Modified bacterial ketoreductase

Abstract

The invention relates to a modified bacterial ketoreductase of Lactobacillus kefir comprising mutations K55L, S102T, L116T, F153C / I, T158N and L205I and to a nucleic acid, an expression vector and a prokaryotic host cell comprising a polynucleotide sequence encoding said modified bacterial ketoreductase. The invention further relates to methods for producing the modified bacterial ketoreductase, or for the enantioselective reduction of the substrate ethyl 4-chloroacetoacetate (COBE) to the product S-4-chloro-3- hydroxybutyrate ((S)-CHBE) using said modified bacterial ketoreductase.

Description

Modified Bacterial KetoreductaseFIELD OF THE INVENTION

[0001] The invention relates to a modified bacterial ketoreductase of Lactobacillus kefir comprising mutations K55L, S102T, L116T, F153C / I, T158N and L205I or K55L, S102T, L116T, F153C / I, T158N, D203N, L205I and E206Q / K and to a nucleic acid, an expression vector and a prokaryotic host cell comprising a nucleic acid sequence encoding said modified bacterial ketoreductase. The invention further relates to methods for producing the modified bacterial ketoreductase, or for the enantioselective reduction of the substrate ethyl 4-chloroacetoacetate (COBE) to the product ethyl S-4-ch Io ro-3-hydroxy butyrate ((S)-CHBE) using said modified bacterial keto reductase.BACKGROUND OF THE INVENTION

[0002] Enantiomerically pure alcohols, such as ethyl S-4-chloro-3-hydroxy butyrate ((S)-CHBE), are important chiral intermediates in the synthesis of active pharmaceutical ingredients (APIs). These optically pure alcohols are commonly generated by biocatalytic stereoselective ketone reduction. In this regard, biocatalysis provides a powerful and competitive method to synthesize chiral chemicals and pharmaceuticals due to its excellent enantioselectivity, high catalytic efficiency, mild reaction conditions and environmentally friendly processes.

[0003] Ketoreductases (KREDs) are the most commonly used enzymes in industrial pharmaceutical synthesis. Ketoreductases are a subclass of the oxidoreductase family, which are capable of catalyzing redox reactions that facilitate the transfer of electrons from a donor molecule to an acceptor molecule. The subclass of ketoreductases can facilitate the reduction of a wide range of prochiral ketones to chemo- and stereoselective alcohols. During this process, electrons are transferred to the carbonyl group (C=O) of the ketone, resulting in the addition of a hydrogen atom to the carbon and another to the oxygen atom. This enzymatic process is coupled to a reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating enzyme, such as glucose dehydrogenase (GDH), formate dehydrogenase or the like.

[0004] Particularly, ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE) is an important raw material for the synthesis of active pharmaceutical ingredients. Compared with chemical synthesis, biocatalytic asymmetric synthesis of (S)-CHBE from ethyl 4-chloroacetoacetate (COBE) has many advantages e.g. high stereoselectivity and yield. KRED from Lactobacillus kefir (designated as L. kefir KRED) uses 2-propanol as a co-substrate to regenerate NADPH in situ and has recently been rationally designed to produceluliconazole chiral intermediate by kilogram-scale, showing great potential for industrial-scale biotransformation (Zheng et al., (2021), Mol. Catal. 509, 111639). An enantioselective triple mutant A94S / S96E / E145A of the Lactobacillus kefir keto reductase (L kefir KRED) showed an increased activity and catalytic efficiency compared to the corresponding wild-type (WT) ketoreductase in the specific biocatalytic production of (S)-2-chloro-1-(2,4-dichlorophenyl) ethanol ((S)-TCPE) with 2, 2’, 4’ -trichloroacetophenone (TCAP).

[0005] Different mutants of Lactobacillus kefir ketoreductase containing mutations A94F, Y190F or E145S, as well as the so-called Sph mutant containing 10 mutations, including G7S, A94T, S96P, R108H, G117S, E145S, N157T, P194N, M206Q, I223V were reported by Noey et al., (Proc. Natl. Acad. Sci. U.S.A, 2015, 112 (51), 7065-7072). The mutated enzymes were enantioselective, but less active than the wild-type enzyme in the reduction of 3-oxocyclopentanone and 3-thiocyclopentanone.

[0006] Further, WO 2009 / 046153 A1 discloses various engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme to produce 2S,3R-methyl-2-benzamidomethyl-3-hydroxybutyrate from methyl-2-benzamidomethyl-3- oxobutyrate.

[0007] Therefore, although different variants of ketoreductases from Lactobacillus kefir harboring mutations have been reported, there is still a need for L. kefir keto reductases with high substrate loading, high conversion, high stereoselectivity and high catalytic efficiency, particularly for the enantioselective enzymatic production of (S)-CHBE.SUMMARY OF THE INVENTION

[0008] The present invention demonstrates different variants of a bacterial ketoreductase from Lactobacillus kefir, harboring at least six and eight site-specific amino acid replacements that can be used in the highly enantioselective reduction of COBE to (S)-CHBE with a high conversion rate, a high chiral purity and a high catalytic efficiency, while also allowing a high substrate loading. These modified bacterial keto reductases are particularly advantageous as they allow the generation of almost enantiomerically pure (S)-CHBE at high product yield utilizing relatively low amounts of enzyme. The product ethyl S-4-chloro-3-hydroxy butyrate ((S)-CHBE), is an important chiral intermediate in the synthesis of 3-hydroxytetrahydrofuran (3-OH THF), which is an intermediate for APIs, such as amprenavir and fosamrenair.

[0009] In one aspect, the present invention relates to a modified bacterial ketoreductase comprising mutations in a bacterial ketoreductase comprising an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1, wherein the mutations comprise K55L, S102T, L116T, F153C / I, T158N and L205I. Preferably, the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 toat least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 13 and comprising amino acids 55L, 102T, 116T, 153C / I, 158N and 205I, preferably amino acids 55L, 102T, 116T, 153C, 158N and 205I.

[0010] In certain embodiments, the modified bacterial ketoreductase further comprises mutations D203N and E206Q or E206K (E206Q / K). Preferably, the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 15, or the modified bacterial ketoreductase comprises an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 15 and comprising amino acids 55L, 102T, 116T, 153C / I, 158N, 203N, 205I, and 206Q / K, preferably amino acids 55L, 102T, 116T, 1531, 158N, 203N, 2051, and 206Q.

[0011] In a further embodiment, the modified bacterial ketoreductase according to the invention further comprises one or more C-terminal and / or N-terminal tag(s), preferably an N-terminal tag, more preferably an N-terminal 6xHis tag.

[0012] In another further embodiment, the modified bacterial ketoreductase according to the invention has a conversion rate of > 90% of ethyl 4-chloroacetoacetate (COBE) to ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE) and 99.9% enantiomeric excess (ee) within 8 hours in a reaction mixture comprising 1.25 g / L modified bacterial keto reductase, 500 g / L COBE, 25% (v / v) isopropanol and 0.1 g / L NADP+in 0.1 M K2HPO4, pH 7.0 at 35°C.

[0013] In another aspect, the invention relates to a nucleic acid molecule comprising a sequence encoding the modified bacterial ketoreductase according to the invention, preferably wherein the sequence of the nucleic acid molecule is codon optimized.

[0014] In yet another aspect, the invention relates to an expression vector comprising a nucleic acid encoding the modified bacterial ketoreductase according to the invention or the nucleic acid molecule according to the invention, operably linked to a promoter, preferably an inducible promoter.

[0015] In yet another aspect, the invention relates to a prokaryotic host cell comprising a nucleic acid encoding the modified bacterial ketoreductase according to the invention, the nucleic acid molecule according to the invention or the expression vector according to the invention.

[0016] The invention further relates to a method of producing a modified bacterial ketoreductase comprising: a) introducing the nucleic acid molecule comprising a sequence encoding a modified bacterial ketoreductase according to the invention or the expression vector according to the invention into a prokaryotic host cell; b) culturing the prokaryotic host cell under conditions that allow producing the modified bacterial ketoreductase; c) lysing the prokaryotic host cell; and d) optionally purifying the modified bacterial ketoreductase. Particularly, the nucleic acid molecule or the expression vector is introduced into the prokaryotic host cell by transformation. The prokaryotic host cell is preferably Escherichia coli, preferably Escherichia coli BL21 or Escherichia coli DH5a, more preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5a.

[0017] In yet another aspect, the invention provides a method for the enantioselective reduction of the substrate ethyl 4-chloroacetoacetate (COBE) according to formula (I)to the product ethyl S-4-ch Io ro-3-hydroxy butyrate ((S)-CHBE) according to formula (II),wherein the method comprises incubating COBE with the modified bacterial ketoreductase according to the invention in the presence of the co-substrate isopropanol and the co-factor NADP+to obtain (S)-CHBE. Preferably, the product is present at an enantiomeric excess (ee) of greater than 99%.

[0018] In yet another aspect, the invention relates to a use of the modified bacterial ketoreductase according to the invention for the enantioselective reduction of ethyl 4-chloroacetoacetate (COBE) to ethyl S-4-chloro-3-hydroxy butyrate ((S)-CHBE).DESCRIPTION OF THE FIGURES

[0019] FIGURE 1 : Illustration of the expression plasmid map of KRED009.

[0020] FIGURE 2: SDS-PAGE of L. kefir KREDs M0-M3 samples in protein purification. S represents supernatant, P represents pellet, E represents purified enzyme protein.

[0021] FIGURE 3: SDS-PAGE of L. kefir KREDs M4-M7 samples in protein purification. S represents supernatant, P represents pellet, E represents purified enzyme protein.

[0022] FIGURE 4: Detection map (I PC chromatogram) of preparation for (S)-CHBE catalyzed by engineered L. kefir KREDs, wherein 11.9 minutes corresponds to the residual substrate COBE and 9.1 minutes corresponds to the product (S)-CHBE. The insert shows the zoomed in peak at 11.9 minutes corresponding to the substrate COBE.DETAILED DESCRIPTION

[0023] The term “comprises” or “comprising” means “including, but not limited to”. The term is intended to be open-ended, to specify the presence of any stated features, elements, integers, steps or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. The term “comprising” thus includes the more restrictive terms “consisting of’ and “essentially consisting of’. With regard to sequences the term “comprising an amino acid sequence of’ include the embodiment “consisting of the amino acid sequence of’. Similarly, the term “encoding” or “encodes” is intended to be open-ended and allows the presence or addition of one or more other features, elements or components. Furthermore, singular and plural forms are not used in a limiting way. As used herein, the singular forms “a”, “an” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0024] The terms “ketoreductase” and “KRED” are used interchangeably herein and refer to a polypeptide having an enzymatic capability of reducing a carbonyl group to its corresponding alcohol. More specifically, the keto reductases of the present invention are capable of reducing the compound (substrate) ethyl 4-chloroacetoacetate (COBE; formula (I)) to the corresponding product ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE; formula (II)), utilizing the co-factor NADPH as the reducing agent. Ketoreductases as used herein includes both naturally occurring (wild-type) as well as non-naturally occurring modified (genetically engineered / mutated) polypeptides generated by human manipulation. The term “modified” as used herein refers to a genetically engineered L. kefir ketoreductase comprising amino acid mutations at specific amino acid positions.

[0025] The term “nucleic acid molecule” is used interchangeably with “polynucleotide” and refers to DNA or RNA of any length. The nucleic acid molecule comprises or consists of a certain sequence that may code for a protein. In the context of the present invention and particularly in the context of an expression vector (such as a plasmid), the person skilled in the art would understand that it refers to a DNA sequence or molecule.

[0026] The terms “protein” as used herein refers to a polypeptide comprising a certain amino acid sequence of any length. In the context of the present invention the protein is a recombinant or heterologous protein. The term recombinant means that the protein is produced using recombinant techniques. A heterologous protein (or nucleic acid) is from a different organism than the host cell, such as a protein from L kefir that is produced in a different bacterium as host cell, such as E. coli.

[0027] The term “encodes” and “codes for” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, the term “encode” describes the process of a DNA sequence of a nucleic acid molecule (DNA molecule) that is transcribed into an RNA molecule and / or an RNA sequence of a nucleic acid molecule (RNA molecule) that is translated into a protein and hence also a DNA sequence that is transcribed and translated into a protein.

[0028] The term “expression” as used herein refers to transcription and / or translation of a nucleic acid sequence, typically a heterologous nucleic acid sequence, within a host cell. The level of expression of a gene product of interest in a host cell may be determined on the basis of either the amount of corresponding RNA that is present in the cell, or the amount of the polypeptide encoded by the selected sequence. For example, RNA transcribed from a selected sequence can be quantified by Northern blot hybridization or by PCR, such as qPCR. Proteins encoded by a selected sequence can be quantitated by various methods, e.g. by ELISA, by SDS-page, Western blotting, by radioimmunoassay, by immunoprecipitation or by assaying for the biological activity of the protein in a cell lysate or following affinity purification.

[0029] The term “prokaryotic cell” as used herein refers to cells that lack a nucleus and other membranebound organelles and comprise a cell envelope and a cytoplasmic region that contains the cell genome, ribosomes and different sorts of inclusions. In the present invention a “prokaryotic cell” or prokaryotic host cell” means a bacterial cell, particularly a cell from Escherichia coli (E. coli).

[0030] The term “transformation” refers to the uptake of exogenous genetic material (e.g. a DNA polynucleotide) from an external medium. In general, bacterial cells can only undergo transformation under specific conditions and cells that are capable of being transformed are referred to as competent bacterial cells. In the laboratory, competence can be artificially induced through methods known in the art, such as electroporation or by treating bacteria with divalent cations (e.g., CaCh) and heat shock.

[0031] The term “mutation” as used herein refers to a substitution of a single amino acid in a nucleic acid sequence. The person skilled in the art will understand that a mutation at a defined amino acid position in a reference sequence is referred to by the original amino acid in the one letter code (e.g., K55 for lysine (K) at position 55) and allows a substitution with any amino acid except the original amino acid (E55X). The substituting amino acid may further be defined in the one letter code, e.g., K55L for a lysine (K) to leucine (L) substitution at position 55 of the reference sequence. For indicating in a certain sequence that may allow some variability that a specific amino acid position is mutated to a specific amino acid substituting amino acid may further be defined in the one letter code without defining the original amino acid in the reference sequence (e.g., 55L for position 55 being L)

[0032] In the context of the present invention, the term "conversion" refers to the activity of a ketoreductase or a modified ketoreductase, which can be assessed by measuring the conversion of ethyl 4-chloroacetoacetate (COBE) into (R / S)-4-chloro-3-hydroxybutyrate ((RZS)-CHBE) in a given time period.

[0033] The term "enantiomeric excess" (ee) as used herein is a measurement of purity used for chiral substances and refers to the degree to which a sample contains one enantiomer (such as e.g. (S)-CHBE) in higher amount than the other enantiomer (such as e.g. (R)-CHBE). The ee can be calculated according to the following formula:ee (%) = ((S)-product - (R)-product) I ((S)-product + (R)-product) x 100

[0034] As used herein, the term "activity" when referring to enzymes denote the enzyme’s catalytic function, which is the capacity to convert a substrate into a product. The “specific activity” as used herein measures the rate at which a substrate is consumed and / or a product is formed within a given time frame and for aspecific amount of protein. It is commonly expressed in pmol of substrate consumed or product produced per minute per milligram of protein. The unit pmol / min is often abbreviated as U (unit). Thus, the unit definitions for specific activity of pmol / min / (mg of protein) or U / (mg of protein) are used interchangeably herein. An enzyme is considered active if it performs its catalytic function in vivo or in vitro, provided a suitable substrate is present. The person skilled in the art knows how to measure enzyme activity, particularly the activity of ketoreductases. Methods for assessing the enzyme's ability to produce (S)-CHBE via the enantioselective reduction of COBE are well-established in the art, and including without being limited thereto the method described in Example 7.Modified bacterial ketoreductase and its expression by a vector or a prokaryotic host cell

[0035] The present invention demonstrates that variants of a ketoreductase from L. kefir, harboring at least six or eight site-specific amino acid replacements can be used for enantioselective reduction of COBE to (S)-CHBE with high substrate loading, high conversion and high catalytic efficiency. Surprisingly, these modified keto reductases increase conversion rates, stereoselectivity, substrate loading and catalytic efficiency compared to the respective wild-type L. kefir ketoreductase (and tested keto reductases from other bacteria), making these modified keto reductases suitable for the large-scale production of enantiomerically pure alcohols which are used as key chiral intermediates in the synthesis of APIs.

[0036] The present invention provides a modified bacterial ketoreductase comprising mutations K55L, S102T, L116T, F153C / I, T158N and L205I or K55L, S102T, L116T, F153C / I, T158N, D203N, L205I and E206Q / K in the bacterial ketoreductase of Lactobacillus kefir comprising the amino acid sequence of amino acids 8-258 of SEQ ID NO: 1 or an amino acid sequence having at least 95%.

[0037] More specifically, in a first aspect, the present invention relates to a modified bacterial ketoreductase comprising mutations in a bacterial ketoreductase comprising an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1, wherein the mutations are K55L, S102T, L116T, F153C / I, T158N and L205I. In certain embodiments, the mutations comprise K55L, S102T, L116T, T158N, L205I and F153C or F153I. Thus, the mutation at amino acid position 153 is F153X, wherein X is C or I. Preferably, the mutation at position 153 is F153C in a modified bacterial ketoreductase with 6 mutations. In one embodiment the mutations are K55L, S102T, L116T, F153C, T158N and L205L

[0038] Preferably, the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 13 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 13 and comprising amino acids 55L, 102T, 116T, 153C / I, 158N and 205I. In certain embodiments the modified bacterial ketoreductase comprises an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 13 and comprises amino acids 55L, 102T, 116T, 153C, 158N and 205I. In certain embodiments, the modified bacterial ketoreductase comprises the mutations in a bacterial ketoreductase having at least 96%, at least 97%, at least 98% or at least 99% sequence identity with amino acids 8 to 258 of SEQ ID NO: 13 and comprises amino acids 55L, 102T, 116T, 153C, 158N and 205I. In certain preferred embodiments the modified bacterial ketoreductasecomprises the sequence of amino acids 8 to 258 of SEQ ID NO: 13. Even more preferably the modified bacterial ketoreductase comprises the sequence of SEQ ID NO: 13.

[0039] The modified bacterial ketoreductase of the invention may further comprise mutations D203N and E206K or E206Q (E206Q / K). Thus, the further mutations are D203N and E206X, wherein X is K or Q (D203N and E206K or D203N or E206Q). Preferably, the mutation E206X is E206Q. Preferably, the mutation at position 153 is F153I and the mutation at position 206 is E206Q in a modified bacterial ketoreductase with 8 mutations. In a preferred embodiment the mutations are K55L, S102T, L116T, F153I, T158N, D203N, L205I and E206Q.

[0040] Preferably, the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 15, or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 15 and comprising amino acids 55L, 102T, 116T, 153C / I, 158N, 203N, 205I, and 206Q / K. Preferably, the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 15, or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 15 and comprising amino acids 55L, 102T, 116T, 1531, 158N, 203N, 205I, and 206Q. Preferably, the modified bacterial ketoreductase comprises the mutations in a bacterial ketoreductase having at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15 and comprising amino acids 55L, 102T, 116T, 1531, 158N, 203N, 205I, and 206Q. In certain preferred embodiments the modified bacterial ketoreductase comprises the sequence of amino acids 8 to 258 of SEQ ID NO: 15. Even more preferably the modified bacterial ketoreductase comprises the sequence of SEQ ID NO: 15.

[0041] In preferred embodiments, the modified bacterial ketoreductase comprises mutations in a bacterial ketoreductase comprising an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1 , wherein the mutations are K55L, S102T, L116T, F153C, T158N and L205I or the mutations are K55L, S102T, L116T, F153I, T158N, D203N, L205I and E206Q.

[0042] The exemplified bacterial L. kefir ketoreductase (also referred to as M0) having the amino acid sequence of SEQ ID NO: 1, as well as the exemplified modified bacterial ketoredutases (also referred to as M1, M2, M3, M4, M5, M6 and M7) having the amino acid sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13 and 15, respectively (see e.g., Table 7) comprise an N-terminal hexahistidine tag (6x histidine tag or 6x His tag). This hexahistidine tag is particularly useful for purification using a nickel column. Thus, amino acids 1 to 7 of the modified keto reductases according to SEQ IDs NO: 1, 3, 5, 7, 9, 11 , 13 and 15 refer to an N-terminal methionine and a 6x histidine tag. In particular, the first amino acid of the respective amino acid sequences refers to a methionine and amino acids 2 to 7 correspond to a subsequent 6x histidine tag. The wild-type ketoreductase of Lactobacillus kefir (GenBank: AAP94029.1) without an N-terminal 6x histidine tag but with an N-terminal methionine has the amino acid sequence of SEQ ID NO: 25. Therefore, amino acids 8 to 258 of SEQ ID NO: 1 correspond to amino acids 2 to 252 of SEQ ID NO: 25. In this regard, both amino acids contain an N-terminal methionine at amino acid position 1. The mutations K55L, S102T, L116T, F1531 / C, T158N, D203N, L205I and E206Q / K based on reference sequence SEQ ID NO: 1 correspond toK49L, S96T, L110T, F147I / C, T152N, D197N, L199I and E200Q / K based on reference sequence SEQ ID NO: 25.

[0043] The modified bacterial ketoreductase according to the invention comprises the mutations in a bacterial ketoreductase comprising an amino acid sequence having at least 95% amino acid sequence identity with amino acids 8 to 258 of SEQ ID NO: 1 (or with amino acids 1-252 of SEQ ID NO: 25). Preferably, the modified bacterial ketoreductase comprises the mutations in a bacterial ketoreductase comprising an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1 (or with amino acids 1-252 of SEQ ID NO: 25). The person skilled in the art will understand that there may be some variations in the amino acid sequence of the bacterial ketoreductase, but within at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1 (or with amino acids 1-252 of SEQ ID NO: 25) and further the bacterial ketoreductase without the recited mutations has a conversion rate and results in an enantiomeric excess that is comparable or better to a bacterial ketoreductase comprising the amino acid sequence of SEQ ID NO: 25 or of amino acids 8 to 258 of SEQ ID NO: 1.

[0044] In the context of the present invention, the term “having at least 95% sequence identity with” or the like refers to a protein that comprises an amino acid sequence which shares at least 95% identity (or the like) of the amino acid residues with a reference sequence. Sequence identity can be easily determined by sequence alignment. The sequence may be a natural sequence, such as of a different species as the reference sequence or an allelic variant of the reference sequence or an engineered sequence comprising one or more modifications over the reference sequence.

[0045] The modified bacterial ketoreductase according to the invention may further comprises one or more C-terminal and / or N-terminal tag(s). Preferably, the modified bacterial ketoreductase comprises a C-terminal or N-terminal tag(s), more preferably an N-terminal tag. Any tag(s) known in the art may be used. For example, the modified bacterial ketoreductase may comprise one or more C-terminal and / or N-terminal affinity tag(s). Exemplary tags may be a polyhistidine tag (e.g. a hexahistidine tag (6xHis) ora heptahistidine tag (7xHis)) or a functional equivalent thereof, a Flag tag having the sequence motif DYKDDDDK (SEQ ID NO: 40), a hemagglutinin tag (HA tag), a myc tag or a streptavidin tag. In certain embodiments the N-terminal tag is a 6x histidine tag (6xHis tag) comprises amino acids 2 to 7 of SEQ ID NO: 1.

[0046] In certain embodiments, the modified bacterial ketoreductase has a conversion rate of > 90% of ethyl 4-chloroacetoacetate (COBE) to ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE) and 99.9% enantiomeric excess (ee) within 8 hours in a reaction mixture comprising 1.25 g / L modified bacterial keto reductase, 500 g / L COBE, 25% (v / v) isopropanol and 0.1 g / L NADP+in 0.1 M K2HPO4, pH 7.0 at 35°C. Preferably, the modified bacterial ketoreductase has a conversion rate of > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99% of COBE to (S)-CHBE within 8 hours, more preferably within > 90%, > 91%, > 92%, > 93%, > 94%, > 95%, > 96%, > 97%, > 98%, > 99% within 5 hours. In further or additional embodiments, the modified bacterial ketoreductase has a specific activity of at least 62 U / mg and an ee of 99.9%, preferably a specific activity of at least 75 U / mg and an ee of 99.9%.

[0047] In a related aspect the invention relates to a use of the modified bacterial ketoreductase according to the invention (first aspect) for the enantioselective reduction of ethyl 4-chloroacetoacetate (COBE) to ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE).

[0048] In a second aspect, the invention relates to a nucleic acid molecule comprising a sequence encoding the modified bacterial ketoreductase of the present invention. Specifically, the encoded modified bacterial ketoreductase is the modified bacterial ketoreductase of the first aspect. Thus, the embodiments and examples specified with regard to the first aspect similarly apply to this aspect. In particular, in certain embodiments the nucleic acid comprises a sequence encoding the modified bacterial ketoreductase comprising mutations K55L, S102T, L116T, F153C / I, T158N and L205I in a bacterial ketoreductase comprising an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1. Preferably, the sequence of the nucleic acid molecule is codon optimized, such as codon optimized for E.coli as host cell. "Codon optimization" refers to modifying the polynucleotide sequence that encodes a protein by replacing codons that are optimized for tRNA pools in a specific organism. This ensures efficient and optimized expression of the protein in the target (production) organism. In certain embodiments, the sequence of the nucleic acid molecule encoding the modified bacterial ketoreductase is codon-optimized for production in E. coli, particularly E. coli BL21 (DE3) or DH5a.

[0049] Thus, the nucleic acid molecule comprising a sequence encoding the modified bacterial ketoreductase according to the invention (first aspect) comprising an amino acid sequence of SEQ ID NO 13 or 15 may have, but is not limited to, the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, respectively.

[0050] In a third aspect, the invention relates to an expression vector comprising a nucleic acid encoding the modified bacterial ketoreductase of the present invention (first aspect) or the nucleic acid molecule of the present invention (second aspect), operably linked to a promoter, preferably operably linked to an inducible promoter. Thus, the embodiments and examples specified with regard to the first aspect and second aspect similarly apply to this aspect. The expression vector according to the invention is for expression of a heterologous sequence in a prokaryotic host cell, preferably a bacterial host cell, such as E. coli. Typically, the expression vector comprises an expression cassette comprising the polynucleotide encoding the modified bacterial ketoreductase operably linked to a promoter.

[0051] Preferably the expression vector is a prokaryotic expression vector and the promoter is a prokaryotic promoter, thus allowing an efficient protein expression in a prokaryotic host cell. More preferably, the expression vector is a bacterial expression vector and the promoter is a bacterial promoter, thus allowing an efficient protein expression in a bacterial host cell. A wide variety of expression vectors have been identified for E. coli and other bacterial hosts and are known in the art. For example, expression vectors of the pBluescripttype, the pET type, the pQE type or the pUC type are commonly used for protein expression in E. coli cells. Preferably, the expression vector comprising the nucleic acid encoding the modified bacterial ketoreductase according to the invention is of the pET type. These vectors typically comprise an origin of replication, a T7 promoter specific to T7 RNA polymerase, a lac operator, a polylinker for cloning the geneencoding the protein to be expressed, a transcription termination sequence, a resistance gene (e.g., a resistance gene for ampicillin or kanamycin), and a lacl gene that encodes the lac repressor protein. In the absence of isopropyl-p-D-thiogalactopyranoside (IPTG) or lactose, the lac repressor binds to the lac operator, inhibiting the T7 promoter and blocking target protein expression. When IPTG or lactose binds to the lac repressor, it causes a conformational change resulting in detachment of the protein from the operator, thereby inducing expression from the T7 promoter. Exemplary pET vectors, without being limited thereto are pET-21 a(+), pET-24a(+), pET-28a(+), pET-29a(+), pET-30a(+), pET-41 a(+), pET-44a(+), pET-21 b(+), pET-24b(+), pET-26b(+), pET-28b(+), pET-29b(+), pET-30b(+), pET-42b(+), and pET-44b(+), with those based on the pET-30a(+) backbone being particularly preferred. Bacterial promoters regulate transcription in bacterial cells. Exemplary bacterial promoters, without being limited thereto are PBAD promoter (systematically araBp), tryptophan promoter (trp; trp operon), lactose promoter (lac; lac operon), and Tac-promoter (Ptac). Preferably, the expression vector comprises an inducible promoter that can trigger expression when an inducer compound is added. The expression vector may further comprise bacterial sequences, such as an origin of replication and resistance genes for vector amplification in bacterial cells.

[0052] In a fourth aspect, the invention relates to a prokaryotic host cell comprising a nucleic acid encoding the modified bacterial ketoreductase of the invention (first aspect), the nucleic acid sequence of the invention (second aspect) or the expression vector of the invention (third aspect). Thus, the embodiments and examples specified with regard to the first, second and third aspect similarly apply to this aspect. Preferably, the prokaryotic host cell is a bacterial host cell. The bacterial host cell may be Escherichia coli, preferably Escherichia coli BL21 or Escherichia coli DH5a. More preferably, the prokaryotic host cell may be Escherichia coli BL21 (DE3) or Escherichia coli DH5a.Method for producing a modified bacterial ketoreductase

[0053] In a fifth aspect, the invention relates to a method for producing the modified bacterial ketoreductase according to the invention (first aspect) comprising a) introducing the nucleic acid molecule comprising a sequence encoding the modified bacterial ketoreductase according to the invention (second aspect) or the expression vector according to the invention (third aspect) into a prokaryotic host cell; b) culturing the prokaryotic host cell under conditions that allow producing the modified bacterial keto reductase; c) lysing the prokaryotic host cell; and d) optionally purifying the modified bacterial ketoreductase. Thus, the embodiments and examples specified with regard to the first, second, third and fourth aspect similarly apply to this aspect.

[0054] In step a) the nucleic acid molecule or the expression vector may be introduced into the prokaryotic host cell by transformation. The nucleic acid molecule or the expression vector may be transformed into the prokaryotic host cell by any suitable method. For example, an expression vector for use in Escherichia coli, preferably Escherichia coli BL21 or Escherichia coli DH5a, more preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5a, may be introduced into the bacterial host cell by methods such as electroporation. Preferably, the prokaryotic host cell is a bacterial host cell. The bacterial host cell may be Escherichia coli,preferably Escherichia coli BL21 or Escherichia coli DH5a, more preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5a.

[0055] In step b) of the method of producing the modified bacterial ketoreductase according to the invention, the prokaryotic host cell is cultured under conditions that allow producing the modified bacterial keto reductase. In this regard, the conditions that allow producing the modified bacterial ketoreductase are dependent on the nucleic acid molecule or the expression vector encoding the modified bacterial ketoreductase as well as on the prokaryotic host cell used in the method. The skilled person can easily select and apply conditions allowing the production of the modified bacterial ketoreductase, such as in Escherichia coli BL21(DE3) or Escherichia coli DH5a. For example, when using an inducible bacterial expression system, such as a pET type expression vector, preferably based on the pET-30a(+) backbone, conditions that enable target protein expression typically involve a culturing temperature in the range of 20 °C to 42 °C, preferably in the range of 25 °C to 37 °C, more preferably in the range of 30 °C to 37 °C. The culture medium generally has a pH in the range of 6.5 and 9.0, preferably in the range of about 7.0 to 8.0, more preferably in the range of about 7.3 to 7.7, most preferably at a pH of about 7.5. The fermentation of the prokaryotic host cell culture may be performed for at least several hours and can be performed for one day or even several days. The prokaryotic host cells may be cultured using either a batch process or a fed-batch process. In a batch process, the culturing time typically ranges from 12 hours to 36 hours, while in a fed-batch process the culturing time can be up to 7 days or even longer depending on the prokaryotic host cell and the culturing process. Preferably, the expression of the modified bacterial ketoreductase is induced in the prokaryotic host cell using an inducible system, such as one that triggers protein production through the addition of an inducer, preferably IPTG or lactose.

[0056] In step c) of the method of producing the modified bacterial ketoreductase according to the invention, the prokaryotic host cells are lysed. Forthat purpose, the prokaryotic host cells are typically harvested prior to lysis. The harvesting of the prokaryotic host cells may be performed by centrifugation of the prokaryotic host cells and subsequent removal of the supernatant. For cell lysis, commonly used procedures are applied which are known to the skilled person including without being limited thereto enzymatic, chemical, osmotic, mechanical and / or physical disruption of the cell membrane and cell wall, can be applied to obtain a cell lysate, i.e. the fluid containing the content of a cell lysed. For example, cell disruption can be achieved by incubating prokaryotic host cells in a lysozyme solution or by ultrasonication.

[0057] In step d) of the method of producing the modified bacterial ketoreductase according to the invention, the modified bacterial ketoreductase is optionally purified from the lysate supernatant. For this purpose, the lysate supernatant is collected and protein purification is performed. Protein purification can be performed by the skilled person using routine methods comprising for example filtration, in particular ultrafiltration or diafiltration, and / or chromatography, in particular affinity chromatography. Purification of the modified bacterial ketoreductase may be performed by methods, such as immobilized metal affinity chromatography (IMAC), nickel affinity chromatography or immunoaffinity chromatography. Preferably, the modified bacterial ketoreductase comprises an affinity tag for purification, such as a polyhistidine tag. The purification of the modified bacterial ketoreductase comprising a polyhistidine tag (e.g. a hexahistidine tag (6xHis) or aheptahistidine tag (7xHis)) or a functional equivalent thereof may be performed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography or by anti-His column chromatography using anti-His antibodies. More preferably, the purification of the modified bacterial ketoreductase comprising a polyhistidine tag (e.g. a hexahistidine tag 6xHis or a heptahistidine tag 7xHis) or a functional equivalent thereof is performed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. After purification, the purified protein may be concentrated by filtration, in particular by ultrafiltration and / or the purified protein may by lyophilized. Following lyophilization the modified bacterial ketoreductase is in the form of a lyophilized enzyme powder.Method for the enantioselective reduction of ethyl 4-chloroacetoacetate to ethyl S-4-chloro-3-hydroxybutyrate

[0058] In a sixth aspect, the invention relates to a method forthe enantioselective reduction ofthe substrate ethyl 4-chloroacetoacetate (COBE) according to formula (I) to the product ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE) according to formula (II), wherein the method comprises incubating COBE with the modified bacterial ketoreductase of the invention (first aspect) in the presence of the co-substrate isopropanol (IPA) and the co-factor isopropanol dehydrogenase (NADP+) to obtain (S)-CHBE. The enantioselective reduction is exemplarily depicted in the following reaction equation:

[0059] For the ketoreductase-catalyzed reduction of COBE to (S)-CHBE, the modified ketoreductase, preferably in a purified form, and the required co-factor (NADP+) are used. The oxidized co-factor NADP+can be continuously regenerated by the co-substrate isopropanol. This allows the reaction to require only catalytic amounts of co-factor.

[0060] In a preferred embodiment, the product (S)-CHBE is present at an enantiomeric excess (ee) greater than 99%. Preferably, the enantiomeric excess (ee) may be greater than 99.5%, more preferably the enantiomeric excess may be 99.9%. By using the modified bacterial ketoreductase according to the invention (first aspect), almost no (R)-CHBE was detectable in the product. In other words, by using the modified bacterial ketoreductase according to the invention (first aspect), the product is essentially free of (R)-CHBE or the product is essentially pure (S)-CHBE (ee > 99.9%).

[0061] In the method forthe enantioselective reduction ofthe substrate ethyl 4-chloroacetoacetate (COBE) according to formula (I) to the product ethyl S-4-chloro-3-hydroxybutyrate ((S)-CHBE) according to formula (II), the modified bacterial ketoreductase according to the invention (first aspect) may be provided in a non-purified form or in a purified form. For example, the non-purified form may be a lysate supernatant containing the modified bacterial ketoreductase, such as the lysate supernatant obtained in step c) of the method of producing the modified bacterial ketoreductase according to the invention (fifth aspect). For example, the purified form may be a modified bacterial ketoreductase obtained in step d) ofthe method ofproducing the modified bacterial ketoreductase according to the invention (fifth aspect), optionally concentrated and / or desalted, or a purified lyophilized enzyme powder of the modified bacterial keto reductase.

[0062] Preferably, the modified bacterial ketoreductase according to the invention is used as purified protein, more preferably as a lyophilized enzyme powder. Particularly, enzyme concentration for use in the method may be in the range of 0.5 g / L to 100 g / L, in the range of about 1 g / L to about 20 g / L or in the range of about 1 g / L to about 10 g / L. Preferably, the substrate loading of COBE is in the range of about 100 g / L to about 1000 g / L, preferably in the range of about 200 g / L to about 500 g / L, more preferably about 500 g / L.

[0063] The reaction is performed in an aqueous buffer. An aqueous buffer may be any buffer using water as solvent, preferable a phosphate buffer, such as a sodium or potassium phosphate (e.g. K2HPO4) buffer, a Tris / HCI buffer, or a triethanolamine (TEA) buffer, and at a pH in the range of about 6 to 9, preferably in the range of about 7 to 8, more preferably at a pH of about 7. In a preferred embodiment, the aqueous buffer is a 0.1 M dipotassium phosphate (K2HPO4) buffer and has a pH of 7.

[0064] The temperature is maintained in the range of 20 °C and 50 °C, preferably in the range of 30 °C and 40 °C, more preferably in a range of 30°C to 37°C. In a preferred embodiment the aqueous buffer is a potassium phosphate buffer (0.1 M K2HPO4, pH 7.0). A suitable co-factor, such as NADP+, and a cosubstrate for the regeneration of the oxidized co-factor, such as isopropanol, are included in the reaction mixture. The concentration of the co-factor, such as NADP+, in the reaction mixture may be in the range of about 0.1 g / L to 1 g / L, preferably in the range of about 0.1 g / L to 0.2 g / L, more preferably about 0.1 g / L. The concentration of the co-substrate, such as isopropanol, may be in the range of about 10 % (v / v) to 40 % (v / v), preferably in the range of about 20 % (v / v) to 30 % (v / v), preferably in the range of about 25% (v / v) to 30% (v / v).

[0065] The reaction may be performed for at least 1 hour and up to 72 hours or even longer. Preferably, the reaction is performed for about 3 hours to 12 hours, more preferably for about 5 to 8 hours. Once a desired conversion level of at least about 90%, preferably at least about 95%, more preferably about 99% is reached, the reaction may be stopped, for example, by addition of acetonitrile. Alternatively, the reaction can be stopped by lowering the pH, by heating, or by addition of an enzyme inhibitor. Subsequently, the resulting enantiomerically pure (S)-CHBE may be extracted with an organic solvent immiscible with water, such as ethyl acetate, and separated from the unreacted COBE or other starting materials using chromatography or distillation. The purity of the enantiomer is typically assessed using GO and / or HPLC with a chiral column, Thin-Layer Chromatography (TLC), or by polarimetry. TLC is particularly useful for high-throughput screening.

[0066] The specific activity of the modified bacterial ketoreductase according to the invention is at least 60 U / mg, 65 U / mg, at least 70 U / mg or at least 80 U / mg. Preferably, the specific activity of the modified bacterial ketoreductase according to the invention is in the range of 70 to 100 U / mg or higher.

[0067] The method for the enantioselective reduction may be carried out in any suitable scale, e.g. laboratory scale or production scale. More preferably, the total reaction volume may be in the range of200 pL to 5000 L or higher, in particular in the range of 2 L to 1000 L, in particular in the range of 10 L to 1000 L.EXAMPLESExample 1 : Wild-Type ketoreductase nucleotide sequence acquisition and synthesis

[0068] The nucleotide sequences of keto reductases KRED001-KRED023 were achieved from literatures based on the chemical reactions they catalyze and were all synthesized by GENEWIZ (Genewiz Biotech Co., Ltd. China) with codons optimized for expression in E. coli. Synthetic KRED genes were further cloned into the pET-30a(+) vector with an N-terminal 6*His-tag (as illustrated for KRED009 in Figure 1). The respective information for KRED001-KRED0023 is listed in Table 1.Table 1: Wild-type ketoreductases from different microorganismsExample 2: Primary screening of wild-type ketoreductases from different microorganisms

[0069] The total screening reaction volume was 200 pL, including 20 pL of 10 mg / mL NADP+, 160 pL of different wild-type ketoreductases from the lysate supernatant and 20 pL of 50 mg / mL COBE dissolved in isopropanol. The reaction was carried out at 30 °C and 1000 rpm for 18 h. Acetonitrile was used to terminate the reaction. Subseguently, the conversion and chiral selectivity of the mixture was measured by ultra-high performance liguid chromatography mass spectrometry (UHPLC-MS). The primary screening results using 5 mg / ml COBE are listed in Table 2.Table 2: Primary screening results>>>> >>> >>>>>>>

[0070] Increasing the substrate COBE in the reaction from 5 g / L to 200 g / L, KRED009 and KRED011 catalyzed COBE to produce (S)-CHBE with a conversion rate of 84.7% and 15.1%, respectively. KRED009 has the ability to catalyze the production of NADPH and acetone from NADP+and isopropanol (IPA). The produced NADPH is then used by KRED009 as hydrogen donor for reacting COBE to (S)-CHBE. In this regard, the usage of NADP+as initial co-factor is particularly advantageous, because it is more stable and cheaper compared to NADPH. Therefore NADP+was used as co-factor in the following examples, except for Example 7.

[0071] KRED009 was chosen for the next directed evolution because of its high conversion rate, high chiral purity and further for the high COBE substrate loading.Example 3: The construction of the mutation library

[0072] The site-saturation mutagenesis library was constructed by PCR using recombinant plasmids pET-30a(+) containing the KRED009 gene as template (Figure 1). Mutagenic primers were designed by Snapgene software. The melting temperature (Tm) difference between two primers was controlled to be within 3 °C and the length to be within 59 bases. The obtained primers were dissolved with ultrapure water to a concentration of 10 pM. The reaction system is listed in Table 3. The PCR amplification was performed using the program listed in Table 4.Table 3: The PCR reaction system of site-directed mutagenesisTable 4: The PCR procedure of site-directed saturation mutation library

[0073] The PCR products were run on a 2% agarose gel for verification, to determine if there is a DNA at the expected size. If not, the missing mutations was recorded and redesigned and / or included in the next round of evolution. The PCR product digested by Dpn\ and purified with ZYMO RESEARCH Kit (ZYMO RESEARCH) was transformed into 50 pL of Escherichia coli BL21 (DE3) competent cells, and colonies after transformation were incubated for DNA sequencing and target protein expression.

[0074] Combinatorial mutagenesis was carried out by PCR according to the reaction system and procedure listed in Table 5 and Table 6, respectively, using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). The other steps were the same as for the site-directed mutagenesis mentioned above.Table 5: The PCR reaction system of combinatorial mutation libraryTable 6: The PCR procedure of combinatorial mutagenesisExample 4: High-throughput screening

[0075] The E. coli BL21 (DE3) colonies prepared in Example 3 were picked and transferred into 96-well plates containing 200 pL autoclaved LB medium (10 g / L tryptone, 5 g / L yeast exact and 5 g / L sodium chloride) containing 50 pg / mL kanamycin per well and cultured overnight at 37 °C and 400 rpm to obtain a primary culture. The next day, 11 pL of primary culture in each well were transferred to a new 96 deep-well plates containing 390 pL autoclaved TB medium (12 g / L Pancreatic digest of casein, 24 g / L yeast extract, 2.2 g / L monopotassium phosphate, 9.4 g / L di potassium phosphate) with 50 pg / mL kanamycin. The cultures were incubated at 37 °C and 400 rpm until the optical density (ODeoo) reached 0.6-0.8, and p-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The cells were incubated at 30 °C and 400 rpm for another overnight fermentation. After fermentation, the cells were centrifuged and the supernatant removed. Subseguently, 250 pL of 2 g / L lysozyme solution were added to each well, and cell disruption was carried out at 30 °C, 1000 rpm for 2 h. After cell disruption, the supernatant containing the enzyme was collected after centrifugation at 4000 rpm for 10 min. The enzyme reaction was performed in 200 pL comprising 32.8-82 pL COBE (200-500 g / L), 20 pL of 10 mg / mL NADP+, 20-40 pL of isopropanol, 10 pL of enzyme from the lysed supernatant, made up with 0.1 M K2HPO4 buffer. The reaction was carried out at 30 °C and 1000 rpm for 18 h and terminated by acetonitrile. 1 pL of reaction solution was loaded to a chromatographic plate and the chromatographic plate was placed into the container with Thin Layer Chromatography (TLC) solvent (Heptane / Ethyl acetate=4 / 1 with 0.1wt% formic acid). Upon TLC solvent reaching a proper height (10-15 cm), the chromatographic plate was taken out and dried with nitrogen. Afterwards, the chromatographic plate was put into a color reaction reagent (1.5 g KMnO4, 10 g K2CO3, 1.25 mL of 10% NaOH solution, add water to 200 mL) for about 5 s, take the chromatographic plate out, and the surface blown and dried with nitrogen. The mutants screened out by TLC were further analyzed by UHPLC-MS. The mutants showing higher conversion rates were incubated for DNA sequencing and retested. The mutation information of the engineered KRED mutants obtained in each round of screening is listed in Table 7.Table 7: Mutation information of the engineered KREDsExample 5: Biocatalytic reduction of COBE by the engineered L. kefir KREDs in shake flasks

[0076] E. coli BL21(DE3) cells carrying the recombinant plasmid were cultivated in 3 mL of LB medium containing 50 pg / mL kanamycin at 37 °C and 200 rpm for overnight culture. 1% (v / v) of the overnight culture was inoculated into 300 mL TB (Terrific Broth) medium with 50 pg / mL kanamycin and grown at 37 °C and 300 rpm. When the optical density (ODeoo) reached 0.6-0.8, 0.1 mM IPTG (lsopropyl-p-D-1-thiogalactopyranoside) was added to induce the enzyme expression at 30 °C for an additional 18-20 h. The cells were harvested by centrifugation at 4000 rpm for 10 min at 4 °C, then resuspended in 25 mL K2HPO4 buffer (0.1 M, pH 7.0). The cells were lysed by ultrasonication in an ice bath, and the supernatant enzyme was collected by centrifugation at 12000 rpm for 10 min at 4 °C.

[0077] Enzyme activities of wild-type L. kefir KRED (M0) and the engineered L. kefir KREDs (M1-M7) in lysate supernatants was assessed by enantioselective enzymatic reduction of COBE using the increasingly stringent conditions as appropriate. In a first reaction mixture, the biocatalytic reduction of COBE by KREDs was conducted on a 10 mL scale in 20 mL flask, containing 1g of COBE, 2.5 mL of isopropanol, 2 mL of 2.5 mg / mL NADP+solved in K2HPO4 buffer (0.1 M, pH 7.0) and 5 mL enzyme from the lysed supernatant (50%), resulting in a final concentration of COBE of 100 g / L, a final concentration of isopropanol of 25% (v / v) and a final concentration of NADP+of 0.5 g / L. The reaction was carried out at 35 °C and 600 rpm for 24 h. 30 pL of the reaction mixture were taken for analysis and diluted with 1 mL of anhydrous acetonitrile. After 25 pm filter membrane filtration, the conversion and enantiomeric excess value were calculated according to the analytical method.

[0078] In a second reaction mixture, the biocatalytic reduction of COBE by KREDs was conducted on a 10 mL scale in 20 mL flask, containing 2 g of COBE, 2.5 mL of isopropanol, 2-5.5 mL of 0.5-1.2 mg / mL NADP+solved by K2HPO4 buffer (0.1 M, pH 7.0) and 1.5-5 mL enzyme from the lysed supernatant (15-50%), resulting in a final concentration of COBE of 200 g / L, a final concentration of isopropanol of 25% (v / v) and a final concentration of NADP+of 0.2 g / L. The reaction was carried out at 35 °C and 600 rpm for 24 h. 30 pL of the reaction mixture was taken for analysis and diluted with 1 mL of anhydrous acetonitrile. After 25 pm filter membrane filtration, the conversion and enantiomeric excess value were calculated according tothe analytical method.

[0079] In a third reaction mixture, the biocatalytic reduction of COBE by KREDs was conducted on a 10 mL scale in 20 mL flask, containing 5 g of COBE, 2.55 mL of isopropanol, 1.85-2.35 mL of 0.5 mg / mL NADP+solved by K2HPO4 buffer (0.1 M, pH 7.0) and 1-1.5 mL enzyme from the lysed supernatant (10-15%) or less. This corresponded to a final concentration of COBE of 500 g / L, a final concentration of isopropanol of 25% (v / v), and a final concentration of NADP+of 0.1 g / L. The reaction was carried out at 35 °C and 600 rpm for 24 h. 30 pL of the reaction mixture was taken for analysis and diluted with 1 mL of anhydrous acetonitrile. After 25 pm filter membrane filtration, the conversion and enantiomeric excess value were calculated according to the analytical method using ion pair chromatography (I PC).

[0080] In the IPC method samples were analyzed using a gradient Ultra High-Performance Liquid Chromatography (UHPLC) apparatus with single quadrupole (SQ) mass selective detector (MSD) and a CHIRALPAK® IG-U column (100 mm x 3.0 mm, 1.6 pm, DAICEL, Part No.87U83). For the mobile phase eluent A (1.0 ml formic acid (mass spectrometry grade, e.g., Honeywell ‘94318) to 1.0 L HPLC water) and eluent B (1.0 mL formic acid to 1.0 L acetonitrile (gradient grade, e-g. Lichrosolv, Merck #1.00030.4008) were used with the following gradient:

[0081] For the preparation of the sample, the sample was filtered with a 0.22 pm filter membrane, phase-separated and the upper layer dissolved in diluent to obtain a clear solution (e.g. 100 pL upper layer were dissolved in 10 mL diluent). In a second step 250 pL of the obtained clear solution was further diluted with 10 mL diluent and 2 pL of the further dilution subjected to UHPLC. The approximate retention times of the (R)-CHBE, (S)-CHBE and COBE are as follows:

[0082] The results of biocatalytic reduction of COBE by wild-type L. kefir KRED and the engineered L. kefirKREDs in shake flasks are listed in Table 8.Table 8: Biocatalytic reduction results<>>>><>Example 6: The protein purification of the engineered KREDs

[0083] E. coli BL21(DE3) cells carrying the recombinant plasmid were cultivated in 3 mL of LB (lysogeny broth or Luria Bertani) medium containing 50 pg / mL kanamycin at 37 °C and 200 rpm for overnight culture.1% (v / v) of the overnight culture was inoculated into 300 mL TB (Terrific Broth) medium with 50 pg / mL kanamycin and grown at 37 °C and 300 rpm. When the optical density (ODeoo) reached 0.6-0.8, 0.1 mM IPTG was added to induce the enzyme expression at 30 °C for an additional 18-20 h. The cells were harvested by centrifugation (4000 rpm, 10 min) at 4 °C and washed with 0.9% NaCI solution twice. Cells were resuspended with Buffer A (2.2 g / L Na2HPO4, 0.54 g / L NaH2PO4, 29.2 g / L NaCI, 0.681 g / L imidazole, pH 7.4), lysed by ultrasonication in an ice bath, and the supernatant was collected by centrifugation at 12000 rpm for 30 min at 4 °C. The supernatant was loaded onto a Ni-NTA column previously equilibrated with Buffer A. The non-target proteins were washed away using a wash buffer formulated by Buffer A and Buffer B (2.2 g / L Na2HPO4, 0.54 g / L NaH2PO4, 29.2 g / L NaCI, 17.02 g / L imidazole, pH 7.4) in a ratio of 19:1 , and the target protein was eluted using an elution buffer formulated by Buffer A and Buffer B in a ratio of 3:2. The purified proteins were concentrated by ultrafiltration and desalted to remove high concentration of imidazole with elution into K2HPO4 buffer (0.1 M, pH 7.0). Then the proteins were stored at -80 °C for further use. The size and purity of proteins were identified by SDS-PAGE (Figures 2 and 3).Example 7: Enzyme activity assay

[0084] Enzyme activities of wild-type L. kefirKRED (M0) and the engineered L. kefir KREDs (M1-M7) were determined by measuring the decrease in the absorbance of NADPH at 340 nm using a BioTek microplate reader Epoch2 (Agilent) at 30 °C. One unit of enzyme activity is defined as the amount of enzyme catalyzing the oxidation of 1 pmol of NADPH per minute. The assay mixture of 200 pL was composed of 100 pL of 0.2mM NADPH, 50 pL of appropriate concentration of enzyme and 50 pL of 8 mM COBE. The final concentration of NADPH was 0.1 mM and the final concentration of COBE was 2 mM. The dilution factor of the enzyme affected the absorbance value and an absorbance value of 0.02-0.04 per minute was appropriate. The linearity between absorbance and concentration of NADPH can be converted from the NADPH standard curve made for each enzyme activity assay. Reaction components were all formulated by K2HPO4 buffer (0.1 M, pH 7.0). Results are listed in Table 9. The respective values represent means with standard deviations.Table 9: Enzyme activity resultsan.d. means non-detectable.Example 8: Kilo-lab scale-up synthesis of (Sl-CHBE by biocatalytic reduction of COBE

[0085] The scale-up of the biocatalytic reduction of COBE by the engineered KREDs was conducted on a 2 L scale in a 10 L reactor, containing 1000 g of COBE, 490 mL of isopropanol, 2.5 g M6 lyophilized enzyme powder and 0.2 g NADP+. The remaining volume was made up with K2HPO4 buffer (0.1 M, pH 7.0). The final concentration of COBE was 500 g / L, the final concentration of isopropanol was 25% (v / v) and the final concentration of NADP+was 0.1 g / L. The reaction was carried out at 35 °C and 300 rpm. After 5 h reaction, the conversion was 99.9%. Isopropyl acetate was added to the reactor for extraction of crude product, after distillation and purification, affording the product (S)-CHBE with > 99.0% purity, > 90.0% yield, and > 99.9% enantiomeric excess (ee).

[0086] Conversion and enantiomeric excess value were calculated according to the analytical method using IPC described above in Example 5. The resulting IPC chromatogram is shown in Fig. 4. The quantification of the peak area is normalized and only peaks of (R)-CHBE, (S)-CHBE and COBE are taken into account for integration. Fig. 4 shows that highly enantiomerically pure (S)-CHBE was obtained (residual COBE and (R)-CHBE were less than 0.05 %).Sequence listing:

Claims

CLAIMS1. A modified bacterial ketoreductase comprising mutations in a bacterial ketoreductase comprising an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 1 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 1 , wherein the mutations comprise K55L, S102T, L116T, F153C / I, T158N and L205I.

2. The modified bacterial ketoreductase of claim 1, wherein the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 13 or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 13 and comprising amino acids 55L, 102T, 116T, 153C / I, 158N and 205I, preferably amino acids 55L, 102T, 116T, 153C, 158N and 205I .

3. The modified bacterial ketoreductase of claim 1 or 2, further comprising mutations D203N and E206Q / K.

4. The modified bacterial ketoreductase of claim 3, wherein the modified bacterial ketoreductase comprises an amino acid sequence of amino acids 8 to 258 of SEQ ID NO: 15, or an amino acid sequence having at least 95% sequence identity with amino acids 8 to 258 of SEQ ID NO: 15 and comprising amino acids 55L, 102T, 116T, 153C / I, 158N, 203N, 205I, and 206Q / K, preferably amino acids 55L, 102T, 116T, 1531, 158N, 203N, 2051, and 206Q.

5. The modified bacterial ketoreductase of any one of claims 1 to 4, wherein the modified bacterial ketoreductase further comprises one or more C-terminal and / or N-terminal tag(s), preferably an N- terminal tag, more preferably an N-terminal 6xHis tag.

6. The modified bacterial ketoreductase of any one of claims 1 to 5, wherein the modified bacterial ketoreductase has a conversion rate of > 90% of ethyl 4-chloroacetoacetate (COBE) to ethyl S-4- chloro-3-hydroxy butyrate ((S)-CHBE) and 99.9% enantiomeric excess (ee) within 8 hours in a reaction mixture comprising 1.25 g / L modified bacterial keto reductase, 500 g / L COBE, 25% (v / v) isopropanol and 0.1 g / L NADP+in 0.1 M K2HPO4, pH 7.0 at 35°C.

7. A nucleic acid molecule comprising a sequence encoding the modified bacterial ketoreductase of any one of claims 1 to 5, preferably wherein the sequence of the nucleic acid molecule is codon optimized.

8. An expression vector comprising a nucleic acid encoding the modified bacterial ketoreductase of any one of claims 1 to 6 or the nucleic acid molecule of claim 7, operably linked to a promoter, preferably operably linked to an inducible promoter.

9. A prokaryotic host cell comprising a nucleic acid encoding the modified bacterial ketoreductase of any one of claims 1 to 6, the nucleic acid molecule of claim 7 or the expression vector of claim 8.2910. A method of producing a modified bacterial ketoreductase comprising:a) introducing the nucleic acid molecule comprising a sequence encoding a modified bacterial ketoreductase according to claim 7 or the expression vector according to claim 8 into a prokaryotic host cell;b) culturing the prokaryotic host cell under conditions that allow producing the modified bacterial keto reductase;c) lysing the prokaryotic host cell; andd) optionally purifying the modified bacterial keto reductase.

11. The method of claim 10, wherein the nucleic acid molecule or the expression vector is introduced into the prokaryotic host cell by transformation.

12. The method of claim 10 or 11 , wherein the prokaryotic host cell is Escherichia coli, preferably Escherichia coli BL21 or Escherichia coli DH5a, more preferably Escherichia coli BL21 (DE3) or Escherichia coli DH5a.

13. A method for the enantioselective reduction of the substrate ethyl 4-chloroacetoacetate (COBE) according to formula (I)to the product ethyl S-4-ch Io ro-3-hydroxy butyrate ((S)-CHBE) according to formula (II)wherein the method comprises incubating COBE with the modified bacterial ketoreductase of any one of claims 1 to 6 in the presence of the co-substrate isopropanol and the co-factor NADP+to obtain (S)-CHBE.

14. The method according to claim 13, wherein the product is present at an enantiomeric excess (ee) greater than 99%.

305. Use of the modified bacterial ketoreductase according to any one of claims 1 to 6 for the enantioselective reduction of ethyl 4-chloroacetoacetate (COBE) to ethyl S-4-chloro-3- hydroxy butyrate ((S)-CHBE).

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