NOVEL 7Beta-HYDROXYSTEROID DEHYDROGENASE MUTANTS AND PROCESS FOR THE PREPARATION OF URSODEOXYCHOLIC ACID

Abstract

The invention relates to novel 7β-hydroxysteroid dehydrogenase mutants, to the sequences which encode these enzyme mutants, to processes for the preparation of the enzyme mutants and to their use in enzymatic reactions of cholic acid compounds, in particular in the preparation of ursodeoxycholic acid (UDCS). The invention also relates to novel processes for the synthesis of UDCS using the enzyme mutants; and to the preparation of UDCS using recombinant, multiply-modified microorganisms.

1 . A 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant, which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the nonmutated enzyme, a decreased substrate inhibition, especially for the 7-ketosteroid substrate, and/or an altered cofactor usage, and the nonmutated enzyme has the amino acid sequence of a 7β-HSDH from a bacterium of the genus  Collinsella  and the mutant has, in comparison with the nonmutated enzyme, 1 to 15 amino acid additions, substitutions, deletions and/or inversions. 
     
     
         2 . The mutant as claimed in  claim 1 , wherein the mutant has at least one mutation in the amino acid sequence according to SEQ ID NO:2 or an amino acid sequence derived therefrom with at least 80% sequence identity to SEQ ID NO:2. 
     
     
         3 . A 7β-HSDH mutant, which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has at least one mutation in the sequence motif VMVGRRE according to position 36 to 42 of SEQ ID NO:2 or in the corresponding sequence motif of an amino acid sequence derived therefrom with at least 80% sequence identity to SEQ ID NO:2. 
     
     
         4 . A 7β-HSDH mutant, which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has at least one mutation in the sequence motif VMVGRRE according to position 36 to 42 of SEQ ID NO:2 or in the corresponding sequence motif of an amino acid sequence derived therefrom with at least 60% sequence identity to SEQ ID NO:2, selected from
 a) the single mutants 039X 1  and R40X 2    
 b) the double mutants 039X 1  R40X 2 ; R40X 2  R41X 3  and 039X 1  R41X 3  or 
 c) the triple mutant 039X 1  R40X 2  R41X 3 , 
 d) or the corresponding single, double or triple mutants of an amino acid sequence derived from SEQ ID NO:2 with at least 60% sequence identity; 
 
       wherein X 1 , X 2  and X 3  stand, in each case independently of one another, for a mutated amino acid residue. 
     
     
         5 . A recombinant microorganism, which carries at least one nucleic acid sequence coding for a mutant as claimed  claim 1  or at least one corresponding expression cassette or at least one corresponding vector and in addition optionally carries the coding sequence for another enzyme, selected from hydroxysteroid dehydrogenases, such as a 3α-hydroxysteroid dehydrogenase (3α-HSDH), and dehydrogenases suitable for cofactor regeneration. 
     
     
         6 . A process for enzymatic or microbial synthesis of 7β-hydroxysteroids, wherein the corresponding 7-ketosteroid is reacted in the presence of a 7β-HSDH mutant according to the definition in  claim 1  or in the presence of a recombinant microorganism expressing this mutant, and at least one reduction product formed is optionally isolated from the reaction mixture. 
     
     
         7 . The process as claimed in  claim 6 , wherein the reduction takes place in the presence of and especially with consumption of NADPH and/or NADH. 
     
     
         8 . The process as claimed in  claim 7 , wherein spent NADPH is regenerated by coupling with an NADPH-regenerating enzyme, wherein this is selected in particular from NADPH dehydrogenases, alcohol dehydrogenases (ADH), and NADPH-regenerating formate dehydrogenases (FDH) and an NADPH-regenerating glucose dehydrogenase (GDH), wherein the NADPH-regenerating enzyme optionally is expressed by a recombinant microorganism; and/or wherein spent NADH is regenerated by coupling with an NADH-regenerating enzyme, wherein this is selected in particular from NADH-dehydrogenases, NADH-regenerating formate dehydrogenases (FDH), NADH-regenerating alcohol dehydrogenases (ADH), NADH-regenerating glucose-6-phosphate-dehydrogenases (G6PDH), NADH-regenerating phosphite dehydrogenases (PtDH) and NADH-regenerating glucose dehydrogenases (GDH), wherein the NADH-regenerating enzyme optionally is expressed in a recombinant microorganism. 
     
     
         9 . The process as claimed in  claim 8 , wherein the NADPH-regenerating enzyme is selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2 , wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor. 
     
     
         10 . The process as claimed in  claim 9 , wherein the NADP + -accepting FDH mutant has at least one mutation in the amino acid sequence of an FDH from  Mycobacterium vaccae  N10 according to SEQ ID NO:36 or an amino acid sequence derived therefrom with at least 60% sequence identity. 
     
     
         11 . The process as claimed in  claim 9 , wherein the NADP + -accepting mutant has at least one mutation in the sequence motif TDRHRL according to position 221 to 226 of SEQ ID NO:36 or in the corresponding sequence motif of an amino acid sequence derived therefrom with at least 60% sequence identity. 
     
     
         12 . A nucleic acid sequence, selected from nucleic acid sequences:
 a) simultaneously coding for an FDH mutant as claimed in one of  claims 9  to  11  and a 7β-HSDH mutant as claimed in  claim 1  and optionally a 3α-HSDH; or   b) simultaneously coding for an FDH mutant as claimed in one of  claims 9  to  11  and a nonmutated 7β-HSDH wild type and optionally a 3α-HSDH; or   c) coding for a fusion protein comprising an FDH mutant selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2 , wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor and a 7β-HSDH mutant as claimed in  claim 1  and optionally a 3α-HSDH; or   d) coding for a fusion protein comprising an FDH mutant selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2  wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor and a nonmutated 7β-HSDH and optionally a 3α-HSDH; or   e) simultaneously coding for FDH wild type and a 7β-HSDH mutant as claimed in  claim 1  and optionally a 3α-HSDH; or   f) coding for a fusion protein, comprising the FDH wild type, a 7β-HSDH mutant as claimed in  claim 1  and optionally a 3α-HSDH; or   g) simultaneously coding for a GDH, a 7β-HSDH wild type and optionally a 3α-HSDH; or   h) coding for a fusion protein, comprising a GDH, a 7β-HSDH wild type and optionally a 3α-HSDH; or   i) simultaneously coding for a GDH, a 7β-HSDH mutant as claimed in  claim 1  and optionally a 3α-HSDH; or   k) coding for a fusion protein, comprising a GDH, a 7β-HSDH mutant as claimed in  claim 1  and optionally a 3α-HSDH.   
     
     
         13 . A recombinant microorganism, which carries at least one nucleic acid sequence as claimed in  claim 12 . 
     
     
         14 . A recombinant microorganism, which is capable of simultaneous expression of 7β-HSDH (wild type), an NADP + -accepting FDH mutant and/or the corresponding FDH wild type and optionally of 3α-HSDH; or which is capable of simultaneous expression of 7β-HSDH wild type, a GDH and optionally of 3α-HSDH. 
     
     
         15 . A recombinant microorganism, which is capable of simultaneous expression of a 7β-HSDH mutant, an NADP + -accepting FDH mutant and/or the corresponding FDH wild type and optionally of 3α-HSDH; or which is capable of simultaneous expression of a 7β-HSDH mutant, a GDH and optionally of 3α-HSDH. 
     
     
         16 . The recombinant microorganism as claimed in  claim 14 , wherein the FDH mutant is a mutant selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2  wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor; and wherein the FDH wild type is an FDH from  Mycobacterium vaccae  N10 according to SEQ ID NO:36 or an FDH derived therefrom with at least 60% sequence identity. 
     
     
         17 . A process for preparing ursodeoxycholic acid (UDCA) of formula (1) 
       
         
           
           
               
               
           
         
         in which 
         R stands for alkyl, NR 1 R 2 , H, an alkali metal ion or N(R 3 ) 4   + , in which the residues R 3  may be identical or different and stand for H or alkyl, 
         wherein
 a) optionally a cholic acid (CA) of formula (2) 
 
       
       
         
           
           
               
               
           
         
         in which R has the meanings given above, is oxidized chemically to dehydrocholic acid (DHCA) of formula (3) 
       
       
         
           
           
               
               
           
         
         in which R has the meanings given above;
 b) DHCA is reduced in the presence of at least one 7β-HSDH mutant according to the definition in  claim 1  and in the presence of at least one 3α-HSDH to the corresponding 12-keto-ursodeoxycholic acid (12-keto UDCA) of formula (5) 
 
       
       
         
           
           
               
               
           
         
         in which R has the meanings given above, especially in the presence of and with consumption of NADH and/or NADPH 
         and then
 c) 12-keto-UDCA of formula (5) is reduced chemically to UDCA; and 
 d) the reaction product optionally is further purified. 
 
       
     
     
         18 . The process as claimed in  claim 17 , wherein at least step b) is carried out in the presence of a recombinant microorganism that carries at least one nucleic acid sequence coding for a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant, which catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the nonmutated enzyme, a decreased substrate inhibition, especially for the 7-ketosteroid substrate, and/or an altered cofactor usage, and the nonmutated enzyme has the amino acid sequence of a 7β-HSDH from a bacterium of the genus  Collinsella  and the mutant has, in comparison with the nonmutated enzyme, 1 to 15 amino acid additions, substitutions, deletions and/or inversions or at least one corresponding expression cassette or at least one corresponding vector and in addition optionally carries the coding sequence for another enzyme, selected from hydroxysteroid dehydrogenases, such as a 3α-hydroxysteroid dehydrogenase (3α-HSDH), and dehydrogenases suitable for cofactor regeneration. 
     
     
         19 . The process as claimed in  claim 17 , wherein step b) is coupled with identical or different cofactor regeneration systems. 
     
     
         20 . The process as claimed in  claim 19 , wherein step b), 7β-HSDH partial step, is coupled to a cofactor regeneration system, in which spent NADPH is regenerated by an NADP + -accepting FDH mutant according to the definition in one of  claims 9  to  11  with consumption of formic acid or a salt thereof; or is coupled to a cofactor regeneration system in which spent NADPH is regenerated by an ADH with consumption of isopropanol; or is coupled to a cofactor regeneration system in which spent NADPH is regenerated by a GDH with consumption of glucose; or is coupled to a cofactor regeneration system in which spent NADH is regenerated by an NADH-regenerating GDH, ADH or FDH. 
     
     
         21 . The process as claimed in  claim 19 , wherein step b), 3α-HSDH partial step, is coupled to a cofactor regeneration step, in which NADPH is regenerated by an NADP + -accepting FDH mutant selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2  wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor with consumption of formic acid or a salt thereof; or wherein step b) is coupled to a cofactor regeneration step, in which NADH is regenerated by an NAD + - and NADP + -accepting FDH mutant selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2  wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor or by the nonmutated FDH in each case with consumption of formic acid or a salt thereof, or by an NAD + -accepting GDH with consumption of glucose. 
     
     
         22 . A process for preparing UDCA of formula (1) 
       
         
           
           
               
               
           
         
         in which 
         R stands for alkyl, NR 1 R 2 , H, an alkali metal ion or N(R 3 ) 4   + , in which the residues R 3  may be identical or different and stand for H or alkyl, 
         wherein
 a) optionally a CA of formula (2) 
 
       
       
         
           
           
               
               
           
         
         in which R has the meanings given above, is oxidized chemically to the DHCA of formula (3) 
       
       
         
           
           
               
               
           
         
         in which R has the meanings given above;
 b) DHCA is reduced in the presence of at least one 7β-HSDH and in the presence of at least one 3α-HSDH to the corresponding 12-keto-UDCA of formula (5) 
 
       
       
         
           
           
               
               
           
         
         in which R has the meanings given above, especially in the presence of and with consumption of NADH and/or NADPH and then
 c) 12-keto-UDCA of formula (5) is reduced chemically to UDCA; and 
 d) the reaction product optionally is further purified; 
 
         wherein the reactions of step b) take place in the presence of a recombinant microorganism that carries at least one nucleic acid sequence as claimed in  claim 12 , or using at least one nucleic acid sequence as claimed in  claim 12 . 
       
     
     
         23 . The process as claimed in  claim 22 , wherein a recombinant microorganism that is capable of simultaneous expression of 7β-HSDH (wild type), an NADP + -accepting FDH mutant and/or the corresponding FDH wild type and optionally of 3α-HSDH; or which is capable of simultaneous expression of 7β-HSDH wild type, a GDH and optionally of 3α-HSDH is used, which simultaneously expresses at least one 7β-HSDH mutant as claimed in  claim 1 , at least one NADP + -accepting FDH mutant selected from mutants of an NAD + -dependent formate dehydrogenase (FDH) which at least catalyzes the enzymatic oxidation of formic acid to CO 2 , wherein the mutant, compared to the nonmutated enzyme, additionally accepts NADP +  as cofactor and at least one 3α-HSDH; or simultaneously expresses at least one 7β-HSDH mutant as claimed in  claim 1 , at least one GDH and at least one 3α-HSDH.