One aspect of the invention provides a nucleic acid encoding a 7β-hydroxysteroid dehydrogenase (7β-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 7β-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 7β-hydroxysteroid dehydrogenase (7β-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 7β-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.

Biotransformation of medroxyprogesterone acetate (MPA) (1) with Cunninghamella blakesleeana (ATCC 8688) yielded five new analogues, i.e. 17α-acetoxy-6α-methylpregn-4-ene-3,11,20-trione (2), 17α-acetoxy-15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 17α-acetoxy-6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 17α-acetoxy-11β,15β-dihydroxy-6α-methylpregn-4-ene-3,20-dione (5), and 17α-acetoxy-6β,11β-dihydroxy-6α-methylpregn-4-ene-3,20-dione (6). In T-cell proliferation assay, metabolites 2, and 5 were found to be potent inhibitors with IC.sub.50<0.5 μM, metabolite 6 showed a significant activity with IC.sub.50=8.64±0.02 μM, while metabolites 3 and 4 were found to be moderately active with IC.sub.50=41.59±8.14, and 40.14±0.12 μM, as compared to substrate 1 (IC.sub.50=6.48±5.18 μM) and standard prednisolone (IC.sub.50=9.75±0.03 μM) in in vitro assay. To establish the binding mode of medroxyprogesterone acetate (MPA) and the bio-transformed derivatives, molecular docking simulations were carried out using Vina.

The present invention concerns a genetically modified bacterium and to its industrial application, in particular in the 1,2-dehydrogenation of steroids.

The present invention concerns a genetically modified bacterium and to its industrial application, in particular in the 1,2-dehydrogenation of steroids.

The invention provides novel 3α-hydroxysteroid dehydrogenase mutants, sequences that code for these enzyme mutants, methods for producing the enzyme mutants, and 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 3α-hydroxysteroid dehydrogenase mutants, sequences that code for these enzyme mutants, methods for producing the enzyme mutants, and 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 present invention belongs to the field of bioengineering technologies, and in particular, to a production method of recombinant Escherichia coli (E. coli) and high-purity ursodeoxycholic acid (UDCA). The present invention constructs novel double-enzyme co-expression gene engineered bacteria, that is, recombinant E. coli. The bacteria simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH). The bacteria is applicable to the production of high-purity UDCA. The yield of a target product is increased through the joint expression and application of 7β-HSDH and GDH. The production method of high-purity UDCA in the present invention is simple, generates a small amount of impurities in a production process, is a green process that satisfies environmental protection requirements, and has significant industrial application value.

The present invention is related to an improved method for production of 7-dehydrocholesterol (7-DHC), an important intermediate towards biotechnological production of vitamin D3 or derivatives/metabolites thereof. The invention features modified host strains expressing enzymes having improved C-5 sterol 5 desaturase activity and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.

The present invention is related to an improved method for production of 7-dehydrocholesterol (7-DHC), an important intermediate towards biotechnological production of vitamin D3 or derivatives/metabolites thereof. The invention features modified host strains expressing enzymes having improved C-5 sterol 5 desaturase activity and their use in a process for production of vitamin D3 or derivatives and/or metabolites thereof.