The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP.sup.+-dependent 7-hydroxysteroid dehydrogenase (7-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP.sup.+-dependent 7-HSDH of E. coli and their application for the oxidation of bile acids.

The present invention relates to a coupled biotransformation process of converting chenodeoxycholic acid (CDCA) and related compounds to ursodeoxycholic acid (UDCA) and related compounds. It also relates to the cloning, expression, and biochemical characterization of a novel NADP.sup.+-dependent 7-hydroxysteroid dehydrogenase (7-HSDH) from Clostridium difficile, cofactor switch mutants thereof, and their application for the oxidation of bile acids. A further aspect of the invention relates to novel NADP-dependent cofactor switch mutants of the NADP.sup.+-dependent 7-HSDH of E. coli and their application for the oxidation of bile acids.

One aspect of the invention provides a nucleic acid encoding a 7-hydroxysteroid dehydrogenase (7-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid. The enzyme includes a mutation at position 64 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 90% sequence identity to SEQ ID NO:2. The mutation at position 64 is the mutation R64X.sub.1, wherein X.sub.1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y. The enzyme shows the following property profile in comparison with the 7-HSDH with SEQ ID NO:2: (a) an increased specific activity (Vmax [U/mg]) for NADPH in the enzymatic reduction of dehydrocholic acid (DHCA) with NADPH as cofactor.

One aspect of the invention provides a nucleic acid encoding a 7-hydroxysteroid dehydrogenase (7-HSDH) that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid. The enzyme includes a mutation at position 64 of SEQ ID NO:2 or in the corresponding sequence positions of an amino acid sequence derived therefrom with at least 90% sequence identity to SEQ ID NO:2. The mutation at position 64 is the mutation R64X.sub.1, wherein X.sub.1 represents E, D, T, L, S, P, V, K, C, A, G, Q, F, W, I or Y. The enzyme shows the following property profile in comparison with the 7-HSDH with SEQ ID NO:2: (a) an increased specific activity (Vmax [U/mg]) for NADPH in the enzymatic reduction of dehydrocholic acid (DHCA) with NADPH as cofactor.

The present invention relates to a genetically-engineered Mycobacterium strain and a use thereof in the preparation of steroidal compounds. The genetically-engineered Mycobacterium strain is a Mycobacteria which lacks of acyl-CoA dehydrogenase genes fadE31, fadE32 and fadE33, wherein acyl-CoA dehydrogenase genes fadE31, fadE32 and fadE33 respectively encode proteins as follows: having amino acid sequences according to SEQ ID NOs 4, 6 and 8; derived by substituting, deleting or inserting one or more amino acids in the amino acid sequence defined by preceding protein and having the same function as that of the preceding protein. The present invention constructs a genetically-engineered Mycobacterium strain and applies it in preparing steroidal compounds, thereby enriching the types of valuable intermediates, improving the production efficiency and product quality of steroid drugs, reducing energy consumption in the steroid drugs production, simplifying production steps, and reducing production costs.

The present invention relates to a genetically-engineered Mycobacterium strain and a use thereof in the preparation of steroidal compounds. The genetically-engineered Mycobacterium strain is a Mycobacteria which lacks of acyl-CoA dehydrogenase genes fadE31, fadE32 and fadE33, wherein acyl-CoA dehydrogenase genes fadE31, fadE32 and fadE33 respectively encode proteins as follows: having amino acid sequences according to SEQ ID NOs 4, 6 and 8; derived by substituting, deleting or inserting one or more amino acids in the amino acid sequence defined by preceding protein and having the same function as that of the preceding protein. The present invention constructs a genetically-engineered Mycobacterium strain and applies it in preparing steroidal compounds, thereby enriching the types of valuable intermediates, improving the production efficiency and product quality of steroid drugs, reducing energy consumption in the steroid drugs production, simplifying production steps, and reducing production costs.

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

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

The invention provides novel 3-hydroxysteroid dehydrogenase mutants, sequences that code for these enzyme mutants, methods for producing the enzyme mutants, and o the use thereof in enzymatic reactions of cholic acid compounds, and in particular in the production of ursodeoxycholic acid (UDCA). The invention further provides processes for the synthesis of UDCA using the enzyme mutants and the production of UDCA using recombinant microorganisms that have been subjected to multiple modifications.

The invention provides novel 3-hydroxysteroid dehydrogenase mutants, sequences that code for these enzyme mutants, methods for producing the enzyme mutants, and o the use thereof in enzymatic reactions of cholic acid compounds, and in particular in the production of ursodeoxycholic acid (UDCA). The invention further provides processes for the synthesis of UDCA using the enzyme mutants and the production of UDCA using recombinant microorganisms that have been subjected to multiple modifications.