The disclosure discloses 17β-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17β-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17β-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17β-hydroxysteroid dehydrogenase (1.66 U/mg).

The disclosure discloses 17β-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17β-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17β-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17β-hydroxysteroid dehydrogenase (1.66 U/mg).

In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the wildtype 7β-HSDH of SEQ ID NO:2, a decreased substrate inhibition and/or an altered cofactor usage, and the mutant has, in comparison with the wildtype 7β-HSDH of SEQ ID NO:2, 1 to 15 amino acid additions, substitutions, deletions and/or inversions in the sequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ ID NO:2.

In various aspects and embodiments, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a 7β-hydroxysteroid dehydrogenase (7β-HSDH) mutant that catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7-hydroxysteroid, wherein the mutant has, compared to the wildtype 7β-HSDH of SEQ ID NO:2, a decreased substrate inhibition and/or an altered cofactor usage, and the mutant has, in comparison with the wildtype 7β-HSDH of SEQ ID NO:2, 1 to 15 amino acid additions, substitutions, deletions and/or inversions in the sequence motif VMVGRRE corresponding to positions 36 to 42 of SEQ ID NO:2.

The invention relates to novel 7-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.

The invention relates to novel 7-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.

The disclosure discloses 17-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17-hydroxysteroid dehydrogenase (1.66 U/mg).

The disclosure discloses 17-hydroxysteroid dehydrogenase mutants and application thereof, and belongs to the technical field of biology. The disclosure provides 17-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y with high specific enzyme activities, and the specific enzyme activities of the 17-hydroxysteroid dehydrogenase mutants V107A, T155N, H164Y and V107A/T155N/H164Y are as high as 1.85, 1.93, 2.06 and 5.15 U/mg, respectively, which are 1.11, 1.16, 1.24 and 3.10 times larger than that of wild-type 17-hydroxysteroid dehydrogenase (1.66 U/mg).

The invention provides a method for increasing the yield of 7-dehydrocholesterol in yeast by using compartmentalization. The method includes steps of: by taking yeast as a starting strain, expressing heterologous sterol delta 24-reductase and cholestenol delta-isomerase, wherein the yield of 7-DHC in Saccharomyces cerevisiae S288C is detected to be 10.15 mg/L. According to the method of the invention, partial enzymes in a 7-DHC synthesis path are positioned in compartments in Saccharomyces cerevisiae by using a peroxisome and a mitochondrial positioning tag, and a relatively independent 7-DHC synthesis path is formed. Meanwhile, the storage space of precursor substances needed by 7-DHC synthesis is increased, the feedback effect is reduced, the conversion efficiency between enzymes is improved in the same compartment, the loss of the acting substrate is reduced, and finally the yield of the 7-DHC is improved by 4 times and reaches 53.31 mg/L.

The invention provides a method for increasing the yield of 7-dehydrocholesterol in yeast by using compartmentalization. The method includes steps of: by taking yeast as a starting strain, expressing heterologous sterol delta 24-reductase and cholestenol delta-isomerase, wherein the yield of 7-DHC in Saccharomyces cerevisiae S288C is detected to be 10.15 mg/L. According to the method of the invention, partial enzymes in a 7-DHC synthesis path are positioned in compartments in Saccharomyces cerevisiae by using a peroxisome and a mitochondrial positioning tag, and a relatively independent 7-DHC synthesis path is formed. Meanwhile, the storage space of precursor substances needed by 7-DHC synthesis is increased, the feedback effect is reduced, the conversion efficiency between enzymes is improved in the same compartment, the loss of the acting substrate is reduced, and finally the yield of the 7-DHC is improved by 4 times and reaches 53.31 mg/L.