7 α-hydroxysteroid dehydrogenase knockout mutants and use therefor

摘要

The invention relates to 12 α-hydroxysteroid dehydrogenases, nucleic acid sequences coding for the same, expression cassettes and vectors, recombinant microorganisms containing the corresponding coding nucleic acid sequences, a method for producing said 12 α-hydroxysteroid dehydrogenases, a method for enzymatic oxidation of 12 α-hydroxysteroids using said enzyme, a method for enzymatic reduction of 12-ketosteroids using said enzyme, a method for qualitative or quantitative determination of 12-ketosteroids and/or 12α-hydroxysteroids using said 12α-hydroxysteroid dehydrogenases and a method for production of ursodesoxycholic acid, comprising the enzyme-catalyzed cholic acid oxidation using said 12 α-hydroxysteroid dehydrogenases.

主权项

1. A recombinant microorganism of the genus Escherichia wherein the enzymatic activity of 7α-hydroxysteroid dehydrogenase (7α-HSDH) is inhibited, while the enzymatic activity of at least one functionally different hydroxysteroid dehydrogenase (HSDH) is contained expressibly wherein:
the expressed HSDH functionally different from the 7α-HSDH contains one of the following amino acid sequences: a) a 3α-HSDH sequence selected from SEQ ID NO: 29 and 31; b) a 7β-HSDH sequence selected from SEQ ID NO: 25; c) a 12α-HSDH sequence selected from SEQ ID NO: 12, 14, 16; or d) an amino acid sequence derived therefrom, coding for an HSDH having a degree of identity of at least 90% to one of the sequences under a), b) and c); andwherein the recombinant microorganism is obtained from a precursor microorganism that endogenously expresses 7α-HSDH.