US 9,096,825 B2Granted

7 α-hydroxysteroid dehydrogenase knockout mutants and use therefor

AssigneePharmaZell GmbH

InventorsRolf Schmid, Michael Braun, Luo Liu, Arno Aigner, Dirk Weuster-Botz

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Abstract

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.

Claims22

Dependent claims

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);
and
wherein the recombinant microorganism is obtained from a precursor microorganism that endogenously expresses 7α-HSDH.

8 more elements

Dependencies are inferred from the claim text.