Oxidoreductases for the stereoselective reduction of keto compounds

Abstract

The invention relates to a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, wherein the keto compound is reduced with an oxidoreductase in the presence of a cofactor, and is characterized in that an oxidoreductase is used which has an amino acid sequence in which (a) at least 70% of the amino acids are identical to the amino acids of one of the amino acid sequences SEQ ID No 1, SEQ ID No 6 and SEQ ID No 8, or (b) at least 55% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 2, or (c) at least 65% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 3, or (d) at least 75% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 4, or (e) at least 65% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 5, or (f) at least 50% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 7, or (g) at least 72% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 129.

1 .- 7 . (canceled) 
     
     
         8 . A process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, the process comprising:
 reducing the keto compound with an oxidoreductase in the presence of a cofactor, characterized in that an oxidoreductase is used which:
 (a) is encoded by a nucleic acid sequence from the group of SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13 and SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 130; or 
 (b) is encoded by a nucleic acid sequence the complementary strand of which hybridizes with one of the nucleic acid sequences mentioned in (a) under highly stringent conditions. 
   
     
     
         9 . A process according to  claim 8 , characterized in that the keto compound has the general Formula I:
   R 1 —C(O)—R 2   (I)   wherein R 1  stands for one of the moieties:   1) —(C 1 -C 20 )-alkyl, wherein alkyl is linear-chain or branched;   2) —(C 2 -C 20 )-alkenyl, wherein alkenyl is linear-chain or branched and optionally contains up to four double bonds;   3) —(C 2 -C 20 )-alkynyl, wherein alkynyl is linear-chain or branched and optionally contains up to four triple bonds;   4) —(C 6 -C 14 )-aryl;   5) —(C 1 -C 8 )-alkyl-(C 6 -C 14 )-aryl;   6) —(C 5 -C 14 )-heterocycle which is unsubstituted or substituted one, two or three times by —OH, halogen, —NO 2  and/or —NH 2 ; or   7) —(C 3 -C 7 )-cycloalkyl;   wherein the moieties mentioned above under 1) to 7) are unsubstituted or substituted one, two or three times, independently of each other, by —OH, halogen, —NO 2  and/or —NH 2 ; and   R 2  stands for one of the moieties:   8) —(C 1 -C 6 )-alkyl, wherein alkyl is linear-chain or branched;   9) —(C 2 -C 6 )-alkenyl, wherein alkenyl is linear-chain or branched and optionally contains up to three double bonds;   10) —(C 2 -C 6 )-alkynyl, wherein alkynyl is linear-chain or branched and optionally contains two triple bonds; or   11) —(C 1 -C 10 )-alkyl-C(O)—O—(C 1 -C 6 )-alkyl, wherein alkyl is linear or branched and is unsubstituted or substituted one, two or three times by —OH, halogen, —NO 2  and/or —NH 2 ;   wherein the moieties mentioned above under 8) to 11) are unsubstituted or substituted one, two or three times, independently of each other, by —OH, halogen, —NO 2  and/or —NH 2 .   
     
     
         10 . A cloning vector comprising:
 one or several nucleic acid sequences coding for the oxidoreductase according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ ID No: 129 or homologues thereof.   
     
     
         11 . An expression vector located in a bacterial, insect, plant or mammalian cell, the expression vector comprising:
 a nucleic acid sequence which codes for the oxidoreductase according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ ID No:129 or homologues thereof and is linked to an expression control sequence.   
     
     
         12 . A recombinant host cell which is a bacterial, insect, plant or mammalian cell and has been transformed or transfected with an expression vector comprising:
 a nucleic acid sequence which codes for the oxidoreductase according to SEQ ID No:1, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4, SEQ ID No:5, SEQ ID No:6, SEQ ID No:7, SEQ ID No:8, SEQ ID No:129 or homologues thereof and is linked to an expression control sequence.   
     
     
         13 . An oxidoreductase for use in a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, said oxidoreductate being characterized by the following:
 the oxidoreductase is encoded by a nucleic acid sequence which is selected from the group consisting of SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16, SEQ ID No 130, and by a nucleic acid sequence the complementary strand of which hybridizes with one of the sequences SEQ ID No 9, SEQ ID No 10, SEQ ID No 11, SEQ ID No 12, SEQ ID No 13, SEQ ID No 14, SEQ ID No 15, SEQ ID No 16 or SEQ ID No 130 under highly stringent conditions.   
     
     
         14 . An oxidoreductase as in  claim 13 , characterized in that the keto compound has the general Formula I:
   R 1 —C(O)—R 2   (I)   wherein R1 stands for one of the moieties:   1) —(C 1 -C 20 )-alkyl, wherein alkyl is linear-chain or branched;   2) —(C 2 -C 20 )-alkenyl, wherein alkenyl is linear-chain or branched and optionally contains up to four double bonds;   3) —(C 2 -C 20 )-alkynyl, wherein alkynyl is linear-chain or branched and optionally contains up to four triple bonds;   4) —(C 6 -C 14 )-aryl;   5) —(C 1 -C 8 )-alkyl-(C 6 -C 14 )-aryl;   6) —(C 5 -C 14 )-heterocycle which is unsubstituted or substituted one, two or three times by —OH, halogen, —NO 2  and/or —NH 2 ; or   7) —(C 3 -C 7 )-cycloalkyl;   wherein the moieties mentioned above under 1) to 7) are unsubstituted or substituted one, two or three times, independently of each other, by —OH, halogen, —NO 2  and/or —NH 2 ; and   R 2  stands for one of the moieties:   8) —(C 1 -C 6 )-alkyl, wherein alkyl is linear-chain or branched;   9) —(C 2 -C 6 )-alkenyl, wherein alkenyl is linear-chain or branched and optionally contains up to three double bonds;   10) —(C 2 -C 6 )-alkynyl, wherein alkynyl is linear-chain or branched and optionally contains two triple bonds; or   11) —(C 1 -C 10 )-alkyl-C(O)—O—(C 1 -C 6 )-alkyl, wherein alkyl is linear or branched and is unsubstituted or substituted one, two or three times by —OH, halogen, —NO 2  and/or —NH 2 ;   wherein the moieties mentioned above under 8) to 11) are unsubstituted or substituted one, two or three times, independently of each other, by —OH, halogen, —NO 2  and/or —NH 2 .   
     
     
         15 . A process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound, wherein the keto compound is reduced with an oxidoreductase in the presence of a cofactor, characterized in that an oxidoreductase is used which has an amino acid sequence in which:
 (a) at least 70% of the amino acids are identical to the amino acids of one of the amino acid sequences SEQ ID No 1, SEQ ID No 6, and SEQ ID No 8;   (b) at least 55% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 2 and which comprises one of the internal partial sequences EYKEAAFTN (SEQ ID No 27), NKVAIITGGISGIGLA (SEQ ID No 28), DVNLNGVFS (SEQ ID No 29), HYCASKGGV (SEQ ID No 30), NCINPGYI (SEQ ID No 31) or LHPMGRLGE (SEQ ID No 32);   (c) at least 65% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 3 and which comprises one of the internal partial sequences CHSDLHAIY (SEQ ID No 35), GYQQYLLVE (SEQ ID No 36), TFDTCQKYV (SEQ ID No 37), LLTPYHAM (SEQ ID No 38), LVSKGKVKP (SEQ ID No 39), GAGGLGVNG (SEQ ID No 40), IQIAKAFGAT (SEQ ID No 41) or LGSFWGTS (SEQ ID No 42);   (d) at least 75% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 4;   (e) at least 65% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 5;   (f) at least 50% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 7; or   (g) at least 72% of the amino acids are identical to the amino acids of the amino acid sequence SEQ ID No 129.