US 2020/0407766 A1Published application

Coupled, Self-Sufficient Biotransformation of Chenodeoxcholic Acid to Ursodeoxycholic Acid and Novel Enzyme Mutants Applicable in said Process

AssigneePharmaZell GmbH

InventorsDaniel Bakonyi, Werner Hummel, Ralf Gross

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Abstract

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+-dependent 7α-hydroxysteroid dehydrogenase (7α-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+-dependent 7α-HSDH of E. coli and their application for the oxidation of bile acids.

Claims24

Dependent claims

A coupled biocatalytic process for preparing an ursodeoxycholic acid (UDCA) compound of the general Formula (1),

wherein
R represents alkyl, H an alkali metal ion or N(R3)4+ wherein residue R3 are the same or different and represent H or alkyl, or wherein group —CO2R is replaced by an acid amide group CONR1R2, wherein R1 and R2 independently of each other represent an alkyl residue;
which method comprises
a) reacting a chenodeoxycholic acid (CDCA) compound of general Formula (2)
wherein
R has the same meanings as defined above for group —CO2R or is replaced by an acid amide group —CONR1R2,
in the presence of a 7α-hydroxysteroid dehydrogenase (7α-HSDH) and a 7ß-hydroxysteroid dehydrogenase (7β-HSDH) and a cofactor selected from NAD+ and NADP+, wherein said 7α-HSDH and said 7β-HSDH have the ability of utilizing the same cofactor system selected from NAD+/NADH and NADP+/NADPH, and wherein
a1) said 7α-HSDH catalyzes the oxidation of said CDCA compound of general Formula (2) to the corresponding intermediate 7-ketolithocholic acid (7-KLCA) compound of general Formula (3)
wherein R is as identified above or wherein group —CO2R or is replaced by an acid amide group —CONR1R2 as defined above,
and
a2) said 7β-HSDH catalyzes the reduction of said 7-KLCA compound of general Formula (3) as formed in reaction step a1) to said UDCA compound of general Formula (1) and under regeneration of the cofactor consumed in reaction step a1);
and
b) optionally further purifying the reaction product.

10 more elements

The process of one of the preceding claims wherein said 7α-HSDH and said 7β-HSDH both utilize the cofactor system NAD+/NADH;

wherein
a) said 7α-HSDH is selected from
(1) a 7α-HSDH comprising an amino acid sequence according to SEQ ID NO:
37 which is isolated from Escherichia coli, which catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids, and a mutant thereof having at least 80% sequence identity to SEQ ID NO:
37 and retaining the ability to utilize NAD+/NADH and the ability to catalyze at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids;
and
(2) a 7α-HSDH which is a mutant of Clostridium difficile 7α-HSDH with SEQ ID NO:
34 which mutant catalyzes at least the stereospecific enzymatic oxidation of 7α-hydroxysteroids to the corresponding 7-ketosteroids under consumption of NAD+ as cofactor, wherein said 7α-HSDH comprises at least one mutation in a position selected from K16, A37 and R38 of SEQ ID NO:
34 and shows a sequence identity of at least 80% to SEQ ID NO:34
b) said 7β-HSDH is selected from
(1) a 7ß-HSDH, which is a mutant of Collinsella aerofaciens 7β-HSDH with SEQ ID NO:
54, which mutant catalyzes at least the stereospecific enzymatic reduction of a 7-ketosteroid to the corresponding 7β-hydroxysteroid under consumption of NADH as cofactor, wherein said 7β-HSDH comprises at least one mutation in a position selected from T17, G39, R40, R41 and K44 of SEQ ID NO:
54 and shows a sequence identity of at least 80% to SEQ ID NO:54;

10 more elements

Dependencies are inferred from the claim text.