be measured by an enzyme assay that measures either the consumption of substrate or the production of product over time. There are numerous different methods for measuring the concentration of substrates and products, and many enzymes can be assayed in several different ways known to those skilled in the art.

[0022] Methods for measuring the enzyme activity of the 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 examine whether the mutant ketoreductase according to the first aspect exhibits increased ketoreductase activity compared to the wild-type ketoreductase, the ketoreductase activities of both ketoreductases are measured using the same method.

[0023] For example, the method for measuring the enzyme activity of ketoreductase may generally be based on a fluorescence assay or a colorimetric assay. Furthermore, the method for measuring the enzyme activity of ketoreductase generally involves the concentration of the product being formed, the detection of the extract being consumed, or the detection of cofactors required for the reaction being formed or consumed, such as NAD + , NADH, NADP + or NADPH.

[0024] The mutant ketoreductase according to the first aspect, which exhibits increased ketoreductase activity compared to the wild-type ketoreductase, shows an increase, for example, greater than 1-fold increase in ketoreductase activity. Those skilled in the art know statistical procedures for evaluating whether one value of enzyme activity is increased compared to another value, such as the Student's t-test or the chi-square test. It is obvious to those skilled in the art that the background signal must be subtracted when analyzing the data.

[0025] In addition to the increased ketoreductase activity, the mutant ketoreductase according to the first aspect may have further increased selectivity with respect to the wild-type ketoreductase. As used herein, the term "selectivity" means the portion of the total reaction substrate that has been converted to the desired target product, taking into account stoichiometry. In general, the selectivity of an enzyme such as ketoreductase can be affected by various reaction parameters such as temperature, pressure, concentration, solvent or reaction time. Methods for measuring the selectivity of the mutant ketoreductase as well as the wild-type ketoreductase, for example, by analyzing the substrate remaining after the enzyme reaction, are well known to those skilled in the art.

[0026] Furthermore, the mutant ketoreductase according to the first aspect of the invention comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (Lactobacillus brevis ATCC 14869 ketoreductase, called Q84EX5 in UniProtKB). Thereby, the amino acid sequence of SEQ ID NO: 1 is derived from the Lactobacillus brevis strain ATCC 14869 ketoreductase, which is called Q84EX5 in UniProtKB.

[0027] As used herein, the term "sequence identity" represents the percentage of characters that exactly match between two different sequences.

[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 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] The mutant ketoreductase may also comprise an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1 (Lactobacillus brevis ATCC 14869 ketoreductase, called Q84EX5 in UniProtKB or L.brevis-Rad).

[0030] The sequence identity according to the present invention can be determined, for example, by a sequence alignment method of a method for comparing sequences. Methods of sequence alignment are well known in the art and include various programs and alignment algorithms. Further, the NCBI Basic Local Alignment Search Tool (BLAST) is available from several information sources including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland) and the Internet, and can be used in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. For example, the percentage of identity of a variant according to the present invention to the amino acid sequence of SEQ ID NO: 1 is typically characterized using NCBI Blast blastp with standard settings. Alternatively, sequence identity can be determined using the software GENEious with standard settings. The alignment results can be derived from Software Geneious (version R8) using, for example, a global alignment protocol with free-end gaps as the alignment type and Blosum62 as the cost matrix.

[0031] The variant ketoreductase according to the present invention has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1. Further, the variant ketoreductase according to the present invention may have at least 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 variant ketoreductase according to the present invention has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu, Ala, Cys, Met or Thr (Leu145, Ala145, Cys145, Met145 or Thr145, respectively), The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Cys, Glu, Ile, Leu or Thr (Cys202, Glu202, Ile202, Leu202 or Thr202, respectively).

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

[0034] Preferably, in the mutant ketoreductase of the first aspect, the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145). Also preferably, in the mutant ketoreductase of the first aspect, the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0035] More preferably, in the mutant ketoreductase of the first aspect, the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0036] In a preferred embodiment of the mutant ketoreductase of the first aspect, - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and / or - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202) or Leu (Leu202), Preferably, the amino acid at the position corresponding to the 145th position of SEQ ID NO: 1 is substituted with Leu (Leu145), and the amino acid at the position corresponding to the 202nd position of SEQ ID NO: 1 is substituted with Ile (Ile202), or the amino acid at the position corresponding to the 145th position of SEQ ID NO: 1 is substituted with Leu (Leu145), and the amino acid at the position corresponding to the 202nd position of SEQ ID NO: 1 is substituted with Leu (Leu202).

[0037] When the mutant ketoreductase according to the first aspect has three or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 1, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the mutant ketoreductase of the first aspect of the present invention preferably includes substitutions at positions corresponding to positions 16, 43, 141, 144 and / or 199 of SEQ ID NO: 1 in addition to the substitutions at positions 145 and 202.

[0038] Mutant Leu145 and Ile202, and - Asn199, - Met199, - Ser199, - Ile141 and Ser199, - Ile141 and Asn199, - Ala144 and Asn199, or - Ala144 and Met199 in combination are particularly preferred, or Mutant Leu145 and Leu202, and - Asn199, - Met199, - Ser199, - Ile141 and Ser199, - Ile141 and Asn199, - Arg144 and Met199, or - Arg144 and Ser199 in combination are particularly preferred.

[0039] Optionally, further mutations defined above or below may be present.

[0040] When the variant ketoreductase according to the first aspect has an amino acid substitution at the position corresponding to position 16 of SEQ ID NO: 1, the amino acid at the position corresponding to position 16 of SEQ ID NO: 1 is preferably substituted with Ala, Cys, Gly, Ile, Met, Ser, Tyr or Val (Ala16, Cys16, Gly16, Ile16, Met16, Ser16, Tyr16 or Val16), respectively. More preferably, in such a case, in the variant ketoreductase of the first aspect, the amino acid at the position corresponding to position 16 is substituted with Ala, Gly, Ile, Ser or Tyr (Ala16, Gly16, Ile16, Ser16 or Tyr16), respectively. Most preferably, in such a case, in the variant ketoreductase of the first aspect, the amino acid at the position corresponding to position 16 is substituted with Ala, Gly or Tyr (Ala16, Gly16, Tyr16), respectively.

[0041] When the variant ketoreductase according to the first aspect has an amino acid substitution at the position corresponding to position 43 of SEQ ID NO: 1, the amino acid at the position corresponding to position 43 is preferably substituted with Ile (Gln43) or Lys (Lys43).

[0042] When the variant ketoreductase according to the first aspect has an amino acid substitution at the position corresponding to position 141 of SEQ ID NO: 1, the amino acid at the position corresponding to position 141 is preferably substituted with Ile (Ile141).

[0043] When the variant ketoreductase according to the first aspect has an amino acid substitution at the position corresponding to position 144 of SEQ ID NO: 1, the amino acid at the position corresponding to position 144 is preferably substituted with Ala, Cys, Ser, Thr or Val (Ala144, Cys144, Ser144, Thr144 or Val144), respectively. More preferably, in such a case, in the variant ketoreductase of the first aspect, the amino acid at the position corresponding to position 144 is substituted with Ala (Ala144).

[0044] When the mutant ketoreductase according to the first aspect has an amino acid substitution at the position corresponding to position 199 of SEQ ID NO: 1, the amino acid at the position corresponding to position 199 is substituted with Phe, Met, Gln, Ser or Val (Phe199, Met199, Gln199, Ser199 or Val199, respectively). More preferably, in such a case, in the mutant ketoreductase of the first aspect, the amino acid at the position corresponding to position 199 is substituted with Gln, Met or Ser (Gln199, Met199 or Ser199, respectively).

[0045] In a more preferred embodiment of the mutant ketoreductase of the first aspect, the amino acid at the position corresponding to position -16 is substituted with Ala, Cys, Gly, Ile, Met, Ser, Tyr or Val (Ala16, Cys16, Gly16, Ile16, Met16, Ser16, Tyr16 or Val16, respectively), preferably substituted with Ala, Gly, Ile, Ser or Tyr (Ala16, Gly16, Ile16, Ser16 or Tyr16, respectively), more preferably substituted with Ala, Gly or Ser (Ala16, Gly16, Ser16, respectively), and / or the amino acid at the position corresponding to position -43 is substituted with Gln or Lys (Gln43 or Lys43), and / or the amino acid at the position corresponding to position -141 is substituted with Ile (Ile141), and / or the amino acid at the position corresponding to position -144 is substituted with Ala, Cys, Ser, Thr or Val (Ala144, Cys144, Ser144, Thr144 or Val144, respectively), preferably the amino acid at the position corresponding to position 144 is substituted with Ala (Ala144), and / or The amino acid at the position corresponding to -199 is substituted with Asn, Phe, Met, Gln, Ser or Val (Phe199, Met199, Gln199, Ser199 or Val199 respectively), preferably Asn, Gln, Met or Ser (Asn199, Gln199, Met199 or Ser199 respectively).

[0046] Increased activity has been demonstrated for variant ketoreductases having the following substitutions. A combination of Leu145 with Cys202, Glu202, Ile202 or Thr202, or Leu145 and Ile202 and - A combination with Asn199, Met199 or Ser199, or Leu145 and Leu202 and - A combination with Asn199, Met199 or Ser199, or Leu145 and Ile202 and - Ala144 and Asn199, - Ala144 and Met199, - A combination of Ile141 and Ser199, or Leu145 and Leu202 and - Ala144 and Met199, - Ala144 and Ser199 in combination.

[0047] When the variant ketoreductase according to the first aspect has 3 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 1, for example, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of the variant ketoreductase of the first aspect of the present invention may optionally include substitutions at positions corresponding to positions 145, 199 and 202 of SEQ ID NO: 1 in combination with position 141.

[0048] In a preferred embodiment of the variant ketoreductase of the first aspect, - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn (Asn199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0049] In a preferred embodiment of the variant ketoreductase of the first aspect, also, - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ser (Ser199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0050] In an even more preferred embodiment of the variant ketoreductase of the first aspect, - The amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn (Asn199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0051] Most preferably, the variant ketoreductase - The amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ser (Ser199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0052] In a more preferred embodiment of the variant ketoreductase of the first aspect, the variant ketoreductase does not contain mutations in one or more of positions 94, 96, 153, 190, 195, 206, and 233. Thus, as used herein, the expression "does not contain mutations in one or more of the positions corresponding to positions 94, 96, 153, 190, 195, 206, and 233 of SEQ ID NO: 1" means that the amino acids of the variant ketoreductase according to the first aspect correspond to the amino acids of SEQ ID NO: 1 at one or more positions corresponding to positions 94, 96, 153, 190, 195, 206, and 233 of SEQ ID NO: 1.

[0053] For example, the variant ketoreductase according to the first aspect does not contain mutations at 1, 2, 3, 4, 5, 6, or 7 positions corresponding to positions 94, 96, 153, 190, 195, 206, and 233 of SEQ ID NO: 1.

[0054] The variant ketoreductase according to the 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, particularly 100% identical, to the amino acid sequences of SEQ ID NOs: 2-17. Methods for determining sequence identity and sequence identity of amino acid sequences of two proteins are well known to those skilled in the art and are also described above.

[0055] In a more preferred embodiment of the variant ketoreductase of the first aspect, the variant ketoreductase consists of, or comprises, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, particularly 100% identical, to any of the amino acid sequences of SEQ ID NOs: 2-17.

[0056] In a preferred embodiment of the variant ketoreductase of the first aspect, the ketoreductase activity relative to the wild-type ketoreductase is increased by at least 2.0, 5.0, or 10-fold. Methods for measuring the ketoreductase activity of a protein, as well as methods for comparing the ketoreductase activities of two or more proteins, are well known to those skilled in the art and are also described above.

[0057] The variant ketoreductase according to the first aspect of the present invention may further have increased conversion compared to the wild-type ketoreductase using a 2-propanol recycling system at a substrate load of 10% and a variant or wild-type ketoreductase load of 1% [w / w] (s / e100).

[0058] As used herein, the term "conversion" means the conversion of any substrate to a product induced by a ketoreductase, such as the variant or wild-type ketoreductase of the present invention. Such conversion may further depend on various reaction parameters such as temperature, pressure, or the amount of substrate or ketoreductase enzyme used. Suitable conditions and methods are described in the examples (see Examples 2-4).

[0059] Preferably, the conversion of the ketoreductase is determined using a 2-propanol recycling system at a substrate load of 10% and a variant or wild-type ketoreductase load of 1% [w / w] (s / e100). In this context, the abbreviation "s / e" represents the term "substrate / enzyme". A load of 1% [w / w] (s / e100) further means that 100 g of substrate is used per 1 g of enzyme, i.e., the substrate and enzyme are used in a ratio of 100 / 1.

[0060] In a more preferred embodiment of the variant ketoreductase of the first aspect, the variant ketoreductase has increased conversion, particularly at least 2.0, 5.0, 7.5, or 10-fold increased conversion compared to the wild-type ketoreductase, using a 2-propanol recycling system at a substrate load of 10% and a variant or wild-type ketoreductase load of 1% [w / w] (s / e100).

[0061] In a preferred embodiment of the variant ketoreductase of the first aspect, the variant ketoreductase can convert a prochiral ketone into a chiral alcohol.

[0062] In a more preferred embodiment, the prochiral ketone has the formula II TIFF2025523053000006.tif27170(wherein R x is hydrogen, C 1~4 alkyl or a halogen atom), and the resulting chiral alcohol has the formula I TIFF2025523053000007.tif27170(wherein Rx is hydrogen, C 1~4 alkyl or a halogen atom).

[0063] Helical bond TIFF2025523053000008.tif11170 is " TIFF2025523053000009.tif6170" or " TIFF2025523053000010.tif6170", that is, it indicates the chirality of the molecule.

[0064] Suitable C 1~4 alkyl groups are selected from methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl or t-butyl, preferably selected from methyl.

[0065] Suitable halogen atoms are fluorine, chlorine, bromine and iodine, preferably bromine.

[0066] In principle, the variant ketoreductase can catalyze the formation of both the S-enantiomer and the R-enantiomer of a chiral alcohol, especially a chiral alcohol of formula I.

[0067] In a preferred embodiment, the variant ketoreductase has the formula Ia TIFF2025523053000011.tif27170 (wherein R x is hydrogen, C 1~4 alkyl or a halogen atom), the formation of the S-enantiomer of the chiral alcohol, more preferably, the formula Ib TIFF2025523053000012.tif27170 catalyzes the formation of the S-enantiomer of the chiral alcohol.

[0068] The enantiomeric excess of the S-enantiomer of the chiral alcohol can reach at least 95%, 96%, 97%, 98% or 99%.

[0069] Furthermore, the mutant ketoreductase according to the first aspect of the present invention can also be made into a fusion protein in combination with a further peptide or protein. Accordingly, the present invention further relates to a fusion protein comprising the mutant ketoreductase of the present invention.

[0070] The fusion protein may further contain a tag. Tags are attached to the protein for various purposes, for example, to facilitate purification, to assist in the proper folding of the protein, to prevent precipitation of the protein, to alter chromatographic properties, to modify the protein, or to mark or label the protein. Several (affinity) tags or (affinity) markers are currently known. Commonly used tags include Arg tag, His tag, Strep tag, Flag tag, T7 tag, S tag, HAT tag, GST tag and MBP tag.

[0071] In a second aspect, the present invention relates to a nucleic acid encoding the mutant ketoreductase according to the first aspect of the present invention. Accordingly, the present invention may also relate to a nucleic acid encoding a fusion protein comprising the mutant ketoreductase according to the first aspect of the present invention.

[0072] As used herein, the term "nucleic acid" generally refers to any nucleotide molecule that encodes a variant ketoreductase of the invention and that can be of variable length. Examples of nucleic acids of the invention include, but are not limited to, plasmids, vectors, or any kind of DNA and / or RNA fragments that can be isolated by standard molecular biology procedures including, for example, ion exchange chromatography. The nucleic acids of the invention can be used for the transfection or transduction of specific cells or organisms.

[0073] The nucleic acid molecules of the 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, for example obtained by cloning or produced by chemical synthesis techniques or combinations thereof. DNA can be triple-stranded, double-stranded or single-stranded. Single-stranded DNA can be the coding strand, also known as the sense strand, or the non-coding strand, also called the antisense strand. As used herein, nucleic acid molecules also refer, inter alia, to single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules that can be single-stranded or more typically double-stranded or triple-stranded and include DNA and RNA, or a mixture of single-stranded and double-stranded regions. Further, as used herein, nucleic acid molecules refer to triple-stranded regions that include RNA or DNA or both RNA and DNA.

[0074] Furthermore, the nucleic acid may contain one or more modified bases. Such nucleic acids may also contain modifications, for example in the ribose - phosphate backbone, in order to increase the stability and half - life of such molecules in a physiological environment. Thus, DNA or RNA having a modified backbone for reasons of stability or otherwise, the characteristics of which are the "nucleic acid molecules" 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 within the context of the present invention. Of course, DNA and RNA have been modified in a wide variety of ways for 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, and in particular the chemical forms of DNA and RNA characteristic of viruses and cells such as simple and complex cells.

[0075] Furthermore, the nucleic acid molecules encoding the variant ketoreductase of the present invention can be functionally linked 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.

[0076] The nucleic acids of the present invention can be formed initially in vitro or in cells in culture, generally by manipulation of the nucleic acid by 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 making nucleic acids.

[0077] The nucleic acids of the present invention may be contained in an expression vector, and the nucleic acid is operably linked to a promoter sequence capable of promoting the expression of the nucleic acid in a host cell.

[0078] In a preferred embodiment of the nucleic acid, it encodes a mutant ketoreductase of the first aspect, and the mutant ketoreductase consists of, or comprises, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, particularly 100% identical, to any of the amino acid sequences of SEQ ID NOs: 2 to 17. Preferably, the nucleic acid has or comprises any of the sequences of SEQ ID NOs: 18 to 34.

[0079] In a third aspect, the present invention relates to a vector comprising a nucleic acid according to the second aspect of the present invention. Thus, the present invention may also relate to a vector comprising a nucleic acid encoding a fusion protein comprising a mutant ketoreductase according to the first aspect of the present invention.

[0080] 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 within a cell (see also the above details regarding the nucleic acids of the present invention). In particular, the vectors of the present invention can be any plasmid or vector known to those skilled in the art that is suitable for expressing proteins in specific host cells such as, but not limited to, mammalian cells, bacterial cells, and yeast cells. The vectors of the present invention can also be nucleic acids that encode the mutant ketoreductase of the present invention and are used for subsequent cloning into their respective vectors 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 such as, for example, Promega (Madison, Wisconsin, USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, California, USA), or MoBiTec (Germany). Methods for 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.

[0081] A vector may further comprise a nucleic acid sequence that enables replication within a host cell, such as an origin of replication, one or more therapeutic genes and / or selectable marker genes, and other gene elements known in the art, such as regulatory elements that direct transcription, translation and / or secretion of the encoded protein. A vector can be used to transduce, transform or infect a cell, thereby causing the cell to express nucleic acids and / or proteins other than those native to the cell. A vector optionally includes something that aids in achieving entry of the nucleic acid into the cell, such as viral particles, liposomes, protein coatings, etc. Numerous types of suitable expression vectors for protein expression are known in the art by standard molecular biology techniques. Such vectors are selected from among 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. supra).

[0082] As detailed above, the nucleic acid encoding the mutant ketoreductase of the present invention is operably linked to a sequence suitable for driving protein expression in a host cell to ensure protein expression. However, when the vector recited in the claims may represent an intermediate product, subsequent cloning into a suitable vector to ensure protein expression is encompassed by the present invention. The vectors of the present invention may further comprise all types of nucleic acid sequences such as, but not limited to, polyadenylation signals, splice donor and splice acceptor signals, intervening sequences, transcriptional enhancer sequences, translational enhancer sequences, drug resistance genes, etc. Optionally, the drug resistance gene may be operably linked to an internal ribosome entry site (IRES) that can be either cell cycle specific or cell cycle independent.

[0083] As used herein, the term "operably linked" generally means that genetic elements are arranged to function in concert for their intended purposes, for example, in that transcription is initiated by a promoter and proceeds through a DNA sequence encoding a variant ketoreductase of the invention. That is, RNA polymerase transcribes the sequence encoding the variant ketoreductase into mRNA, which is then spliced and translated into protein.

[0084] As used in the context of the present invention, the term "promoter sequence" generally refers to any type of regulatory DNA sequence operably linked to a downstream coding sequence, where the promoter can bind RNA polymerase and initiate transcription of the encoded open reading frame in a cell, thereby driving expression of the downstream coding sequence. The promoter sequences of the present invention can 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.

[0085] Furthermore, the present invention also includes host cells comprising a variant ketoreductase of the invention or a fusion protein thereof, a nucleic acid of the second aspect of the invention, or a vector of the third aspect of the invention.

[0086] The "host cell" of the present invention can be any type of organism suitable for application in recombinant DNA technology, including, but not limited to, any type of bacterial strain and yeast strain suitable for expressing one or more recombinant proteins. Examples of host cells include, for example, various Bacillus subtilis strains or Escherichia coli strains. Various Escherichia coli bacterial host cells are known to those skilled in the art and can be commercially purchased from various suppliers such as Stratagene (California, USA), Promega (Wisconsin, USA) or Qiagen (Hilden, Germany), including, but not limited to, strains such as DH5-alpha, HB101, MV1190, JM109, JM101 or XL-1 Blue. A particularly suitable host cell, namely Escherichia coli BL21(DE3) cells, is also described in the examples. Examples of Bacillus subtilis strains that can be used as host cells include, for example, 1012 wild type: leuA8 metB5 trpC2 hsdRM1 and 168 Marburg: trpC2 (Trp-), which are commercially available, for example, from MoBiTec (Germany).

[0087] The culturing of host cells according to the present invention is a standard procedure known to those skilled in the art. That is, nucleic acids encoding the mutant ketoreductases of the present invention can be introduced into appropriate host cells, and the respective proteins can be produced by recombinant means. These host cells can be any suitable type of cell that can be cultured in culture, preferably bacterial cells such as Escherichia coli. In a first step, this approach can involve cloning each gene into an appropriate vector, such as a vector according to a second aspect of the present invention. Vectors are widely used in gene cloning and can be easily introduced, i.e., transfected, into bacterial cells that have been made transiently permeable to DNA. After the protein has been expressed in the respective host cells, the cells can be harvested and serve as starting material for the preparation of a cell extract containing the protein of interest. The cell extract containing the protein of interest is 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 include, but are not limited to, hypotonic salt treatment, homogenization, or sonication.

[0088] In a fourth aspect, the present invention relates to a method for the enzymatic reduction of prochiral ketones and the formation of chiral alcohols in the presence of the mutant ketoreductases of the present invention.

[0089] In a preferred embodiment, the prochiral ketone has the formula II TIFF2025523053000013.tif27170(wherein R x is hydrogen, C 1~4 alkyl or a halogen atom), and the resulting chiral alcohol has the formula I TIFF2025523053000014.tif27170(wherein R x is hydrogen, C 1~4 alkyl or a halogen atom).

[0090] In a more preferred embodiment, the formula Ia The S-enantiomer of the chiral alcohol of TIFF2025523053000015.tif27170 (wherein Rx is hydrogen, C1-4 alkyl or a halogen atom), more preferably the formula Ib Prepare the S-enantiomer of the chiral alcohol of TIFF2025523053000016.tif27170.

[0091] 2-Bromo-1-(4-nitro-phenyl)ethanone is the ketone of formula II that is particularly used.

[0092] Enzymatic reduction by the mutant ketoreductase usually occurs in the presence of the cofactor NADP, which is regenerated in situ.

[0093] The oxidized cofactor is in principle continuously regenerated using the secondary alcohol, which is a cosubstrate. Typical cosubstrates 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, and preferably can be selected from 2-propanol.

[0094] Preferably, the cofactor is regenerated using the cosubstrate with the same enzyme that also catalyzes the target reaction. Acetone formed when 2-propanol is used as the cosubstrate can, in a more preferred embodiment, be continuously removed from the reaction mixture.

[0095] The cofactor loading, i.e., the ratio of the substrate (prochiral ketone) to the cofactor (s / c), can vary between 10 and 3000, preferably between 50 and 1000, and most preferably between 100 and 500.

[0096] In a particular embodiment of the present invention, the enzymatic reduction is carried out in an aqueous buffer medium in the presence of a cosubstrate, i.e., preferably in the presence of 2-propanol. The concentration of the cosubstrate is typically in the range of 5%v to 40%v.

[0097] A suitable buffer can be selected from acidic to neutral buffers such as 2-morpholine-4-ethanesulfonic acid-, ammonium acetate, acetate, phosphate, 1,4-piperazinediethanesulfonic acid, etc. that enable the pH of the reaction to be maintained in the range of pH 5.2 to pH 7.2.

[0098] The substrate loading, i.e., the loading of the prochiral ketone, can be selected between 1% wt and 20% wt, preferably between 10% wt and 20% wt, and the ratio of substrate to enzyme (s / e) can be selected between 25 and 200, preferably between 100 and 200.

[0099] The reaction temperature is usually maintained in the range of 20°C to 50°C, preferably in the range of 25°C to 45°C.

[0100] When the reaction is completed, the obtained chiral alcohol can be post-treated conventionally by extraction or preferably by filtration.

[0101] In a fifth aspect, the present invention relates to the use of a mutant ketoreductase in the enzymatic reduction of a prochiral ketone and the formation of a chiral alcohol in the synthesis of a morpholine compound of the formula TIFF2025523053000017.tif36170(wherein R 1 is aryl or heteroaryl, and the aromatic ring is optionally substituted by one or two C 1~7 -alkyl substituents).

[0102] The term "C 1~7 -alkyl" relates to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical consisting of 1 to 6 carbon atoms, preferably 1 to 4, more preferably 1 to 2 carbon atoms. This term is further exemplified by radicals such as methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl or t-butyl, pentyl and its isomers, hexyl and its isomers, and heptyl and its isomers.

[0103] The term "aryl" relates to an aromatic carbocyclic ring such as a phenyl or naphthyl ring, preferably a phenyl ring.

[0104] The term "heteroaryl" refers to an aromatic 5- to 6-membered monocyclic ring or 9- to 10-membered bicyclic ring that may contain 1, 2, or 3 heteroatoms selected from nitrogen, oxygen, and / or sulfur, for example, pyridinyl, pyrazolyl, pyrimidinyl, benzimidazolyl, quinolinyl, and isoquinolinyl.

[0105] Preferably, R 1 is two C 1~7 -alkyl, more preferably heteroaryl substituted with two C 1~2 -alkyl substituents, and even more preferably pyrazolyl.

[0106] Even more preferably, the morpholine compound is TAARI clinical candidate lalmidaron having the formula X TIFF2025523053000018.tif38170.

[0107] As described in WO 2017 / 157873, Scheme 1, page 5, the synthesis of lalmidaron can be carried out as follows. TIFF2025523053000019.tif81170

[0108] The use according to the fifth aspect of the present invention relates to the preparation of the 2-(4-aminophenyl)morpholine intermediate 2.

[0109] Intermediate 2 is a chiral 2-(4-aminophenyl)morpholine of the formula TIFF2025523053000020.tif31170 (wherein R 2 is a Boc amino protecting group).

[0110] The preparation of Intermediate 2 can be achieved according to the method disclosed in International Publication No. WO 2015 / 086495, in which the enzymatic reduction in step a) is replaced by the enzymatic reduction of a prochiral ketone and the formation of a chiral alcohol in the presence of a mutant ketoreductase according to the fourth aspect of the present invention. The synthetic steps b) - e) can then be carried out according to the disclosure of International Publication No. WO 2015 / 086495.

[0111] With respect to the use of the present invention, reference is made to the terms, examples and specific embodiments used in the context of other aspects of this disclosure, which are also applicable to this aspect. In particular, the mutant ketoreductase or its fusion protein according to the present invention can be used as detailed with respect to the method of the present invention.

[0112] Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. 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, 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).

[0113] The present invention is not limited to the specific methodologies, protocols, and reagents described herein because these can vary. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but the preferred methods and materials are described herein. Further, the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention.

[0114] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "comprise," "contain," and "encompass" are to be construed inclusively rather than exclusively. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plural" refers to two or more.

[0115] The following figures and examples are intended to illustrate various embodiments of the present invention. Accordingly, the specific modifications described are not to be construed as limitations on the scope of the present 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 present invention, and thus it is to be understood that such equivalent embodiments are included herein.

Examples

[0116] 1 General Procedure 1.1 Production of Biocatalyst For the acquisition of the ketoreductase gene and the construction of the expression vector, the ketoreductase (KRED) open reading frame was designed and synthesized for expression in Escherichia coli (E. coli) based on the reported amino acid sequence of ketoreductase, and the desired variant sequences (provided in the section sequences), and the codon optimization algorithm of Twist Bioscience (San Francisco, USA). In all cases, a stop codon was added at the end. Restriction sites for subsequent cloning in the target vector pET-29b(+) were added to the nucleotide sequence, an NdeI restriction was added to the 5' end, and an XhoI restriction sequence was added to the 3' end. The vector contains a coding sequence for kanamycin resistance (Kmr gene). According to the cloning strategy, the expression is under the control of the lac promoter. The obtained plasmid was transformed into E. coli BL21(DE3) using standard methods. The sequences of the codon-optimized gene and the encoded polypeptide are shown in the section "Sequences".

[0117] 1.2 Preparation of Ketoreductase The plasmid from Twist Bioscience was resuspended in sterile water. Inoculation of E. coli BL21(DE3) cells was achieved by heating (45 seconds at 42°C).

[0118] The preculture was incubated overnight at 37°C on a Luria Bertani (LB) agar plate containing 25 μg / mL of kanamycin. A single microbial colony was picked and incubated overnight according to the protocol.

[0119] Terrific broth medium containing 25 μg / mL of kanamycin was added to the culture. After incubation at 28 °C for 3.5 hours, isopropyl β-D-thiogalactoside (IPTG) was added at a final concentration of 1 mM to induce the expression of KRED. Incubation was continued at 28 °C overnight. Cells were harvested by centrifugation (3220 rcf, 45 minutes, 4 °C), and the supernatant was discarded. The cells were resuspended in KPI buffer (100 mM, pH 7), 2 mM MgCl2, 1 mg / ml lysozyme, 0.75 mg / ml polymyxin, 0.2 mg / ml DNase I, and incubated for 60 minutes. Then they were centrifuged (3220 rcf, 45 minutes, 4 °C), the lysate was frozen and stored at -20 °C.

[0120] 2 Biocatalyst 2.1 Process Development Enzyme reduction occurs at a defined temperature (23 - 45 °C) in a reaction mixture of buffer (e.g., using 2-morpholino-4-ylethane sulfonic acid MES, 0.5 M stock solution, pH 6.5) and 2-propanol (final reducing agent). The buffer and 2-propanol (5 - 40% by volume) vary in the experiment, and in reactions with a substrate load exceeding 1%, the 2-PrOH concentration is at least 20%. The load of ketoreductase and cofactor, nicotinamide adenine dinucleotide phosphate cofactor (NADP), is defined depending on the substrate load. Substrates are added at different concentrations of 1 - 20% by weight. The enzyme load varies between experiments and corresponds to a substrate-to-enzyme ratio (s / e) of 33 - 200. The cofactor load varies between experiments and corresponds to a substrate-to-cofactor ratio (s / c) between 10 - 1000.

[0121] 2.2 Analytical Methods Chiral HPLC method (IPC) The production of alcohol is measured by HPLC analysis on a C18 XP column (3.0 × 75 mm, particle size 2.5 μm and 311 bar at 50 °C) after 18 hours. Phase A contains 5% acetonitrile and 0.1% formic acid in water, and phase B contains acetonitrile and 0.1% formic acid. Flow rate 1 ml / min, 90% of phase A at time 0, 60% at 7 minutes, 90% of phase A at 7.5 minutes. Detection wavelength 280 nm.

[0122] For sample preparation, the sample is diluted to a concentration of 1 mg / ml with acetonitrile / water 4:1, and the total injection volume is 1 μl. The retention times are 4.89 minutes for the extract, 3.65 minutes for the product, and 3.46 minutes for the epoxide. The conversion is calculated as the ratio of the product peak area to the total peak area.

[0123] Chiral HPLC method (OP) The enantiomeric excess of the generated compound is measured by chiral analysis on an IE-3 column (4.6 × 150 mm, 3 μm particle size and 250 bar at 40 °C). Phase A contains 5% acetonitrile in water, and phase B contains water, ethanol, and isopropyl alcohol in a ratio of 30:35:35. Flow rate 0.7 ml / min, 50% phase A in 30 minutes. Detection wavelength 264 nm.

[0124] For sample preparation, the sample is diluted to a concentration of 1 mg / ml with ethanol, and the total injection volume is 5 μl. The retention times are 8.8 minutes for the S product, 9.7 minutes for the R product, 15.8 minutes for the epoxide, and 24.8 minutes for the free form. The enantiomeric excess is calculated as the ratio of the value obtained by subtracting the peak of the product S from the peak of the product R to the sum of the areas of the two peaks.

[0125] Design of mutants for improving the KRED-specific activity of 3-ketone I The specific activities of the presented mutants were measured according to the reaction conditions described in Section 2. These positions were identified in L. brevis R-specific alcohol dehydrogenase (Uniprot ID Q84EX5, PDB structure 1ZK4) by structural analysis. Positions near the substrate and cofactor were selected for mutation.

[0126] The conversions of the wild-type (WT) activities are reported in Table 1 respectively and defined as the parent (under given conditions). The improvement factor (FIOP) relative to the parent is 1. For mutants, the FIOP is recorded with the wild-type as the reference (parent).

[0127] FIOP is calculated as follows. TIFF2025523053000021.tif13170·C mut The conversion% (activity) of the area achieved by the use of mutain, and C WT The conversion achieved by the use of WT-ketoreductase ·s / e mut The s / e of the experiment (substrate to mutain ratio), and s / e WT The s / e of the experiment using WT-ketoreductase ·C mut , C WT The reaction time (t WT / t mut )

[0128] The determination of FIOP ideally requires a comparison of similar conversion levels, which may require different substrate concentrations (c) and / or enzyme loads (S / E values) related to the individual activities of the enzyme mutants under the reaction conditions.

Table 1

[0129] The two single mutants T16S and E145L showed an improvement nearly twice that of the parent (here the wild type). The mutant E145L showed complete conversion in the reaction quench, indicating that the conversion was completed earlier in time.

Table 2

[0130] Table 2 shows that positions 16, 141, 144, 145, and 199 were further investigated as single mutants or in combination with beneficial mutations inferred from the results of Table 1. For each position, a subset of amino acids showed increased activity relative to the parent. In particular, the combination of M141I, I144A, E145L, A202C, A202I, and A202L showed the highest conversion.

Table 3

[0131] Table 3 shows further investigation of the combinations of the variants in Table 2. Preferred variants are reported in Table 3 and include the combinations of M141I, I144A, E145L, L199M / N / S, A202I / L.

Table 4

[0132] Table 4 shows that the effective variants from Table 3 were successfully tested with higher substrate concentrations and high substrates for higher enzyme loads, i.e., under technical scale conditions.

[0133] Characterization of 4 Mutant Keto-Reductases 4.1 General Screening Procedure - 1% [w / v] Substrate Load 10 mg (0.1 mmol) of 2-bromo-1-(4-nitrophenyl)ethanone is mixed with a mixture of 100 μl of MES buffer pH 6.5 (0.5 M), 650 μl of water, 200 μl of 2-propanol, and 20 μl of magnesium bromide hexahydrate (0.1 M). The reaction was initiated by adding 200 μg of NADP and 100 μg of keto-reductase at room temperature. After about 1 day, the reaction conversion was determined by achiral HPLC method (IPC) and chiral HPLC method (OP).

[0134] 4.2 General Screening Procedure - 5% [w / v] Substrate Load 50 mg (0.2 mmol) of 2-bromo-1-(4-nitrophenyl)ethanone is mixed with a mixture of 100 μl of MES buffer pH 6.5 (0.5 M), 650 μl of water, 355 μl of 2-propanol, and 20 μl of magnesium bromide hexahydrate (0.1 M). The reaction was initiated by adding 1.0 mg of NADP and 250 μg of keto-reductase at room temperature. After 4 hours and after about 21 hours, the reaction conversion was determined by achiral HPLC method (IPC) and chiral HPLC method (OP).

[0135] 4.3 Temperature stability screening - 5% [w / v] substrate loading 0.25 mg of ketoreductase and 1.0 mg of NADP were dissolved in a mixture of 0.1 ml of MES buffer pH 6.5 (0.5 M), 355 μl of water, and 20 μl of magnesium bromide hexahydrate (0.1 M) at different temperatures in the range of 25 °C to 45 °C (see Table 5). After incubation for 16.5 hours under shaking (1000 rpm), the reaction was initiated by adding 50 mg (0.2 mmol) of 2-bromo-1-(4-nitrophenyl)ethanone dissolved in 0.4 ml of 2-propanol. After 4 hours and 23 hours, the reaction conversion was determined by achiral HPLC method (IPC). All the results are summarized in Table 5.

Table 5

[0136] Table 5 shows that three selected mutants exhibit similar performance at 23 hours at 25 °C and 30 °C temperatures, despite different initial activities (at 4 hours). The quadruple mutant shows the best performance at 35 °C.

[0137] 4.4 Upscaling production - 10% [w / v] substrate loading 3 g of 2-bromo-1-(4-nitrophenyl)ethanone (12.3 mmol) was suspended in a mixture of 3 ml of MES buffer pH 6.5 (0.5 M), 11.4 ml of water, 12 ml of 2-propanol, and 0.6 ml of magnesium bromide hexahydrate (0.1 M) with stirring. The reaction was initiated by adding 30 mg of NADP and 15 mg of ketoreductase at room temperature. At the end of the reaction, the final pH was 6.1. After 4 hours and 21 hours, the reaction conversion was determined by achiral HPLC method (IPC), and the enantiomeric excess at 21 hours was determined by chiral HPLC method (OP). All the results are summarized in Table 6.

Table 6

[0138] Table 6 shows that three selected mutants show complete conversion after 21 hours in a larger scale experiment.

[0139] 4.5 Upscaling production - 20% [w / v] substrate load 6 g of 2-bromo-1-(4-nitrophenyl)ethanone (24.6 mmol) is suspended in a mixture of 3 ml of MES buffer pH 6.5 (0.5 M), 8.4 ml of water, 12 ml of 2-propanol (final reducing agent) and 0.6 ml of magnesium bromide hexahydrate (0.1 M) while stirring. The reaction is initiated by adding 60 mg of NADP and 30 mg of ketoreductase (see Table 7) at room temperature. At the end of the reaction, the final pH was 5.8. After 4 hours, 21 hours and 2 days, the reaction conversion was determined by achiral HPLC method (IPC), and the enantiomeric excess after 2 days was determined by chiral HPLC method (OP). All results are summarized in Table 7.

Table 7

[0140] Table 7 shows that complete conversion of the product and excellent enantiomeric excess can be achieved even with a 20% substrate load within 2 days. Sequence Amino acid sequence > Lactobacillus brevis ATCC 14869 ketoreductase called Q84EX5 in UniProtKB (SEQ ID NO: 1) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQ HDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGT RLGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRV NTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_A202L (SEQ ID NO: 2) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_A202C (SEQ ID NO: 3) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGCEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_A202E (SEQ ID NO: 4) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGEEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_A202I (SEQ ID NO: 5) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_A202T (SEQ ID NO: 6) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGTEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_L199M_A202I (SEQ ID NO: 7) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_L199M_A202L (SEQ ID NO: 8) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_L199N_A202L (SEQ ID NO: 9) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDNPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_L199N_A202I (SEQ ID NO: 10) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDNPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_L199S_A202L (SEQ ID NO: 11) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_E145L_L199S_A202I (Sequence number 12) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_M141I_E145L_L199S_A202I(Sequence number 13) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINISSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_I144A_E145L_L199M_A202I(Sequence number 14) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_I144A_E145L_L199N_A202I (Accession No. 15) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDNPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_I144A_E145L_L199M_A202L (Accession No. 16) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ >Q84EX5_I144A_E145L_L199S_A202L (Accession No. 17) MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ Nucleic acid sequence >Lactobacillus brevis ATCC 14869 ketoreductase, called Q84EX5 in UniProtKB (SEQ ID NO: 18) ATGAGCAACCGTCTGGACGGCAAGGTGGCGATCATTACCGGTGGCACCCTGGGTATTGGTCTGGCGATTGCGACCAAGTTCGTGGAGGAAGGTGCGAAAGTTATGATCACCGGCCGTCACAGCGACGTGGGCGAGAAGGCGGCGAAAAGCGTTGGCACCCCGGACCAGATTCAATTCTTTCAGCACGATAGCAGCGACGAGGATGGTTGGACCAAGCTGTTCGATGCGACCGAAAAAGCGTTTGGCCCGGTTAGCACCCTGGTTAACAACGCGGGTATTGCGGTGAACAAGAGCGTTGAGGAAACCACCACCGCGGAGTGGCGTAAACTGCTGGCGGTGAACCTGGATGGTGTTTTCTTTGGCACCCGTCTGGGTATCCAACGTATGAAGAACAAAGGTCTGGGCGCGAGCATCATTAACATGAGCAGCATTGAAGGTTTCGTTGGCGACCCGAGCCTGGGTGCGTACAACGCGAGCAAGGGTGCGGTTCGTATCATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAGGACTACGATGTGCGTGTTAACACCGTGCACCCGGGCTATATTAAAACCCCGCTGGTTGACGATCTGCCGGGTGCGGAGGAAGCGATGAGCCAGCGTACCAAGACCCCGATGGGTCACATCGGCGAACCGAACGACATCGCGTACATTTGCGTTTATCTGGCGAGCAACGAGAGCAAATTCGCGACCGGTAGCGAATTTGTGGTTGATGGTGGCTATACCGCGCAATAA >Q84EX5_E145L_A202L (SEQ ID NO: 19) ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCCTGGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_A202C (SEQ ID NO: 20) ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCTGCGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_A202E (SEQ ID NO: 21) ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCGAAGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_A202I (SEQ ID NO: 22) ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCATTGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_A202T (SEQ ID NO: 23) ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCACCGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_L199M_A202I (SEQ ID NO: 24) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_L199M_A202L (SEQ ID NO: 25) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_L199N_A202L (SEQ ID NO: 26) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAACCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_L199N_A202I (SEQ ID NO: 27) ATGAGCAATCGTCTGGATGGAAAGGTAGCAATTATTACCGGCGGGACTCTGGGCATTGGACTCGCGATTGCGACAAAATTCGTGGAAGAAGGCGCGAAAGTGATGATTACGGGTCGCCATTCGGACGTAGGGGAAAAAGCTGCGAAAAGTGTTGGCACTCCGGACCAGATTCAGTTTTTTCAACATGATTCCTCCGATGAGGATGGCTGGACGAAATTATTCGACGCGACCGAAAAAGCATTTGGGCCGGTCTCAACATTGGTCAATAATGCTGGCATCGCCGTCAATAAATCTGTCGAAGAAACCACCACCGCTGAATGGCGCAAACTGCTGGCCGTCAATCTGGATGGCGTTTTCTTTGGTACGCGGCTCGGGATTCAGCGGATGAAGAACAAAGGGCTGGGGGCAAGTATCATTAATATGTCGAGCATCCTTGGGTTTGTCGGCGACCCCTCATTAGGGGCCTACAACGCTAGCAAAGGTGCCGTACGCATCATGAGCAAATCTGCGGCGTTGGACTGCGCCCTGAAAGATTACGATGTGCGCGTTAATACCGTCCATCCGGGTTATATTAAAACGCCGTTGGTAGATGATAACCCAGGTATCGAGGAAGCAATGTCCCAGCGCACCAAAACCCCAATGGGACATATTGGCGAACCGAACGATATTGCCTATATTTGTGTATACCTGGCGTCAAATGAGTCTAAATTTGCGACGGGGAGCGAATTTGTGGTAGATGGCGGCTACACCGCGCAATAA >Q84EX5_E145L_L199S_A202L (SEQ ID NO: 28) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_E145L_L199S_A202I (SEQ ID NO: 29) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTGCGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_M141I_E145L_L199S_A202I(SEQ ID NO: 30) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATTAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_I144A_E145L_L199M_A202I(SEQ ID NO: 31) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_I144A_E145L_L199N_A202I(SEQ ID NO: 32) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAACCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_I144A_E145L_L199M_A202L(SEQ ID NO: 33) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA >Q84EX5_I144A_E145L_L199S_A202L(SEQ ID NO: 34) ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

Claims

1. A mutant ketoreductase having increased ketoreductase activity compared to wild-type ketoreductase, wherein said mutant ketoreductase comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1 (Lactobacillus brevis ATCC 14869 ketoreductase), wherein said mutant ketoreductase has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu, Ala, Cys, Met or Thr (Leu145, Ala145, Cys145, Met145 or Thr145, respectively), - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Cys, Glu, Ile, Leu or Thr (Cys202, Glu202, Ile202, Leu202 or Thr202, respectively), A mutant ketoreductase.

2. - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and / or - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202) or Leu (Leu202), Preferably, the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202), or the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Leu (Leu202). The mutant ketoreductase according to Claim 1.

3. - the amino acid at the position corresponding to position 16 of SEQ ID NO: 1 is Ala, Cys, Gly, Ile, Met, Ser, Tyr or Val (Ala16, Cys16, Gly16, Ile16, Met16, Ser16, Tyr16 or Val16, respectively), preferably substituted with Ala, Gly, Ile, Ser16 or Tyr (Ala16, Gly16, Ile16, Ser16 or Tyr16, respectively), preferably the amino acid at the position corresponding to position 16 of SEQ ID NO: 1 is substituted with Ala, Gly or Tyr (Ala16, Gly16, Tyr16, respectively), and / or - The amino acid at the position corresponding to position 43 of SEQ ID NO: 1 is substituted with Gln or Lys (Gln43 or Lys43), and / or - The amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), and / or, - The amino acid at the position corresponding to position 144 of SEQ ID NO: 1 is substituted with Ala, Cys, Ser, Thr or Val (respectively, Ala144, Cys144, Ser144, Thr144 or Val144), preferably the amino acid at the position corresponding to position 144 of SEQ ID NO: 1 is substituted with Ala (Ala144), and / or - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn, Phe, Met, Gln, Ser or Val (respectively, Asn199, Phe199, Met199, Gln199, Ser199 or Val199), preferably substituted with Asn, Gln, Met or Ser (respectively, Asn199, Gln199, Met199 or Ser199). The variant ketoreductase according to claim 1 or 2.

4. - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn (Asn199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202), or or - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ser (Ser199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202). The variant ketoreductase according to any one of claims 1 to 3.

5. - The amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), - The amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - The amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn (Asn199), - The amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202), or or preferably - The amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), - the amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ser (Ser199), - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202), The variant ketoreductase according to any one of claims 1 to 3.

6. The variant ketoreductase according to any one of claims 1 to 5, which does not contain mutations at one or more of the positions corresponding to positions 94, 96, 153, 190, 195, 206 and 233 of SEQ ID NO:

1.

7. The variant ketoreductase according to any one of claims 1 to 6, which consists of, or comprises, an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, particularly 100% identical, to any of the amino acid sequences of SEQ ID NOs: 2 to 14.

8. The variant ketoreductase according to any one of claims 1 to 7, wherein the ketoreductase activity is increased by at least 2.0-fold, 5.0-fold, or 10-fold compared to the wild-type ketoreductase.

9. Using a 2-propanol recirculation system, having an increased conversion compared to the wild-type ketoreductase at a 10% substrate load and a 1% [w / w] (s / e100) variant or wild-type ketoreductase load, Particularly, having a conversion increased by at least 1.05-fold, 1.10-fold, 1.20-fold, 1.30-fold, 1.40-fold, 1.50-fold, 1.75-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, or 10-fold compared to the wild-type ketoreductase. The variant ketoreductase according to any one of claims 1 to 8.

10. The variant ketoreductase according to any one of claims 1 to 9, which is capable of converting a prochiral ketone into a chiral alcohol.

11. Said prochiral ketone is of formula II (wherein R ｘ is hydrogen, C １～４ alkyl or a halogen atom), and the obtained chiral alcohol has the formula I (wherein R ｘ is hydrogen, C １～４ alkyl or a halogen atom), the mutant ketoreductase according to claim 10.

12. The variant ketoreductase according to claim 11, which has the ability to convert the ketone of formula (II) into the S-enantiomer of the chiral alcohol of formula (I) with an enantiomeric excess of at least 95%, 96%, 97%, 98% or 99%.

13. A nucleic acid encoding the variant ketoreductase according to any one of claims 1 to 12, optionally contained in a vector.

14. A method for the enzymatic reduction of a prochiral ketone and the formation of a chiral alcohol in the presence of a variant ketoreductase according to any one of claims 1 to 12.

15. wherein the prochiral ketone has the formula II (wherein R ｘ is hydrogen, C １～４ alkyl or a halogen atom), and the obtained chiral alcohol has the formula I (wherein R ｘ is hydrogen, C １～４ alkyl or a halogen atom), the method according to claim 14.

16. The method according to claim 15, wherein the obtained chiral alcohol is the S-enantiomer.

17. Formula (wherein R １ is aryl or heteroaryl, and the aromatic ring is optionally substituted by one or two C １～７ -alkyl substituents) Use of the method according to any one of claims 14 to 16 for preparing a morpholine compound.

18. R １ is pyrazolyl substituted with two C １～７ -alkyl substituents, the use according to claim 17. Use according to claim 17 or 18 for the preparation of

19. .

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