KETOREDUCTASES

Abstract

The invention relates to ketoreductases and the use thereof. The ketoreductases of the invention are particularly useful for enzymatically catalyzing the reduction of ketones to chiral secondary alcohols.

Claims

1. A ketoreductase comprising an amino acid sequence with at least 72% homology to SEQ ID NO:2.

2. The ketoreductase according to claim 1, which is capable of stereoselectively reducing a keto substrate of general formula (I) ##STR00017## to a secondary alcohol; or which is capable of reducing an aldehyde substrate of general formula (I′) ##STR00018## to a primary alcohol; wherein X and Y are each independently selected from saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic or alicyclic C.sub.1-12-hydrocarbon residues; unsubstituted or mono- or polysubstituted C.sub.6-10-aromatic hydrocarbon residues, optionally being bridged to the CO-moiety through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted heteroaromatic hydrocarbon residues, optionally being bridged to the CO-moiety through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; and sugar residues or desoxysugar residues in each case comprising mono-, di- or oligosaccharides; wherein mono- or polysubstituted means independently substituted with one or more functional groups selected from -halo, —OH, ═O, —OC.sub.1-12-alkyl, —OC.sub.6-10-aryl, —O-heteroaryl, —OCOC.sub.1-12-alkyl, —OCOC.sub.6-10-aryl, —OCO-heteroaryl, —SH, —SC.sub.1-12-alkyl, —SC.sub.6-10-aryl, —S-heteroaryl, —S(═O).sub.1-2OH, —NO, —NO.sub.2, —N.sub.3, —NH.sub.2, —NH(C.sub.1-12-alkyl), —N(C.sub.1-12-alkyl).sub.2, —NH(C.sub.6-10-aryl), —N(C.sub.6-10-aryl).sub.2, —NH(heteroaryl), —N(heteroaryl).sub.2, —CN, —CHO, —CO.sub.2H, CO—C.sub.1-2-alkyl, —CO—C.sub.6-10-aryl and —CO-heteroaryl.

3. The ketoreductase according to claim 2, wherein the keto substrate is selected from the group consisting of (i) 3-aryl-3-ketopropanamine-derivatives according to general formula (II) ##STR00019## wherein R.sub.1 and R.sub.2 are each independently selected from the group consisting of —H; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.3-8-cycloalkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; or unsubstituted or mono- or polysubstituted heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; or alternatively, wherein R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached form an unsubstituted or mono- or polysubstituted C.sub.2-8-heterocycloalkyl ring or an unsubstituted or mono- or polysubstituted heteroaryl ring; R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are each independently selected from —H; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; or wherein R.sub.3 and R.sub.4 together are ═O; R.sub.7 is unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl; or unsubstituted or mono- or polysubstituted -heteroaryl; (ii) 5-hydroxy-3-oxo-hexanoate-derivatives according to general formula (III) ##STR00020## wherein R.sub.8 is unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; or unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; R.sub.9 is —H; -halo; —CN; or —OR.sub.11, wherein R.sub.11 is hydrogen or a protecting group; R.sub.10 is —H; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; or unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; (iii) acetophenone-derivatives according to general formula (IV) ##STR00021## wherein R.sub.12, R.sub.13, R.sub.14, R.sub.15, and R.sub.16 are each independently selected from the group consisting of —H; -halo; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted-heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; and —OR.sub.18, wherein R.sub.18 is —H, unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl, or unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; R.sub.17 is —H; -halo; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted-heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; —OR.sub.19, —NH.sub.2, —NHR.sub.19, or —NR.sub.19R.sub.20, wherein R.sub.19 and R.sub.20 are each independently selected from unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; or unsubstituted or mono- or polysubstituted-heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; (iv) benzoyl-derivatives according to general formula (V) ##STR00022## wherein R.sub.21 and R.sub.22 are each independently selected from unsubstituted or mono- or polysubstituted C.sub.6-10-aryl and unsubstituted or mono- or polysubstituted heteroaryl; (v) secodione-derivatives according to general formula (VI) ##STR00023## wherein R.sub.23 and R.sub.24 are each independently selected from the group consisting of —H and —C.sub.1-12-alkyl; and (vi) 3-quinuclidone; (vii) ethyl-4-chloro-3-oxo-butanoate; and (viii) ethyl-3-oxo-3-phenyl-propanoate; (ix) ketose; or wherein the aldehyde substrate is selected from the group consisting of (x) 2-butanal; and (xi) 1-heptanal; wherein in each case mono- or polysubstituted means independently substituted with one or more functional groups selected from -halo, ═O, —OH, —OC.sub.1-12-alkyl, —OC.sub.6-10-aryl, —O-heteroaryl, —OCOC.sub.1-12-alkyl, —OCOC.sub.6-10-aryl, —OCO-heteroaryl, —SH, —SC.sub.1-12-alkyl, —SC.sub.6-10-aryl, —S-heteroaryl, —S(═O).sub.1-20 H, —NO, —NO.sub.2, —N.sub.3, —NH.sub.2, —NH(C.sub.1-12-alkyl), —N(C.sub.1-12-alkyl).sub.2, —NH(C.sub.6-10-aryl), —N(C.sub.6-10-aryl).sub.2, —NH(heteroaryl), —N(heteroaryl).sub.2, —CN, —CHO, —CO.sub.2H, CO—C.sub.1-2-alkyl, —CO—C.sub.6-10-aryl and —CO-heteroaryl.

4. The ketoreductase according to claim 1, which (i) converts isopropyl alcohol to acetone at a rate of 0.01-100 U/mg lyophilisate of the ketoreductase; and/or (ii) after incubation for 48 h in 50% of aqueous isopropyl alcohol at 30° C. exhibits a residual activity of at least 1%, relative to its activity before incubation.

5. The ketoreductase according to claim 1, which is not identical with the peptide of SEQ ID NO:2 and which exhibits improved specific activity, temperature stability, and/or stereoselectivity compared to the peptide of SEQ ID NO:2.

6. The ketoreductase according to claim 5, wherein SEQ ID NO:2 is engineered in at least one or more positions selected from the group consisting of the positions Y21, V23, S33, L39, R40, A43, P68, V89, G95, P97, T98, D103, G109, V119, L121, V124, Y125, I149, L150, S154, E155, T157, A158, T163, H190, Y193, L198, L199, A201, A206, Y207, V229, and V247.

7. The ketoreductase according to claim 5 wherein SEQ ID NO: 2 is engineered in at least one or more positions selected from the group consisting of positions TABLE-US-00003 Y21Q; D103E; T163A or S; V23T; G109Y; H190C; S33A; V119Y; Y193A, F, G, P, T or V; L39V; L121Q; L198M; R40C; V124I; L199A, F, I or T; A43E or G; Y125F; A201G; P68S; I149A, G, L, M, Q, T or V; A206G; V89F; L150A, F, H or S; Y207R or L; G95A, E, M, Q, S154G; E155A, D, F, G, K, V229I; and S or V; L or S; V247I. P97A, E, K, N, T157Y V or Y; A158G, L, P, Q, S, V or T98A or G; W;

8. The ketoreductase according to any of claim 5 (i) wherein the specific activity of the ketoreductase is higher than the specific activity of the wild type ketoreductase of SEQ ID NO:2; and/or (ii) wherein the temperature stability of the ketoreductase is higher than the temperature stability of the wild type ketoreductase of SEQ ID NO:2; and/or (iii) wherein the stereoselectivity of the ketoreductase is higher than the stereoselectivity of the wild type ketoreductase of SEQ ID NO:2.

9. The ketoreductase according to claim 1, which comprises an amino acid sequence of at least 85% homology to the SEQ ID NO:4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, or 93.

10. A method for reduction and/or oxidation of substrates comprising: providing a keto substrate, an aldehylde substrate, a primary or a secondary alcohol substrate; providing the ketoreductase according to claim 1 and a cofactor, and, in presence of the ketoreductase and the cofactor reducing the keto substrate to a secondary alcohol; reducing the aldehyde substrate to a primary alcohol; oxidizing the secondary alcohol substrate to a keto product; oxidizing the primary alcohol substrate to an aldehyde; and/or oxidizing the aldehyde substrate to a carboxylic acid.

11. The method according to claim 10, wherein the keto substrate, aldehylde substrate, primary or secondary alcohol substrate are the keto substrate, aldehylde substrate, primary or secondary alcohol substrate of claim 2.

12. The method according to claim 11, wherein (i) the substrate is ethyl-4-chloro-3-oxo-butanoate which is stereoselectively reduced to ethyl (3S)-4-chloro-3-hydroxy-butanoate with a ketoreductase comprising the amino acid sequence of SEQ ID NO:2; (ii) the substrate is tert-butyl (5R)-6-cyano-5-hydroxy-3-oxo-hexanoate which is stereoselectively reduced to tert-butyl (3R,5R)-6-cyano-3,5-dihydroxy-hexanoate with a ketoreductase comprising the amino acid sequence of SEQ ID NO:91; (iii) the substrate is N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropanamine which is stereoselectively reduced to (1S)-3-(dimethylamino)-1-(2-thienyl)-propan-1-ol with a ketoreductase comprising the amino acid sequence of SEQ ID NO:58; (iv) The substrate is (5S)-6-chloro-5-hydroxy-3-oxohexanoate which is stereoselectively reduced to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxy-hexanoate with a ketoreductase comprising the amino acid sequence of SEQ ID NO:62 or 91; (v) the substrate is ethylsecodion (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14,17-dione) which is stereoselectively reduced to 17-β-Seconol (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14-on-17-β-ol) with a ketoreductase comprising the amino acid sequence of SEQ ID NO:70; or (vi) the substrate is N-monomethyl-3-keto-3-(2-thienyl)-1-ketopropanamine which is stereoselectively reduced (1S)-3-(methylamino)-1-(2-thienyl)-propan-1-ol with a ketoreductase comprising the amino acid sequence of SEQ ID NO:58 or 87.

13. (canceled)

14. (canceled)

15. A method for increasing thermo stability of a ketoreductase having an amino acid sequence that is alignable to SEQ ID NO:2 which method comprises: providing the ketoreductase of claim 1, and engineering the ketoreductase in at least one amino acid position selected from the group consisting of positions that correspond to positions V89, Y125, and V229 of SEQ ID NO:2.

16. The ketoreductase of claim 1, wherein the ketoreductase has been engineered relative to a non-engineered ketoreductase consisting of the SEQ ID NO: 2.

17. The ketoreductase of claim 2, wherein the ketoreductase has been engineered relative to a non-engineered ketoreductase consisting of the SEQ ID NO: 2.

18. The ketoreductase of claim 3, wherein the ketoreductase has been engineered relative to a non-engineered ketoreductase consisting of the SEQ ID NO: 2.

19. The ketoreductase of claim 4, wherein the ketoreductase has been engineered relative to a non-engineered ketoreductase consisting of the SEQ ID NO: 2.

20. A method of comprising: providing the ketoreductase according to claim 16; providing a keto substrate of general formula (I) ##STR00024## and stereoselectively reducing the keto substrate of general formula (I) with the ketoreductase to a secondary alcohol or providing a aldehyde substrate of general formula (I′) ##STR00025## and reducing the aldehyde substrate of general formula (I′) with the ketoreductase to a primary alcohol; wherein X and Y are each independently selected from saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic or alicyclic C.sub.1-12-hydrocarbon residues; unsubstituted or mono- or polysubstituted C.sub.6-10-aromatic hydrocarbon residues, optionally being bridged to the CO-moiety through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted heteroaromatic hydrocarbon residues, optionally being bridged to the CO-moiety through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; and sugar residues or desoxysugar residues in each case comprising mono-, di- or oligosaccharides; wherein mono- or polysubstituted means independently substituted with one or more functional groups selected from -halo, —OH, ═O, —OC.sub.1-12-alkyl, —OC.sub.6-10-aryl, —O-heteroaryl, —OCOC.sub.1-12-alkyl, —OCOC.sub.6-10-aryl, —OCO-heteroaryl, —SH, —SC.sub.1-12-alkyl, —SC.sub.6-10-aryl, —S-heteroaryl, —S(═O).sub.1-20 H, —NO, —NO.sub.2, —N.sub.3, —NH.sub.2, —NH(C.sub.1-12-alkyl), —N(C.sub.1-12-alkyl).sub.2, —NH(C.sub.6-10-aryl), —N(C.sub.6-10-aryl).sub.2, —NH(heteroaryl), —N(heteroaryl).sub.2, —CN, —CHO, —CO.sub.2H, CO—C.sub.1-2-alkyl, —CO—C.sub.6-10-aryl and —CO-heteroaryl.

21. The method of claim 20, wherein the keto substrate is selected from the group consisting of (i) 3-aryl-3-ketopropanamine-derivatives according to general formula (II) ##STR00026## wherein R.sub.1 and R.sub.2 are each independently selected from the group consisting of —H; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.3-8-cycloalkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; or unsubstituted or mono- or polysubstituted heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; or alternatively, wherein R.sub.1 and R.sub.2 together with the nitrogen atom to which they are attached form an unsubstituted or mono- or polysubstituted C.sub.2-8-heterocycloalkyl ring or an unsubstituted or mono- or polysubstituted heteroaryl ring; R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are each independently selected from —H; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; or wherein R.sub.3 and R.sub.4 together are ═O; R.sub.7 is unsubstituted or mono- or polysubstituted —C.sub.8-10-aryl; or unsubstituted or mono- or polysubstituted -heteroaryl; (ii) 5-hydroxy-3-oxo-hexanoate-derivatives according to general formula (III) ##STR00027## wherein R.sub.8 is unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; or unsubstituted or mono- or polysubstituted —C.sub.8-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; R.sub.9 is —H; -halo; —CN; or —OR.sub.11, wherein R.sub.11 is hydrogen or a protecting group; R.sub.10 is —H; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; or unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; (iii) acetophenone-derivatives according to general formula (IV) ##STR00028## wherein R.sub.12, R.sub.13, R.sub.14, R.sub.15, and R.sub.16 are each independently selected from the group consisting of —H; -halo; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted-heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; and —OR.sub.18, wherein R.sub.18 is —H, unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl, or unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; R.sub.17 is —H; -halo; unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; unsubstituted or mono- or polysubstituted-heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; —OR.sub.19, —NH.sub.2, —NHR.sub.19, or —NR.sub.19R.sub.20, wherein R.sub.19 and R.sub.20 are each independently selected from unsubstituted or mono- or polysubstituted —C.sub.1-12-alkyl; unsubstituted or mono- or polysubstituted —C.sub.6-10-aryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; or unsubstituted or mono- or polysubstituted-heteroaryl, optionally being bridged through a saturated or unsaturated, unsubstituted or mono- or polysubstituted aliphatic C.sub.1-12-hydrocarbon residue; (iv) benzoyl-derivatives according to general formula (V) ##STR00029## wherein R.sub.21 and R.sub.22 are each independently selected from unsubstituted or mono- or polysubstituted C.sub.6-10-aryl and unsubstituted or mono- or polysubstituted heteroaryl; (v) secodione-derivatives according to general formula (VI) ##STR00030## wherein R.sub.23 and R.sub.24 are each independently selected from the group consisting of —H and —C.sub.1-12-alkyl; and (vi) 3-quinuclidone; (vii) ethyl-4-chloro-3-oxo-butanoate; and (viii) ethyl-3-oxo-3-phenyl-propanoate; (ix) ketose; or wherein the aldehyde substrate is selected from the group consisting of (x) 2-butanal; and (xi) 1-heptanal; wherein in each case mono- or polysubstituted means independently substituted with one or more functional groups selected from -halo, ═O, —OH, —OC.sub.1-12-alkyl, —OC.sub.6-10-aryl, —O-heteroaryl, —OCOC.sub.1-12-alkyl, —OCOC.sub.6-10-aryl, —OCO-heteroaryl, —SH, —SC.sub.1-12-alkyl, —SC.sub.6-10-aryl, —S-heteroaryl, —S(═O).sub.1-20 H, —NO, —NO.sub.2, —N.sub.3, —NH.sub.2, —NH(C.sub.1-12-alkyl), —N(C.sub.1-12-alkyl).sub.2, —NH(C.sub.6-10-aryl), —N(C.sub.6-10-aryl).sub.2, —NH(heteroaryl), —N(heteroaryl).sub.2, —CN, —CHO, —CO.sub.2H, CO—C.sub.1-2-alkyl, —CO—C.sub.6-10-aryl and —CO-heteroaryl.

22. The method of claim 20, wherein the ketoreductase (iii) converts isopropyl alcohol to acetone at a rate of 0.01-100 U/mg lyophilisate of the ketoreductase; and/or (iv) after incubation for 48 h in 50% of aqueous isopropyl alcohol at 30° C. exhibits a residual activity of at least 1%, relative to its activity before incubation.

Description

EXAMPLE 1

[0214] Detection of the New Ketoreductase Gene Corresponding to SEQ ID NO:1

[0215] The gene of the new ketoreductase was detected during a screening for new ketoreductases in a genomic library derived from microbial communities living in deadwood on the top of small-leaved lime tree (Tilia cordada). The DNA of microorganisms selectively grown in a 96-well format was isolated, mechanically fragmented to the desired size range and cloned into the two-promoter expression vector system pF2F4 (WO2010/075956 A1). The resulting plasmids were transformed to E. coli BL21(DE3)placI(+) cells. Screening of the library was done with cluster screening (WO2005/040376 A2) with cluster sizes of 5,000 to 350,000 clones per plate.

[0216] For expression of the genomic library cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) and chloramphenicol (34 mg/1)). Expression of the genes of the genomic library was induced at logarithmic phase either by IPTG (0.1 mM) or arabinose (0.1 (v/v)). Cultivations were carried out at 30° C. for 16 hours.

[0217] Cells were harvested by centrifugation (3220×g, 20 min, 4° C.) and disrupted with cell lysis buffer (50 mM Tris-HCl pH 7.0; 2 mM MgCl.sub.2, 1× CelLytic B (Sigma); DNA nuclease 0.02 U, lysozyme 0.5 mg/ml). The crude extracts were separated from cell debris by centrifugation (3220×g 30 min, 4° C.).

[0218] The crude extracts of the genomic library were investigated regarding their ability to reduce the mixture of substrates: ethyl-4-chloro-3-oxo-butanoate and 1-(4-chloro-phenyl)ethanone by measuring a decrease in absorbance at 340 nm resulting from the oxidation of NAD(P)H.

EXAMPLE 2

[0219] Expression of the New Ketoreductase Gene Corresponding to SEQ ID NO:1

[0220] The gene of the newly found ketoreductase corresponding to SEQ ID NO:1 was cloned into the expression vector pLE1A23 (derivative of pRSF-1b, Novagen). The gene was moreover codon optimized for E. coli expression while simultaneously decreasing the GC-content (see SEQ ID NO:2). The gene was cloned into the expression vector pLE1A27 (derivative of pRSF-1b, Novagen). The resulting plasmid was used for transformation of E. coli BL21(DE3) cells.

[0221] For expression of the new ketoreductase gene corresponding to SEQ ID NO:1 cells were cultivated in ZYM505 medium (F. William Studier, Protein Expression and Purification 41 (2005) 207-234) supplemented with kanamycin (50 mg/1) at 37° C. Expression of the gene was induced at logarithmic phase by IPTG (0.1 mM) and carried out at 30° C. for 16-18 hours.

[0222] Cells were harvested by centrifugation (3220×g, 20 min, 4° C.) and disrupted with cell lysis buffer (50 mM Tris-HCl pH 7.0; 2 mM MgCl.sub.2, 1× CelLytic B (Sigma); DNA nuclease 0.02 U, lysozyme 0.5 mg/ml). The crude extracts were separated from cell debris by centrifugation (3220×g 30 min, 4° C.).

[0223] The crude extract was investigated regarding the level of ketoreductase expression via denaturing SDS-PAGE and its ability to reduce ethyl-4-chloro-3-oxo-butanoate by measuring a decrease in absorbance at 340 nm resulting from the oxidation of NAD(P)H.

EXAMPLE 3

[0224] Preparative Scale Reduction of Ethyl-4-Chloro-3-Oxo-Butanoate to Ethyl (3S)-4-Chloro-3-Hydroxy-Butanoate by the New Ketoreductase of SEQ ID NO:2

[0225] 24.08 g D(+)-Glucose monohydrate was dissolved in 0.1 M sodium phosphate buffer pH 6.5 to a final volume of 45 ml in a 250 ml round bottom flask equipped with a magnetic stirrer. The pH of the solution was adjusted to pH 6.5-6.6 with NaOH. 10.7 mg of the new ketoreductase of SEQ ID NO:2, 22.9 mg glucose dehydrogenase (GDH-03, commercially available at c-LEcta GmbH) and 39.8 mg NAD.sup.+, each dissolved in 5 ml 0.1 M sodium phosphate buffer pH 6.5 were added. The flask was connected to a pH Stat titration device and tempered to 35° C. while stirring. The reaction was started by a stepwise controlled addition of a solution of 18.18 g ethyl-4-chloro-3-oxo-butanoate in 9.375 ml n-butyl acetate. During the complete reaction time the mixture was stirred and tempered to 35° C. The pH was automatically controlled by NaOH addition by the pH-stat device (setpoint: pH=6.5). Reaction progress is controlled by tracking the amount of 5 M NaOH that was titrated automatically by pH Stat. After 22 hours the reaction is completed resulting in an overall conversion of >99.9% analyzed by GC analytics. The reduction product was shown to have an enantiomeric excess of >99 for the ethyl (3 S)-4-chloro-3-hydroxy-butanoate.

EXAMPLE 4

[0226] Evaluation of Ketoreductase Variants Regarding their Thermal Stability

[0227] Several ketoreductase variants that had been generated were analyzed regarding their thermal stability. Melting profiles were recorded by incubation of the ketoreductase containing crude extract for 15 minutes at different temperatures in a PCR cycler. Afterwards the crude extracts were incubated on ice for 30 minutes. Insoluble proteins were separated by centrifugation and the supernatants were analyzed regarding their remaining ketoreductase activity in a standard ketoreductase assay. In this standard assay isopropyl alcohol is oxidized to acetone by the ketoreductase with concomitant reduction of NAD.sup.+ to NADH. The increase of ketoreductase is monitored by measuring the absorption at 340 nm in a standard photometer.

[0228] It was found, that the ketoreductase corresponding to SEQ ID NO:4 exhibits a melting temperature (Tm), that is 15° C. higher than the Tm of the wild type ketoreductase of SEQ ID NO:2

EXAMPLE 5

[0229] Reduction of Tert-Butyl (5R)-6-Cyano-5-Hydroxy-3-Oxo-Hexanoate by Engineered Ketoreductases Derived from Ketoreductase of SEQ ID NO:2

[0230] Numerous engineered ketoreductases that had been generated were analyzed regarding their capacity to reduce the substrate tert-butyl (5R)-6-cyano-5-hydroxy-3-oxo-hexanoate to tert-butyl (5R)-6-cyano-3,5-dihydroxy-hexanoate. Screening assays were performed in a 96-well plate scale with a final volume of 150 μl per well in 0.1 M sodium phosphate buffer pH 6.5 and a final concentration of 0.1 M purified tert-butyl (5R)-6-cyano-5-hydroxy-3-oxo-hexanoate and 1 mM cofactor NAD.sup.+. Reactions were started by adding 10 μl of a 1 to 300 dilution of crude extract in 0.05 M Tris-HCl buffer pH 7.0, 2 mM MgCl.sub.2 to each well. Activities of the ketoreductase variants were determined by measuring the decrease of absorbance at 340 nm in a microplate reader at 30° C. It was found that the ketoreductase variant corresponding to SEQ ID NO:91 reduced the substrate tert-butyl (5R)-6-cyano-5-hydroxy-3-oxo-hexanoate at a 55 fold higher rate than ketoreductase of SEQ ID NO:2.

EXAMPLE 6

[0231] Preparative Scale Reduction of Tert-Butyl (5R)-6-Cyano-5-Hydroxy-3-Oxo-Hexanoate by Engineered Ketoreductase Derived from Ketoreductase of SEQ ID NO:2

[0232] 4.3 g of a crude batch of tert-butyl (5R)-6-cyano-5-hydroxy-3-oxo-hexanoate (purity ˜70%) was weighed in a glass beaker (corresponds to 3 g of pure tert-butyl (5R)-6-cyano-5-hydroxy-3-oxo-hexanoate). A solution of 3.7 g D(+)-Glucose monohydrate in water (final volume 6.8 ml), 1.5 ml of 1 M sodium phosphate buffer pH 6.5 and 10 mg of NAD.sup.+ dissolved in water were added. The pH of the solution was adjusted to 6.5-6.6 with NaOH. The reaction mixture was connected to a pH Stat titration device and tempered to 30° C. while stirring. The reaction was started by addition of a solution of the engineered ketoreductase corresponding to SEQ ID NO:91 (21 mg) and glucose dehydrogenase (GDH-03, 10 mg) in water. During the complete reaction time the mixture was stirred and tempered to 30° C. The pH was automatically controlled by NaOH addition by the pH-stat device (setpoint: pH=6.5). Reaction progress was controlled by tracking the amount of 5 M NaOH that was titrated automatically by pH Stat. After 12 hours reaction is finished resulting in an overall conversion of >95% analyzed by HPLC analytics (detection at 212 nm and 200 nm, quantification by calibration curves of substrate and product). The reduction product was shown to have a diastereomeric excess of >99 for the syn product (tert-butyl (3R,5R)-6-cyano-3,5-dihydroxy-hexanoate) over the corresponding anti product (tert-butyl (3S,5R)-6-cyano-3,5-dihydroxy-hexanoate) as measured by chiral HPLC.

EXAMPLE 7

[0233] Reduction of N,N-Dimethyl-3-Keto-3-(2-Thienyl)-1-Ketopropanamine by Engineered Ketoreductases Derived from ADH97.

[0234] Numerous engineered ketoreductases that had been generated were analyzed regarding their capacity to reduce the substrate N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropanamine to N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine. Screening assays were performed in a 96-well plate scale with a final volume of 300 μl per well in 0.1 M Triethanolamine/HCl buffer pH 9.0; 50 isopropanol (v/v) and a final concentration of 0.5 M N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropanamine and 1 mM cofactor NAD.sup.+. Reactions were started by adding 10 μl of a 1 to 10 dilution of crude extract in 0.1 M Triethanolamine-HCl buffer per well. Reactions were incubated at 30° C. for 20 h. Activities of the ketoreductase variants were determined by HPLC analysis (detection at 230 nm/245 nm; determination of conversion by calibration curves of substrates and product) of substrate and product. It was found that the ketoreductase variant corresponding to SEQ ID NO:58 was able to reduce the substrate N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropanamine. After 20 hours of reaction a conversion of 23% was achieved. Ketoreductase of SEQ ID NO:2 showed no conversion under the given conditions.

EXAMPLE 8

[0235] Preparative Scale Reduction of N,N-Dimethyl-3-Keto-3-(2-Thienyl)-1-Ketopropanamine by Engineered Ketoreductase Derived from Ketoreductase of SEQ ID NO:2

[0236] A solution of 25% NaOH (110 ml), isopropanol (41.5 ml) and N,N-dimethyl-3-keto-3-(2-thienyl)-1-ketopropanamine hydrochloride (60 g) were added to a 500 ml round bottom flask equipped with a magnetic stirrer. The resulting slurry was stirred at room temperature until complete dissolution and phase separation occurred. Water (40 ml) was added to 27.3 ml of the upper layer in a glass beaker. The pH of the solution was adjusted to 9.0 by addition of concentrated sulfuric acid. 37.5 ml isopropanol was added to the mixture, which was subsequently mixed with 66.5 mg of NAD.sup.+ in a 250 ml round bottom flask. The flasks neck was connected to a rotary evaporator and the solution was tempered to 40° C. by rotating the flask in a preheated (40° C.) oil bath. A solution of the engineered ketoreductase corresponding to SEQ ID NO:58 (1.35 g) dissolved in 22.5 ml water was added to start the reaction. During the complete reaction time the flask was rotated and tempered to 40° C. in an oil bath and vacuum (110 mbar, 82.5 mm Hg) was applied to remove mainly acetone and isopropanol. A preheated (40° C.) mixture of isopropanol and water (80:20) was added periodically every half hour to the reaction mixture. Samples were taken every hour for control of the reaction progress and analyzed by HPLC (detection at 230 nm/245 nm; determination of conversion by calibration curves of substrates and product). After 8 hours the reaction is completed resulting in an overall conversion of >98%. The reduction product was shown to have an enantiomeric excess of >99.5 in favor of the enantiomer (1S)-3-(dimethylamino)-1-(2-thienyl)-propan-1-ol.

EXAMPLE 9

[0237] Reduction of Ethylsecodion (Ethyl-3-Methoxy-8,14-Seco-Gona-1,3,5(10),9(11)-Tetraen-14,17-Dione) by Engineered Ketoreductases Derived from Ketoreductase of SEQ ID NO:2

[0238] Numerous engineered ketoreductases that had been generated were analyzed regarding their capacity to reduce the substrate ethylsecodion (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14,17-dione). Screening assays were performed in a 96-well plate scale with a final volume of 500 μl per well in 0.1 M Triethanolamine/HCl buffer pH 7.0; 2 mM MgCl.sub.2, 50% isopropanol (v/v), 1% Triton™ X-100 (v/v), 3% DMSO (v/v) and a final concentration of 10 g/l ethylsecodion (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14,17-dione) and 1 mM cofactor NAD.sup.+. Reactions were started by adding 100 μl of a 1 to 10 dilution of crude extract per well. Reactions were incubated at 30° C. for 4 h while stirring. Activities of the ketoreductase variants were determined by HPLC analysis (detection at 265 nm; determination of conversion by calibration curves of substrate and product). It was found that the ketoreductase variant corresponding to SEQ ID NO:70 was able to reduce the substrate ethylsecodion (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14,17-dione) with a conversion of 94% and a stereomeric excess for the 17-β-Seconol (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14-on-17-β-ol) of >99.5% under given conditions. Ketoreductase of SEQ ID NO:2 showed no conversion under the given conditions.

[0239] The engineered ketoreductase corresponding to SEQ ID NO:70 may also be used for preparative scale reduction of ethylsecodion (ethyl-3-methoxy-8,14-seco-gona-1,3,5(10),9(11)-tetraen-14,17-dione) under similar conditions as described in this example, wherein a substrate feed is applied.

EXAMPLE 10

[0240] Reduction of Tert-Butyl (5S)-6-Chloro-5-Hydroxy-3-Oxohexanoate by Engineered Ketoreductases Derived from Ketoreductase of SEQ ID NO:2

[0241] Numerous engineered ketoreductases that had been generated were analyzed regarding their capacity to reduce the substrate (5S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (5S)-6-chloro-3,5-dihydroxy-hexanoate. Screening assays were performed in a 96-well plate scale with a final volume of 150 μl per well in 0.1 M sodium phosphate buffer pH 6.5 and a final concentration of 25 mM purified tert-butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate and 1 mM cofactor NAD.sup.+. Reactions were started by adding 10 μl of several dilutions of crude extract in 0.05 M Tris-HCl buffer pH 7.0, 2 mM MgCl.sub.2 to each well. Activities of the ketoreductase variants were determined by measuring the decrease of absorbance at 340 nm in a microplate reader at 30° C. It was found that ketoreductase variants corresponding to SEQ ID NO:62 and 91 reduced the substrate (5S)-6-chloro-5-hydroxy-3-oxohexanoate at a 3 fold higher rate than ketoreductase of SEQ ID NO:2.

[0242] The engineered ketoreductases corresponding to SEQ ID NO:62 and 91 may also be used for preparative scale reduction of (5S)-6-chloro-5-hydroxy-3-oxohexanoate to tert-butyl (3R,5S)-6-chloro-3,5-dihydroxy-hexanoate under conditions comparable to the conditions described in example 6.

EXAMPLE 11

[0243] Reduction of N-Monomethyl-3-Keto-3-(2-Thienyl)-1-Ketopropanamine by Engineered Ketoreductases Derived from Ketoreductase of SEQ ID NO:2

[0244] Numerous engineered ketoreductases that had been generated were analyzed regarding their capacity to reduce the substrate N-monomethyl-3-keto-3-(2-thienyl)-1-ketopropanamine to N-monomethyl-3-hydroxy-3-(2-thienyl)-1-propanamine. Screening assays were performed in a 96-well plate scale with a final volume of 200 per well in 0.1 M Triethanolamine/HCl buffer pH 7.0; 10 isopropanol (v/v) and a final concentration of 0.1 M N-monomethyl-3-keto-3-(2-thienyl)-1-ketopropanamine and 1 mM cofactor NAD.sup.+. Reactions were started by adding 10 μl crude extract per well. Reactions were incubated at 30° C. for 4 h and 24 h. Activities of the ketoreductase variants were determined by HPLC analysis (detection at 230 nm/245 nm; determination of conversion by calibration curves of substrates and product) of substrate and product. It was found that the ketoreductase variants corresponding to SEQ ID NO:58 and SEQ ID NO:87 were able to reduce the substrate N-monomethyl-3-keto-3-(2-thienyl)-1-ketopropanamine best. After 4 hours and 24 hours of reaction conversions of 40-43% and 87-89%, respectively were achieved. Ketoreductase of SEQ ID NO:2 showed no conversion under the given conditions.

[0245] The engineered ketoreductases corresponding to SEQ ID NO:58 and 87 may also be used for preparative scale reduction of N-monomethyl-3-keto-3-(2-thienyl)-1-ketopropanamine to (1S)-3-(methylamino)-1-(2-thienyl)-propan-1-ol under conditions comparable to the conditions described in example 6 or example 8.