Mutant ketoreductases with increased ketoreductase activity and methods and uses involving same

Abstract

The present invention relates to a mutant ketoreductase, a nucleic acid encoding the mutant ketoreductase, a vector comprising the nucleic acid, a method of enzymatic reduction of a prochiral ketone with the mutant ketoreductase and formation of a chiral alcohol, the use of said mutant ketoreductase for the preparation of chiral alcohols and the use of said method for the preparation of pharmaceutically active morpholine compounds.

Description

[0001] The present invention relates to a mutant ketoreductase, a nucleic acid encoding the mutant ketoreductase, a vector comprising the nucleic acid, a method for enzymatically reducing a prochiral ketone with the mutant ketoreductase and forming a chiral alcohol, the use of the mutant ketoreductase for preparing a chiral alcohol, and the use of the method for preparing a pharmaceutically active morpholine compound.

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

[0003] A subclass of ketoreductases has the unique 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 keto group (C=O), resulting in the addition of a first hydrogen to the carbon of the keto group and a second hydrogen 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] Ketoreductases are also commonly used to date for the enzymatic reduction of prochiral keto compounds and thus for the preparation of intermediates for various pharmaceutical compounds, such as for the preparation of compounds with good affinity for trace amine associated receptors (TAARs).

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

[0006] Chiral alcohol of formula I

[0007]

[0008] Where R X Represents hydrogen, C 1-4 Alkyl or represents a halogen atom, is a key intermediate for preparing compounds with good affinity for TAAR, especially TAAR1, as summarized in, for example, PCT publications WO 2012 / 016879, WO 2012 / / 126922, and WO 2017 / 157873. Such compounds can be used, for example, to treat psychiatric disorders, such as schizophrenia and mood disorders.

[0009] One particularly promising TAARI clinical candidate is ralmitaront, which has the formula X.

[0010]

[0011] The preparation of chiral alcohols of the formula I is described, for example, in WO 2015 / 086495.

[0012] The object of the present invention is to design improved mutant ketoreductases having increased ketoreductase activity relative to wild-type ketoreductases, in particular ketoreductases of Lactobacillus brevis, especially the ketoreductase of SEQ ID NO: 1. These mutants can be used for the production of chiral alcohols, such as chiral alcohols of formula I, including their production in scale-up processes.

[0013] Surprisingly, mutant ketoreductases have been found 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, referred to as Q84EX5 in UniProtKB); and wherein the mutant ketoreductase 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), 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 15 or Thr202, respectively) relative to the wild-type ketoreductase, in particular the ketoreductase of Lactobacillus brevis, in particular the ketoreductase of SEQ ID NO: 1. The ketoreductase of NO:1 shows increased ketoreductase activity.

[0014] As shown in the examples, various single and double mutations introduced into the ketoreductase of Lactobacillus brevis, in particular SEQ ID NO: 1, increased ketoreductase activity relative to the wild type, which can be derived from the fold improvement (FIOP) values ​​relative to the parent (see Tables 1 and 2). The combination of mutations at positions 145 and 202 has proven to be particularly suitable. Substitution at position 145 with Leu, Ala, Cys, Met or Thr (respectively Leu145, Ala145, Cys145, Met145 or Thr145) significantly increased ketoreductase activity (>8 times as shown in Table 2). Additional mutations at position 202, i.e. substitution with Cys, Glu, Ile, Leu or Thr (respectively Cys202, Glu202, Ile202, Leu202 or Thr202) further increased activity (>11 times as shown in Table 2). Further mutations may be added, such as substitutions at positions 141, 144 or 199 (see Tables 3 to 7). In addition, mutations at positions 16 and 43 also showed beneficial effects (see Tables 1 and 2).

[0015] Furthermore, such ketoreductases have been found to be highly active in catalyzing the enzymatic reduction of prochiral ketones and the formation of chiral alcohols that can be used as intermediates in the preparation of compounds with good affinity for TAARs such as TAAR1.

[0016] Furthermore, it has been found that the mutant ketoreductases of the present invention catalyze the enzymatic reduction of prochiral ketones with increased selectivity and increased conversion compared to wild-type ketoreductases.

[0017] In particular, ketones of the formula

[0018]

[0019] Where R x For hydrogen, C 1-4 Alkyl or halogen atom, enzymatic reduction with mutant ketoreductase to form a chiral alcohol of the formula

[0020]

[0021] Where R x As described above, it has a very high enantiomeric excess. Therefore, the ketoreductase according to the present invention is extremely useful for the preparation of key intermediates, especially for the preparation of key intermediates in compounds with good affinity for TAARs (such as TAARI), and more particularly for TAARI clinical candidates of formula X

[0022]

[0023] Thus, in a first aspect, the present invention relates to a mutant ketoreductase having increased ketoreductase activity relative to a wild-type ketoreductase,

[0024] 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); and

[0025] The mutant ketoreductase has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, wherein

[0026] - 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

[0027] - 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).

[0028] As used herein, the term "ketoreductase" means any protein that has the ability to catalyze the selective conversion of a prochiral ketone to the enantiomer of the corresponding secondary alcohol.

[0029] As used herein, the term "wild-type ketoreductase" means any ketoreductase that exists per se in nature. As used herein, the term "mutant ketoreductase" means any ketoreductase that is derived from the corresponding wild-type ketoreductase and whose amino acid sequence has been 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 in terms of amino acid substitution. Methods for generating mutations in amino acid sequences, such as amino acid substitutions, are well known to those skilled in the art. For example, such mutations may have been introduced at the nucleic acid level, resulting in the expression of the desired mutant amino acid sequence. Therefore, suitable methods are well known to those skilled in the art and are described in part below, for example in the context of nucleic acids according to the second aspect of the invention.

[0030] Suitable mutant ketoreductases according to the first aspect may be derived from wild-type ketoreductases of any organism. A preferred source is Lactobacillus brevis. Particularly preferred is the Lactobacillus brevis ATCC 14869 ketoreductase, referred to as Q84EX5.

[0031] According to the present invention, the mutant ketoreductase is ketoreductase active. This means that the mutant ketoreductase is able to convert a prochiral ketone into the corresponding secondary alcohol under appropriate conditions, as described above and below. Methods for determining ketoreductase activity are described herein and given in the Examples.

[0032] The mutant ketoreductase according to the first aspect exhibits increased ketoreductase activity relative to the wild-type ketoreductase.

[0033] Activity can be determined in an enzyme assay that measures the consumption of a substrate or the production of a product over time. There are many different methods to measure substrate and product concentrations, and many enzymes can be assayed in several different ways as known to those skilled in the art.

[0034] Methods for determining the enzymatic activity of a mutant ketoreductase or a 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. In order to determine whether the mutant ketoreductase according to the first aspect exhibits increased ketoreductase activity relative to the wild-type ketoreductase, the ketoreductase activity of the two ketoreductases is measured using the same method.

[0035] For example, methods for determining the enzymatic activity of a ketoreductase may generally be based on a fluorescence or colorimetric assay. In addition, methods for determining the enzymatic activity of a ketoreductase may generally include detecting the concentration of a formed product, a consumed educt, or detecting the formation or consumption of a cofactor required for the reaction, such as NAD + , NADH, NADP + or the concentration of NADPH.

[0036] For example, a mutant ketoreductase showing increased ketoreductase activity according to the first aspect shows an increase in ketoreductase activity of more than one-fold relative to a wild-type ketoreductase. Statistical procedures are known to those skilled in the art to assess whether one value of enzyme activity is increased relative to another value, such as a Student's t-test or a chi-square test. It will be apparent to those skilled in the art that any background signal must be subtracted when analyzing the data.

[0037] In addition to the increased ketoreductase activity, the mutant ketoreductase according to the first aspect may further have an increased selectivity relative to the wild-type ketoreductase. As used herein, the term "selectivity" means the portion of the total reaction substrate that has been converted into the desired target product, taking into account the stoichiometry. In general, the selectivity of an enzyme such as a ketoreductase may be affected by various reaction parameters such as temperature, pressure, concentration, solvent or reaction time. Methods for determining the selectivity of a mutant ketoreductase as well as a wild-type ketoreductase are well known to those skilled in the art, such as by analyzing the substrate remaining after the enzyme reaction.

[0038] Further, the mutant ketoreductase according to the first aspect of the present 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, referred to as Q84EX5 in UniProtKB). Therefore, the amino acid sequence of SEQ ID NO: 1 is derived from the ketoreductase of Lactobacillus brevis strain ATCC 14869, which is referred to as Q84EX5 in UniProtKB.

[0039] As used herein, the term "sequence identity" describes the percentage of characters that exactly match between two different sequences.

[0040] For example, as used herein, the term "at least 80% identical to the amino acid sequence of SEQ ID NO: 1" 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.

[0041] 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, referred to as Q84EX5 in UniProtKB, or as L. brevis-Rad).

[0042] Sequence identity according to the present invention can be determined for example by the sequence alignment method of sequence comparison form.The method for sequence alignment is well known to those skilled in the art, and comprises various programs and alignment algorithms.In addition, NCBI basic local alignment search tool (BLAST) can be obtained from several sources, comprises National Center for Biotechnology Information (NCBI, Bethesda, MD) and the Internet, is used to be connected with sequence analysis program blastp, blastn, blastx, tblastn and tblastx.Mutant according to the present invention is characterized with respect to the identity percentage ratio of the amino acid sequence of for example SEQ ID NO:1 usually using NCBI Blast blastp with standard settings.Alternatively, the software GENEious with standard settings can be used to determine sequence identity.Comparison result can for example be derived from software Geneious (R8 version), uses the global alignment scheme with free end gap as alignment type and uses Blosum62 as cost matrix.

[0043] The mutant ketoreductase according to the present invention has at least two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1. In addition, the mutant ketoreductase according to the present invention may have at least three, four, five, six, seven, eight, nine, ten or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1.

[0044] In particular, the mutant 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

[0045] - 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

[0046] - 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).

[0047] Methods for preparing the mutant ketoreductase according to the first aspect of the 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 known to those skilled in the art to be suitable for preparing recombinant enzymes, such as recombinantly expressing a modified nucleic acid of the mutant ketoreductase in cell culture, followed by protein isolation and purification.

[0048] 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).

[0049] 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).

[0050] In a preferred embodiment of the mutant ketoreductase of the first aspect,

[0051] - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145); and / or

[0052] - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202) or with Leu (Leu202),

[0053] Preferably, wherein 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 wherein 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).

[0054] If the mutant ketoreductase according to the first aspect has more than two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, such as three, four, five, six, seven, eight, nine, ten or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, then the amino acid sequence of the mutant ketoreductase of the first aspect of the invention preferably comprises 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.

[0055] Particularly preferred are mutations Leu145 and Ile202, with

[0056] -Asn199;

[0057] -Met199;

[0058] -Ser199;

[0059] -Ile141 and Ser199;

[0060] -Ile141 and Asn199;

[0061] -Ala144 and Asn199; or

[0062] - Ala144 and Met199 combination, or

[0063] Mutations Leu145 and Leu202, with

[0064] -Asn199;

[0065] -Met199;

[0066] -Ser199;

[0067] -Ile141 and Ser199;

[0068] -Ile141 and Asn199;

[0069] - Arg144 and Met199; or

[0070] - Arg144 and Ser199 combination.

[0071] Further mutations may be present, optionally as defined above or below.

[0072] If the mutant ketoreductase according to the first aspect has an amino acid substitution at the position corresponding to position 16 of SEQ ID NO: 1, 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 mutant 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 mutant 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).

[0073] If the mutant 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 with Lys (Lys43).

[0074] If the mutant 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).

[0075] If the mutant 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 mutant ketoreductase of the first aspect, the amino acid at the position corresponding to position 144 is substituted with Ala (Ala144).

[0076] If 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).

[0077] In a further preferred embodiment of the mutant ketoreductase of the first aspect,

[0078] - the amino acid at the position corresponding to position 16 is substituted by Ala, Cys, Gly, Ile, Met, Ser, Tyr or Val (Ala16, Cys16, Gly16, Ile16, Met16, Ser16, Tyr 16 or Val16, respectively), preferably by Ala, Gly, Ile, Ser or Tyr (Ala16, Gly16, Ile16, Ser16 or Tyr16, respectively), more preferably by Ala, Gly or Ser (Ala16, Gly16, Ser16, respectively); and / or

[0079] - the amino acid at the position corresponding to position 43 is substituted with Gln or Lys (Gln43 or Lys43); and / or

[0080] - the amino acid at the position corresponding to position 141 is substituted by Ile (Ile141); and / or

[0081] - 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 wherein the amino acid at the position corresponding to position 144 is substituted with Ala (Ala144); and / or

[0082] - the amino acid at the position corresponding to position 199 is substituted with Asn, Phe, Met, Gln, Ser or Val (Phe199, Met199, Gln199, Ser199 or Val199, respectively), preferably with Asn, Gln, Met or Ser (Asn199, Gln199, Met199 or Ser199, respectively).

[0083] Increased activity has been demonstrated for mutant ketoreductases having the following substitutions:

[0084] Leu145 in combination with Cys202, Glu202, Ile202 or Thr202; or

[0085] Leu145 and Ile202, with

[0086] - Asn199, Met199 or Ser199 combination; or

[0087] Leu145 and Leu202, with

[0088] - Asn199, Met199 or Ser199 combination; or

[0089] Leu145 and Ile202, with

[0090] -Ala144 and Asn199,

[0091] -Ala144 and Met199,

[0092] - Ile141 and Ser199 combination; or

[0093] Leu145 and Leu202, with

[0094] -Ala144 and Met199,

[0095] -Ala144 and Ser199 combination.

[0096] If the mutant ketoreductase according to the first aspect has more than two amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, such as three, four, five, six, seven, eight, nine, ten or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 1, then the amino acid sequence of the mutant ketoreductase of the first aspect of the invention may include substitutions at positions corresponding to positions 145, 199 and 202 of SEQ ID NO: 1, optionally in combination with position 141.

[0097] In a preferred embodiment of the mutant ketoreductase of the first aspect,

[0098] - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and

[0099] - the amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn (Asn199), and

[0100] - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0101] In a further preferred embodiment of the mutant ketoreductase of the first aspect,

[0102] - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and

[0103] - the amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ser (Ser199), and

[0104] - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0105] In an even more preferred embodiment of the mutant ketoreductase of the first aspect,

[0106] - the amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), and

[0107] - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and

[0108] - the amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Asn (Asn199), and

[0109] - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0110] Most preferably, the mutant ketoreductase is characterized by

[0111] - the amino acid at the position corresponding to position 141 of SEQ ID NO: 1 is substituted with Ile (Ile141), and

[0112] - the amino acid at the position corresponding to position 145 of SEQ ID NO: 1 is substituted with Leu (Leu145), and

[0113] - the amino acid at the position corresponding to position 199 of SEQ ID NO: 1 is substituted with Ser (Ser199), and

[0114] - the amino acid at the position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ile (Ile202).

[0115] In another preferred embodiment of the mutant ketoreductase of the first aspect, the mutant ketoreductase does not comprise a mutation at one or more of the portions 94, 96, 153, 190, 195, 206 and 233. Therefore, as used herein, the expression "does not comprise a mutation at 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 acid at one or more of the positions corresponding to positions 94, 96, 153, 190, 195, 206 and 233 of the mutant ketoreductase according to the first aspect corresponds to the amino acid of SEQ ID NO: 1.

[0116] For example, the mutant ketoreductase according to the first aspect does not comprise mutations at one, two, three, four, five, six or seven positions corresponding to positions 94, 96, 153, 190, 195, 206 and 233 of SEQ ID NO:1.

[0117] The mutant ketoreductase according to the first aspect of the invention may further comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, in particular 100% identical to the amino acid sequence of SEQ ID NO: 2 to 17. Sequence identity and methods for determining sequence identity between the amino acid sequences of two proteins are well known to those skilled in the art and are also described above.

[0118] In another preferred embodiment of the mutant ketoreductase of the first aspect, the mutant ketoreductase consists of or comprises the following: an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, in particular 100% identical to the amino acid sequence of any one of SEQ ID NO: 2 to 17.

[0119] In preferred embodiments of the mutant ketoreductase of the first aspect, the ketoreductase activity is increased at least 2.0, 5.0 or 10 times relative to the 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 also described above.

[0120] The mutant ketoreductase according to the first aspect of the invention may further have increased conversion relative to the wild-type ketoreductase at 10% substrate loading and at 1% [w / w] (s / e 100) loading of the mutant or wild-type ketoreductase using a 2-propanol recovery system.

[0121] As used herein, the term "conversion" means the conversion of any substrate to a product induced by a ketoreductase, such as a mutant ketoreductase or a 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 used. Suitable conditions and methods are described in the Examples (see Examples 2 to 4).

[0122] Preferably, the conversion of the ketoreductase is determined at a substrate loading of 10% and a mutant or wild-type ketoreductase loading of 1% [w / w] (s / e 100) using a 2-propanol recovery system. In the present invention, the abbreviation "s / e" describes the term "substrate / enzyme". A loading of 1% [w / w] (s / e 100) further means that 100 g of substrate is used per 1 g of enzyme, i.e. substrate and enzyme are used in a ratio of 100 / 1.

[0123] In another preferred embodiment of the mutant ketoreductase of the first aspect, the mutant ketoreductase has an increased conversion relative to the wild-type ketoreductase at 10% substrate loading and 1% [w / w] (s / e 100) of the mutant or wild-type ketoreductase using a 2-propanol recovery system, in particular an increased conversion rate of at least 2.0, 5.0, 7.5 or 10 times.

[0124] In a preferred embodiment of the mutant ketoreductase of the first aspect, the mutant ketoreductase is capable of converting a prochiral ketone into a chiral alcohol.

[0125] In a further preferred embodiment, the prochiral ketone has the formula II

[0126]

[0127] Where R x For hydrogen, C 1-4 alkyl or halogen atom and the resulting chiral alcohol has the formula I

[0128]

[0129] Where Rx is hydrogen, C 1-4 an alkyl group or a halogen atom.

[0130] Helical Key

[0131]

[0132] represent Thus indicating the chirality of the molecule.

[0133] Suitable C 1-4 The alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, preferably from methyl.

[0134] Suitable halogen atoms are fluorine, chlorine, bromine and iodine, but preferably bromine.

[0135] The mutant ketoreductase can in principle catalyze the formation of both the S-enantiomer and the R-enantiomer of a chiral alcohol, in particular a chiral alcohol of formula I.

[0136] In a preferred embodiment, the mutant ketoreductase catalyzes the formation of the S-enantiomer of the chiral alcohol of Formula Ia

[0137]

[0138] Where R x For hydrogen, C 1-4 An alkyl or halogen atom, more preferably catalyzes the formation of a chiral alcohol of formula Ib

[0139]

[0140] The enantiomeric excess of the S-enantiomer of the chiral alcohol may be at least 95%, 96%, 97%, 98% or 99%.

[0141] In addition, the mutant ketoreductase according to the first aspect of the present invention can also be combined with another peptide or protein to form a fusion protein. Therefore, the present invention further relates to a fusion protein comprising the mutant ketoreductase of the present invention.

[0142] Fusion protein can further comprise label. Label is attached to protein for various purposes, for example, for the convenience of purification, to help the correct folding of protein, to prevent protein precipitation, to change chromatographic properties, to modify protein or to identify or label protein. Currently known to exist many (affinity) labels or (affinity) markers. Commonly used labels include Arg label, His label, Strep label, Flag label, T7 label, S label, HAT label, GST label and MBP label.

[0143] In a second aspect, the present invention relates to a nucleic acid encoding a mutant ketoreductase according to the first aspect of the invention. Thus, the present invention may also relate to a nucleic acid encoding a fusion protein comprising a mutant ketoreductase according to the first aspect of the invention.

[0144] As used herein, the term "nucleic acid" generally refers to any nucleotide molecule encoding a 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 kind of DNA and / or RNA fragments, which may be isolated by standard molecular biology procedures, including, for example, ion exchange chromatography. The nucleic acids of the present invention may be used for transfection or transduction of specific cells or organisms.

[0145] Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA or cRNA, or in the form of DNA, including, for example, cDNA and genomic DNA, for example, obtained by cloning or produced by chemical synthesis technology or by a combination thereof. DNA can be triple-stranded, double-stranded or single-stranded. Single-stranded DNA can be a coding strand, also referred to as a sense strand, or can be a non-coding strand, also referred to as an antisense strand. As used herein, nucleic acid molecules, among others, also refer to single-stranded and double-stranded DNA, DNA for a mixture of single-stranded and double-stranded RNA, and RNA for a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA, which hybrid molecules can be single-stranded, or more typically double-stranded, or triple-stranded, or a mixture of single-stranded and double-stranded regions. In addition, as used herein, nucleic acid molecules refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA.

[0146] In addition, nucleic acid may contain one or more modified bases. Such nucleic acids may also contain modifications, such as in the ribose phosphate backbone, to increase the stability and half-life of such molecules in a physiological environment. Therefore, due to stability or due to other reasons, the DNA or RNA whose backbone is modified is a "nucleic acid molecule", as referred to herein by this feature. In addition, to give only two examples, DNA or RNA comprising abnormal bases such as inosine or modified bases such as trityl bases are nucleic acid molecules in the context of the present invention. It should be understood that various modifications have been carried out to DNA and RNA, which are used for many useful purposes known to those skilled in the art. The term "nucleic acid molecule" as used herein includes such chemical, enzymatic or metabolically modified forms of nucleic acid molecules, and DNA and RNA chemical forms unique to viruses and cells (especially including simple and complex cells).

[0147] Furthermore, nucleic acid molecules encoding mutant ketoreductases of the 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.

[0148] Nucleic acids of the invention may be initially formed in vitro or in cultured cells, typically by manipulation of nucleic acids by endonucleases and / or exonucleases and / or polymerases and / or ligases and / or recombinases or other methods of producing nucleic acids known to those skilled in the art.

[0149] The nucleic acid of the present invention may be contained in an expression vector, wherein the nucleic acid is operably linked to a promoter sequence capable of promoting expression of the nucleic acid in a host cell.

[0150] In a preferred embodiment of the nucleic acid encoding the mutant ketoreductase of the first aspect, 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%, in particular 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2 to 17. Preferably, the nucleic acid has or comprises the sequence of any one of SEQ ID NOs: 18 to 34.

[0151] In a third aspect, the present invention relates to a vector comprising a nucleic acid according to the second aspect of the 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 invention.

[0152] As used herein, the term "vector" generally refers to any type of nucleic acid molecule that can be used to express a target protein in a cell (see also above for details on the nucleic acids of the present invention). In particular, the vector of the present invention may be any plasmid or vector known to those skilled in the art, which is suitable for expressing proteins in specific host cells, including but not limited to mammalian cells, bacterial cells and yeast cells. The vector of the present invention may also be a nucleic acid encoding a mutant ketoreductase of the present invention and used for subsequent cloning into a corresponding vector to ensure expression. Plasmids and vectors for protein expression are well known in the art and are commercially available from different suppliers, including, for example, Promega (Madison, WI, USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, CA, 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, 3rd edition.

[0153] The vector may further include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication, one or more therapeutic genes and / or selective marker genes, and other genetic elements known in the art, such as regulatory factors that direct the transcription, translation, and / or secretion of the encoded protein. The vector may be used to transduce, transform, or infect cells so that the cells express nucleic acids and / or proteins other than the cell's native nucleic acids and / or proteins. The vector optionally includes materials that contribute to achieving the entry of nucleic acids into cells, such as viral particles, liposomes, protein coatings, etc. Various types of suitable expression vectors are known in the art for protein expression by standard molecular biology techniques. Such vectors are selected from conventional vector types, including insects, such as baculovirus expression, or yeast, fungi, bacteria, or viral expression systems. Many of these types are other suitable vectors known in the art that may also be used for this purpose. Methods for obtaining such vectors are well known (see, e.g., Sambrook et al., supra).

[0154] As described above, the nucleic acid encoding the mutant ketoreductase of the present invention is operably linked to a sequence suitable for driving the expression of the protein in the host cell to ensure the expression of the protein. However, the vectors included in the present invention may represent intermediates, which are then cloned into a suitable vector to ensure the expression of the protein. The vectors of the present invention may further comprise all kinds of nucleic acid sequences, including but not limited to polyadenylation signals, splice donor and splice acceptor signals, insertion sequences, transcription enhancer sequences, translation enhancer sequences, drug resistance genes, etc. Optionally, the drug resistance gene can be operably linked to an internal ribosome entry site (IRES), which can be cell cycle specific or cell cycle independent.

[0155] As used herein, the term "operably linked" generally means that genetic elements are arranged so that they function in concert for their intended purpose, for example, because transcription is initiated by a promoter and carried out by a DNA sequence encoding a mutant ketoreductase of the present invention. That is, RNA polymerase transcribes the sequence encoding the mutant ketoreductase into mRNA, which is then spliced ​​and translated into protein.

[0156] The term "promoter sequence" as used in the context of the present invention generally refers to any type of regulatory DNA sequence that is operably linked to a downstream coding sequence, wherein the promoter is capable of binding RNA polymerase and initiating transcription of an open reading frame encoded in a 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.

[0157] In addition, the present invention also includes a host cell comprising the mutant ketoreductase of the present invention or a fusion protein thereof, the nucleic acid of the second aspect of the present invention, or the vector of the third aspect of the present invention.

[0158] The "host cell" of the present invention can be any kind of organism suitable for use in recombinant DNA technology, including but not limited to all kinds of bacteria and yeast strains suitable for expressing one or more recombinant proteins. Examples of host cells include, for example, various Bacillus subtilis or Escherichia coli strains. A variety of Escherichia coli bacterial host cells are known to those skilled in the art, including but not limited to strains such as DH5-α, HB101, MV1190, JM109, JM101 or XL-1 blue, which are commercially available from different suppliers, including, for example, Stratagene (CA, USA), Promega (WI, USA) or Qiagen (Hilden, Germany). Particularly suitable host cells, i.e., Escherichia coli BL21 (DE3) cells, are also described in the examples. 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).

[0159] The cultivation of host cells according to the present invention is a routine 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 a suitable host cell to produce the corresponding protein by recombinant means. The host cell can be any kind of suitable cell, preferably a bacterial cell such as Escherichia coli, which can be cultured in culture. In the first step, the method may include cloning the corresponding gene into a suitable vector, such as a vector according to the 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 transiently infiltrated with DNA. After the protein is expressed in the corresponding host cell, the cells can be harvested and used as a starting material for preparing a cell extract containing the target protein. A cell extract containing the target protein is obtained by lysing the cells. Methods for preparing cell extracts by means of chemical or mechanical cell lysis are well known to those skilled in the art, and include, but are not limited to, for example, hypotonic salt treatment, homogenization or ultrasonic treatment.

[0160] In a fourth aspect, the present invention relates to a method for the enzymatic reduction of a prochiral ketone and the formation of a chiral alcohol in the presence of a mutant ketoreductase of the present invention.

[0161] In a preferred aspect, the prochiral ketone has formula II

[0162]

[0163] Where R x For hydrogen, C 1-4 alkyl or halogen atom and the resulting chiral alcohol has the formula I

[0164]

[0165] Where R x For hydrogen, C 1-4 an alkyl group or a halogen atom.

[0166] In a further preferred embodiment, the S-enantiomer of the chiral alcohol of formula Ia is prepared,

[0167]

[0168] Where R x For hydrogen, C 1-4 an alkyl group or a halogen atom, more preferably the S-enantiomer of a chiral alcohol of formula Ib,

[0169]

[0170] 2-Bromo-1-(4-nitro-phenyl)ethanone is a ketone of formula II which is particularly useful.

[0171] The enzymatic reduction of the mutant ketoreductases generally occurs in the presence of NADP as cofactor, which is regenerated in situ.

[0172] The oxidized cofactor is usually continuously regenerated with a secondary alcohol as 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, preferably 2-propanol.

[0173] Preferably, the cofactor is regenerated with the aid of the cosubstrate in the presence of the same enzyme which also catalyzes the target reaction. In a further preferred embodiment, the acetone formed when 2-propanol is used as cosubstrate can be removed continuously from the reaction mixture.

[0174] The cofactor loading, ie the ratio of substrate (prochiral ketone) to cofactor (s / c) can vary between 10 and 3000, preferably between 50 and 1000, most preferably between 100 and 500.

[0175] In a particular embodiment of the invention, the enzymatic reduction is carried out in the presence of a cosubstrate, ie preferably in the presence of 2-propanol, in an aqueous buffered medium. The concentration of the cosubstrate is typically in the range of 5%v to 40%v.

[0176] Suitable buffers can be chosen from acidic to neutral buffers such as 2-morpholine-4-ethanesulfonic acid-, ammonium acetate, acetate, phosphate, 1,4-piperazine dimethanesulfonic acid, which allow the pH of the reaction to be kept in the range between pH 5.2 and pH 7.2.

[0177] 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 substrate to enzyme ratio (s / e) can be selected between 25 and 200, preferably between 100 and 200.

[0178] The reaction temperature is usually kept between 20°C and 50°C, preferably between 25°C and 45°C.

[0179] After termination of the reaction, the chiral alcohol obtained can be processed conventionally by extraction or preferably by filtration.

[0180] In a fifth aspect, the present invention relates to the use of the enzymatic reduction of prochiral ketones and the formation of chiral alcohols in the presence of mutant ketoreductases in the synthesis of morpholine compounds of the formula

[0181]

[0182] Where R 1 is aryl or heteroaryl, wherein the aromatic ring is optionally substituted by one or two C 1-7 -substituted with an alkyl substituent.

[0183] The term "C 1-7 "-alkyl" refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to six carbon atoms, preferably one to four, more preferably one to two carbon atoms. The term is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl or tert-butyl, pentyl and its isomers, hexyl and its isomers and heptyl and its isomers.

[0184] The term "aryl" relates to an aromatic carbon ring, such as a benzene ring or a naphthalene ring, preferably a benzene ring.

[0185] The term "heteroaryl" refers to an aromatic 5- to 6-membered monocyclic or 9- to 10-membered bicyclic ring which may contain 1, 2 or 3 heteroatoms selected from nitrogen, oxygen and / or sulfur, such as pyridyl, pyrazolyl, pyrimidinyl, benzimidazolyl, quinolinyl and isoquinolinyl.

[0186] Preferably, R 1 It is composed of two C 1-7 -alkyl, more preferably two C 1-2 -heteroaryl substituted with an alkyl substituent, more preferably pyrazolyl.

[0187] Even more preferably, the morpholino compound is a TAARI clinical candidate lamironte having the formula X

[0188]

[0189] As described in PCT Publication WO 2017 / 157873, Scheme 1, page 5, the synthesis of lamiron can be carried out as follows:

[0190]

[0191] The use according to the fifth aspect of the present invention relates to the preparation of 2-(4-aminophenyl)morpholine intermediate 2.

[0192] Intermediate 2 is a chiral 2-(4-aminophenyl)morpholine of the formula

[0193]

[0194] Where R 2 It is a Boc amino protecting group.

[0195] The preparation of intermediate 2 can be accomplished according to the method disclosed in PCT Publication WO 2015 / 086495, wherein according to the fourth aspect of the present invention, the enzymatic reduction in step a) is replaced by the enzymatic reduction of the prochiral ketone and the formation of the chiral alcohol in the presence of a mutant ketoreductase. The subsequent synthetic steps b) to e) can follow the disclosure in PCT Publication WO 2015 / 086495.

[0196] Regarding the use of the present invention, the terms, examples and specific embodiments used in the context of other aspects of the present disclosure are mentioned, which also apply to this aspect. In particular, the mutant ketoreductase or its fusion protein according to the present invention can be used as detailed in relation to the method of the present invention.

[0197] Unless otherwise defined, all technical and scientific terms and any abbreviations used herein have the same meaning as commonly understood by those skilled in the art in the field of the present invention. Definitions of commonly used 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).

[0198] The present invention is not limited to the particular methods, protocols and reagents described herein, as they are variable. Although any methods and materials similar or equivalent to those disclosed herein can be used in the practice of the present invention, preferred methods and materials are disclosed herein. Further, the terms used herein are only used for the purpose of disclosing specific embodiments, rather than attempting to limit the scope of the present invention.

[0199] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the words "include," "comprise," and "comprising" are to be construed as inclusive rather than exclusive. Likewise, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plurality" means two or more.

[0200] The following drawings and examples are intended to illustrate various embodiments of the present invention. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present invention. For those skilled in the art, it is apparent that various equivalent forms, changes and modifications can be made without departing from the scope of the present invention, and therefore it should be understood that such equivalent embodiments will be included herein.

[0201] Examples

[0202] 1 General Procedure

[0203] 1.1 Biocatalyst production

[0204] In order to obtain the ketoreductase gene and construct an expression vector, a ketoreductase (KRED) open reading frame for expression in Escherichia coli (E. coli) was designed and synthesized based on the reported amino acid sequence of ketoreductase and the desired mutant sequence (provided in the section sequence), and the codon optimization algorithm of Twist Bioscience (South San Francisco, USA). A stop codon was added at the end in all cases. Restriction sites for subsequent cloning in the target vector pET-29b (+) were added to the nucleotide sequence; NdeI restrictions were added at the 5' end, and XhoI restriction sequences were added at the 3' end. The vector contains the coding sequence for kanamycin resistance (Kmr gene). According to the cloning strategy, expression is controlled by the lac promoter. The resulting plasmid was transformed into E. coli BL21 (DE3) using standard methods. The sequences of the codon-optimized genes and the encoded polypeptides are provided in the section "Sequence".

[0205] 1.2 Ketoreductase production

[0206] Plasmids from Twist Bioscience were resuspended in sterile water. Inoculation in E. coli BL21 (DE3) cells was achieved by thermal heating (42°C for 45 s).

[0207] The preculture was incubated overnight on Luria Bertani medium agar plates containing 25 μg / mL kanamycin at 37° C. According to the protocol, a single microbial colony was picked and incubated overnight.

[0208] Add the Terrific broth containing 25 μ g / mL kanamycin to the culture. After 3.5 hours of incubation at 28°C, add isopropyl βD-thiogalactoside (IPTG) to induce the expression of KRED at a final concentration of 1 mM. Continue to incubate overnight at 28°C. Harvest cells via centrifugation (3220 rcf, 45 min, 4°C), and discard the supernatant. Resuspend the cells 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 incubate for 60 min. Then they are centrifuged (3220 rcf, 45 min, 4°C), and the lysate is frozen and stored at -20°C.

[0209] 2 Biocatalysis

[0210] 2.1 Process Development

[0211] The enzymatic reduction takes place in a reaction mixture of buffer (e.g. 2-morpholin-4-ylethylsulfonate MES; 0.5 M stock solution, pH 6.5 is used) and 2-propanol (final reducing agent) at a defined temperature (23°C–45°C). Buffer and 2-propanol (5 vol%–40 vol%) were varied among experiments, and for reactions with substrate loadings above 1%, the 2-PrOH concentration was at least 20%. The loading of ketoreductase and cofactor nicotinamide adenine dinucleotide phosphate cofactor (NADP) was defined in dependence on the substrate loading. Substrate was added at variable concentrations ranging from 1 wt% to 20 wt%. Enzyme loading varied between experiments, corresponding to substrate to enzyme ratios (s / e) between 33 and 200. Cofactor loading varied between experiments, corresponding to substrate to cofactor ratios (s / c) between 10 and 1000.

[0212] 2.2 Analysis methods

[0213] Achiral HPLC Method (IPC)

[0214] After 18 hours, the production of alcohol was measured by HPLC analysis on a C18 XP column (3.0 x 75 mm, 2.5 μm particle size at 50° C. and 311 bar). Phase A contained 5% acetonitrile and 0.1% formic acid in water; phase B contained acetonitrile and 0.1% formic acid. Flow rate 1 ml / min; 90% phase A at time 0, 60% at 7 minutes, and 90% at 7.5 minutes. The detection wavelength was 280 nm.

[0215] For sample preparation, samples were diluted in acetonitrile / water (4:1) to a concentration of 1 mg / ml and a total injection volume of 1 μl. The retention times of the educt were 4.89'; 3.65' for the product; and 3.46' for the epoxide. The conversion was calculated as the ratio of the product peak area to the total peak area.

[0216] Chiral HPLC Method (OP)

[0217] The enantiomeric excess of the product compound was measured by chiral analysis on an IE-3 column (4.6 x 150 mm, 3 μm particle size at 40° C. and 250 bar). Phase A contained 5% acetonitrile in water; phase B contained water, ethanol and isopropanol in a ratio of 30:35:35. The flow rate was 0.7 ml / min; 50% phase A at 30 minutes. The detection wavelength was 264 nm.

[0218] For sample preparation, the sample was diluted in ethanol to a concentration of 1 mg / ml and the total injection volume was 5 μl. The retention time of the S product was 8.8'; the R product was 9.7'; the epoxide was 15.8', and the educt was 24.8. The enantiomeric excess was calculated as the ratio of the product R peak area minus the product S peak area divided by the sum of the two peak areas.

[0219] 3. Mutant design for improving the KRED specific activity of keto I

[0220] The specific activities of the displayed mutants were determined according to the reaction conditions described in Section 2. These positions were identified in the Lactobacillus brevis R-specific alcohol dehydrogenase (Uniprot ID Q84EX5, PDB structure 1ZK4) by structural analysis. Positions near substrates and cofactors were selected for mutation.

[0221] The respective conversion of wild type (WT) activity is reported in Table 1 and is defined as the parent (under given conditions). Its fold improvement (FIOP) over the parent is 1. For mutants, the FIOP is reported, with the wild type as reference (parent).

[0222] FIOP is calculated as follows:

[0223]

[0224] ·C mut % conversion area (activity) achieved by the mutant protein used; C WT One of the WT-ketoreductases used

[0225] ·s / e mut The s / e (ratio of substrate to mutant protein) used for the experiment, and s / e WT s / e for experiments using WT-ketoreductase

[0226] · Reach C mut and C WT The required reaction time (t WT / t mut )

[0227] Ideally, FIOP determination requires comparison of similar conversion extent levels, which may require different substrate concentrations (c) and / or enzyme loadings (S / E values), related to the respective activities of the enzyme variants under the reaction conditions.

[0228] Table 1: Area % conversion and FIOP for the investigated ketoreductases (WT and single mutants) at 1% substrate loading and 33S / E loading.

[0229]

[0230] *Since the time of complete (100%) conversion has not yet been determined, FIOP is a given value at a minimum.

[0231] Two single mutants, T16S and E145L, showed nearly 2-fold improvement compared to the parent (here wild type). Mutant E145L showed complete conversion at the time of reaction quenching, indicating that the conversion was completed earlier in time.

[0232] Table 2: Area % conversion and FIOP for the investigated ketoreductases (WT and single or double mutants) at different S / E loadings at 1% substrate concentration.

[0233]

[0234]

[0235] **FIOP for T16S was taken from the experiment with Lyophylisate (Table 1) - others were calculated on this basis according to Equation 1.

[0236] Table 2 shows that positions 16, 141, 144, 145, 199 were further studied 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.

[0237] Table 3 Conversion (area %) and FIOP of the investigated ketoreductases (WT and triple or quadruple mutants) at different S / E loadings at 1% substrate concentration.

[0238] Ketoreductase at c = 1% Enzyme loading 17h conversion area% FIOP ee[%] I144A_E145L_L199M_A202L s / e 66 32 1.9 WT s / e 33 33.72 1 I144A_E145L_L199S_A202L s / e 66 28.8 1.7 I144A_E145L_L199M_A202L s / e 66 32 1.9 E145L_L199N_A202L s / e 66 45.3 2.7 I144A_E145L_L199N_A202I s / e 66 48.8 2.9 I144A_E145L_L199M_A202I s / e 66 60.9 3.6 E145L_L199S_A202L s / e 66 63.5 3.8 E145L_L199M_A202L s / e 66 84.4 5.0 E145L_L199M_A202I s / e 66 100* >5.9 E145L_L199S_A202I s / e 66 100* >5.9 99.9 M141I_E145L_L199S_A202I s / e 66 100* >5.9 99.9 E145L_L199N_A202I s / e 66 100* >5.9 100

[0239] *Since the time of complete (100%) conversion has not yet been determined, FIOP is a given value at a minimum.

[0240] Table 3 illustrates further investigation of combinations of mutants from Table 2. Preferred mutants are reported in Table 3 and include the combination of M141I, I144A, E145L, L199M / N / S, A202I / L.

[0241] Table 4 Conversion (area %) and FIOP of the investigated ketoreductases (WT, single, triple or quadruple mutants) at 10% substrate concentration.

[0242]

[0243] Table 4 illustrates that the effective mutants from Table 3 were successfully tested at higher substrate concentrations and high substrate to enzyme loadings, ie, under technical scale conditions.

[0244] 4 Characterization of mutant ketoreductase

[0245] 4.1 General Screening Procedure – 1% [w / v] Substrate Loading

[0246] 10 mg (0.1 mmol) of 2-bromo-1-(4-nitrophenyl)ethanone was mixed in a mixture of 100 μl MES-buffer pH 6.5 (0.5 M), 650 μl water, 200 μl 2-propanol and 20 μl magnesium bromide hexahydrate (0.1 M). The reaction was started by adding 200 μg NADP and 100 μg ketoreductase at room temperature. After about 1 d, the reaction conversion was determined by non-chiral HPLC method (IPC) and chiral HPLC method (OP).

[0247] 4.2 General Screening Procedure – 5 Percent [w / v] Substrate Loading

[0248] 50 mg (0.2 mmol) 2-bromo-1-(4-nitrophenyl)ethanone was mixed in a mixture of 100 μl MES-buffer pH 6.5 (0.5 M), 650 μl water, 355 μl 2-propanol and 20 μl magnesium bromide hexahydrate (0.1 M). The reaction was started by adding 1.0 mg NADP and 250 μg ketoreductase at room temperature. After 4 h and about 21 h, the reaction conversion was determined by achiral HPLC method (IPC) and chiral HPLC method (OP).

[0249] 4.3 Temperature stability screening – 5% [w / v] substrate loading

[0250] At different temperatures between 25°C and 45°C, 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) (see Table 5). After incubation for 16.5 h under vibration (1000 rpm), the reaction was started by adding 50 mg (0.2 mmol) of 2-bromo-1-(4-nitrophenyl) ethanone dissolved in 0.4 ml of 2-propanol. After about 4 h and 23 h, the reaction conversion was determined by non-chiral HPLC method (IPC). All results are summarized in Table 5.

[0251] Table 5: Temperature stability screening of the investigated ketoreductases.

[0252]

[0253] Table 5 illustrates that despite different initial activities (4 hour time point), the three selected mutants showed similar potency at the 23 hour time point at temperatures of 25°C and 30°C. The quadruple mutant showed the best potency at 35°C.

[0254] 4.4 Scale-up – 10% [w / v] substrate loading

[0255] Under stirring, 3g (12.3mmol) 2-bromo-1-(4-nitrophenyl) ethanone was suspended in a mixture of 3ml MES-buffer pH 6.5 (0.5M), 11.4ml water, 12ml 2-propanol and 0.6ml magnesium bromide hexahydrate (0.1M). The reaction was started by adding 30mg NADP and 15mg ketoreductase at room temperature. The final pH was 6.1 when the reaction was completed. After about 4h and 21h, the reaction conversion was determined by non-chiral HPLC method (IPC), and the enantiomeric excess was determined by chiral HPLC method (OP) after 21h. All results are summarized in Table 6.

[0256] Table 6: Characterization of the ketoreductases studied at 10% [w / v] substrate loading in a large-scale process.

[0257]

[0258] Table 6 illustrates that in the larger scale experiment, the three selected mutants showed complete transformation after 21 hours.

[0259] 4.5 Scale-up – 20 percent [w / v] substrate loading

[0260] Under stirring, 6g (24.6mmol) 2-bromo-1-(4-nitrophenyl) ethanone is suspended in a mixture of 3ml MES-buffer pH 6.5 (0.5M), 8.4ml water, 12ml 2-propanol (final reducing agent) and 0.6ml magnesium bromide hexahydrate (0.1M). The reaction is started by adding 60mg NADP and 30mg ketoreductase (see Table 7) at room temperature. The final pH is 5.8 when the reaction is completed. After 4h, 21h and 2d, the reaction conversion is determined by non-chiral HPLC method (IPC), and after 2d, the enantiomeric excess is determined by chiral HPLC method (OP). All results are summarized in Table 7.

[0261] Table 7: Characterization of the ketoreductases studied at 20% [w / v] substrate loading in a large-scale process.

[0262]

[0263]

[0264] Table 7 shows that complete conversion of the product and excellent enantiomeric excess can also be achieved within 2 days at 20% substrate loading.

[0265] sequence

[0266] Amino acid sequence

[0267] >Lactobacillus brevis ATCC 14869 ketoreductase, known as Q84EX5 in UniProtKB

[0268] (SEQ ID NO:1)

[0269] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSIEGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGAEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0270] >Q84EX5_E145L_A202L (SEQ ID NO:2)

[0271] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0272] >Q84EX5_E145L_A202C (SEQ ID NO:3)

[0273] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGCEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0274] >Q84EX5_E145L_A202E (SEQ ID NO:4)

[0275] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGEEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0276] >Q84EX5_E145L_A202I (SEQ ID NO:5)

[0277] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0278] >Q84EX5_E145L_A202T (SEQ ID NO:6)

[0279] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDQGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDLPGTEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0280] >Q84EX5_E145L_L199M_A202I (SEQ ID NO:7)

[0281] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0282] >Q84EX5_E145L_L199M_A202L (SEQ ID NO:8)

[0283] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0284] >Q84EX5_E145L_L199N_A202L (SEQ ID NO:9)

[0285] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDNPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0286] >Q84EX5_E145L_L199N_A202I (SEQ ID NO:10)

[0287] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDNPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0288] >Q84EX5_E145L_L199S_A202L (SEQ ID NO:11)

[0289] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0290] >Q84EX5_E145L_L199S_A202I (SEQ ID NO:12)

[0291] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0292] >Q84EX5_M141I_E145L_L199S_A202I (SEQ ID NO:13)

[0293] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINISSILGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0294] >Q84EX5_I144A_E145L_L199M_A202I (SEQ ID NO:14)

[0295] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0296] >Q84EX5_I144A_E145L_L199N_A202I (SEQ ID NO:15)

[0297] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDNPGIEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0298] >Q84EX5_I144A_E145L_L199M_A202L (SEQ ID NO:16)

[0299] MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDMPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ

[0300] >Q84EX5_I144A_E145L_L199S_A202L (SEQ ID NO:17)MSNRLDGKVAIITGGTLGIGLAIATKFVEEGAKVMITGRHSDVGEKAAKSVGTPDQIQFFQHDSSDEDGWTKLFDATEKAFGPVSTLVNNAGIAVNKSVEETTTAEWRKLLAVNLDGVFFGTRLGIQRMKNKGLGASIINMSSALGFVGDPSLGAYNASKGAVRIMSKSAALDCALKDYDVRVNTVHPGYIKTPLVDDSPGLEEAMSQRTKTPMGHIGEPNDIAYICVYLASNESKFATGSEFVVDGGYTAQ Nucleic acid sequence

[0301] >Lactobacillus brevis ATCC 14869 ketoreductase, known as Q84EX5 in UniProtKB

[0302] (SEQ ID NO:18)

[0303] ATGAGCAACCGTCTGGACGGCAAGGTGGCGATCATTACCGGTGGCACCCTGGGTATTGGTCTGGCGATTGCGACCAAGTTCGTGGAGGAAGGTGCGAAAGTTATGATCACCGGCCGTCACAGCGACGTGGGCGAGAAGGCGGCGAAAAGCGTTGGCACCCCGGACCAGATTCAATTCTTTCAGCACGATAGCAGCGACGAGGATGGTTGGACCAAGCTGTTCGATGCGACCGAAAAAGCGTTTGGCCCGGTTAGCACCCTGGTTAACAACGCGGGTATTGCGGTGAACAAGAGCGTTGAGGAAACCACCACCGCGGAGTGGCGTAAACTGCTGGCGGTGAACCTGGATGGTGTTTTCTTTGGCACCCGTCTGGGTATCCAACGTATGAAGAACAAAGGTCTGGGCGCGAGCATCATTAACATGAGCAGCATTGAAGGTTTCGTTGGCGACCCGAGCCTGGGTGCGTACAACGCGAGCAAGGGTGCGGTTCGTATCATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAGGACTACGATGTGCGTGTTAACACCGTGCACCCGGGCTATATTAAAACCCCGCTGGTTGACGATCTGCCGGGTGCGGAGGAAGCGATGAGCCAGCGTACCAAGACCCCGATGGGTCACATCGGCGAACCGAACGACATCGCGTACATTTGCGTTTATCTGGCGAGCAACGAGAGCAAATTCGCGACCGGTAGCGAATTTGTGGTTGATGGTGGCTATACCGCGCAATAA

[0304] >Q84EX5_E145L_A202L (SEQ ID NO:19) ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCCTGGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0305] >Q84EX5_E145L_A202C (SEQ ID NO:20)

[0306] ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCTGCGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0307] >Q84EX5_E145L_A202E (SEQ ID NO:21)

[0308] ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCGAAGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0309] >Q84EX5_E145L_A202I (SEQ ID NO:22)

[0310] ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCATTGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0311] >Q84EX5_E145L_A202T (SEQ ID NO:23)

[0312] ATGTCCAATCGCTTGGACGGGAAGGTTGCGATTATTACCGGTGGCACCCTGGGCATCGGCCTGGCGATCGCTACTAAATTTGTGGAAGAAGGTGCCAAGGTCATGATTACCGGCCGTCACAGCGATGTAGGCGAAAAAGCAGCAAAGTCCGTCGGGACCCCTGATCAGATTCAATTCTTTCAACACGATTCGAGCGACGAGGATGGATGGACTAAATTGTTTGATGCCACCGAAAAGGCATTCGGTCCTGTAAGTACCTTGGTCAACAATGCAGGCATCGCTGTAAACAAAAGCGTCGAGGAGACTACTACGGCAGAATGGCGCAAACTTCTGGCCGTCAACTTGGACGGCGTTTTTTTTGGCACGCGTCTGGGCATTCAACGTATGAAAAACAAAGGTTTGGGAGCGTCCATCATCAATATGAGCAGCATCCTTGGATTCGTAGGGGACCCGTCGCTGGGTGCATACAACGCCTCGAAAGGGGCGGTGCGCATTATGTCAAAAAGCGCGGCCCTGGACTGTGCCTTAAAAGATTATGATGTACGCGTGAACACAGTTCATCCCGGTTACATTAAAACCCCGCTTGTCGATGATCTCCCCGGCACCGAGGAAGCGATGTCTCAGCGCACCAAAACGCCGATGGGCCACATTGGCGAACCTAACGATATCGCATATATTTGCGTTTACCTGGCAAGCAATGAATCTAAATTTGCGACCGGCTCAGAGTTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0313] >Q84EX5_E145L_L199M_A202I (SEQ ID NO:24)

[0314] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0315] >Q84EX5_E145L_L199M_A202L (SEQ ID NO:25)

[0316] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0317] >Q84EX5_E145L_L199N_A202L (SEQ ID NO:26)

[0318] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAACCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0319] >Q84EX5_E145L_L199N_A202I (SEQ ID NO:27)

[0320] ATGAGCAATCGTCTGGATGGAAAGGTAGCAATTATTACCGGCGGGACTCTGGGCATTGGACTCGCGATTGCGACAAAATTCGTGGAAGAAGGCGCGAAAGTGATGATTACGGGTCGCCATTCGGACGTAGGGGAAAAAGCTGCGAAAAGTGTTGGCACTCCGGACCAGATTCAGTTTTTTCAACATGATTCCTCCGATGAGGATGGCTGGACGAAATTATTCGACGCGACCGAAAAAGCATTTGGGCCGGTCTCAACATTGGTCAATAATGCTGGCATCGCCGTCAATAAATCTGTCGAAGAAACCACCACCGCTGAATGGCGCAAACTGCTGGCCGTCAATCTGGATGGCGTTTTCTTTGGTACGCGGCTCGGGATTCAGCGGATGAAGAACAAAGGGCTGGGGGCAAGTATCATTAATATGTCGAGCATCCTTGGGTTTGTCGGCGACCCCTCATTAGGGGCCTACAACGCTAGCAAAGGTGCCGTACGCATCATGAGCAAATCTGCGGCGTTGGACTGCGCCCTGAAAGATTACGATGTGCGCGTTAATACCGTCCATCCGGGTTATATTAAAACGCCGTTGGTAGATGATAACCCAGGTATCGAGGAAGCAATGTCCCAGCGCACCAAAACCCCAATGGGACATATTGGCGAACCGAACGATATTGCCTATATTTGTGTATACCTGGCGTCAAATGAGTCTAAATTTGCGACGGGGAGCGAATTTGTGGTAGATGGCGGCTACACCGCGCAATAA

[0321] >Q84EX5_E145L_L199S_A202L (SEQ ID NO:28)

[0322] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0323] >Q84EX5_E145L_L199S_A202I (SEQ ID NO:29)

[0324] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCATTGCGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0325] >Q84EX5_M141I_E145L_L199S_A202I (SEQ ID NO:30)

[0326] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATTAGCAGCATTCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0327] >Q84EX5_I144A_E145L_L199M_A202I (SEQ ID NO:31)

[0328] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0329] >Q84EX5_I144A_E145L_L199N_A202I (SEQ ID NO:32)

[0330] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAACCCGGGCATTGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0331] >Q84EX5_I144A_E145L_L199M_A202L (SEQ ID NO:33)

[0332] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATATGCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

[0333] >Q84EX5_I144A_E145L_L199S_A202L (SEQ ID NO:34)

[0334] ATGAGCAACCGCCTGGATGGCAAAGTGGCGATTATTACCGGCGGCACCCTGGGCATTGGCCTGGCGATTGCGACCAAATTTGTGGAAGAAGGCGCGAAAGTGATGATTACCGGCCGCCATAGCGATGTGGGCGAAAAAGCGGCGAAAAGCGTGGGCACCCCGGATCAGATTCAGTTTTTTCAGCATGATAGCAGCGATGAAGATGGCTGGACCAAACTGTTTGATGCGACCGAAAAAGCGTTTGGCCCGGTGAGCACCCTGGTGAACAACGCGGGCATTGCGGTGAACAAAAGCGTGGAAGAAACCACCACCGCGGAATGGCGCAAACTGCTGGCGGTGAACCTGGATGGCGTGTTTTTTGGCACCCGCCTGGGCATTCAGCGCATGAAAAACAAAGGCCTGGGCGCGAGCATTATTAACATGAGCAGCGCGCTGGGCTTTGTGGGCGATCCAAGCTTGGGCGCGTATAACGCGAGCAAAGGCGCGGTGCGCATTATGAGCAAAAGCGCGGCGCTGGATTGCGCGCTGAAAGATTATGATGTGCGCGTGAACACCGTGCATCCGGGCTATATTAAAACCCCGCTGGTGGATGATAGCCCGGGCCTGGAAGAAGCGATGAGCCAGCGCACCAAAACCCCGATGGGCCATATTGGCGAACCGAACGATATTGCGTATATTTGCGTGTATCTGGCGAGCAACGAAAGCAAATTTGCGACCGGCAGCGAATTTGTGGTGGATGGCGGCTATACCGCGCAGTAA

Claims

1. A mutant ketoreductase having increased ketoreductase activity relative to a 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); and The mutant ketoreductase 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), 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).

2. The mutant ketoreductase according to claim 1, wherein - 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 with Leu (Leu202), Preferably, wherein 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 wherein 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 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).

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

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

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

6. The mutant ketoreductase according to any one of claims 1 to 5, wherein the mutant ketoreductase does not comprise a mutation 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 mutant ketoreductase according to any one of claims 1 to 6, wherein the mutant ketoreductase consists of or comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99%, in particular 100% identical to the amino acid sequence of any one of SEQ ID NOs: 2 to 14.

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

9. The mutant ketoreductase according to any one of claims 1 to 8, wherein the mutant ketoreductase has increased conversion relative to the wild-type ketoreductase at 10% substrate loading and at 1% [w / w] (s / e 100) loading of the mutant or wild-type ketoreductase using a 2-propanol recovery system, In particular, there is at least a 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 increased conversion.

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

11. The mutant ketoreductase according to claim 10, wherein the prochiral ketone has formula II Where R x For hydrogen, C 1-4 alkyl or halogen atom and the resulting chiral alcohol has the formula I Where R x For hydrogen, C 1-4 an alkyl group or a halogen atom.

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

13. A nucleic acid encoding the mutant 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 formation of a chiral alcohol in the presence of a mutant ketoreductase according to claims 1 to 12.

15. The method of claim 14, wherein the prochiral ketone has formula II Where R x For hydrogen, C 1-4 alkyl or halogen atom and the resulting chiral alcohol has the formula I Where R x For hydrogen, C 1-4 an alkyl group or a halogen atom.

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

17. Use of the method according to claims 14 to 16 for preparing a morpholine compound of the formula Where R 1 is aryl or heteroaryl, wherein the aromatic ring is optionally substituted by one or two C 1-7 -substituted with an alkyl substituent.

18. The use according to claim 17, wherein R 1 is pyrazolyl, which is 1-7 -substituted with an alkyl substituent.

19. The use according to claim 17 or 18, for preparing

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