title: Novel whole-cell biocatalysts with recombinant hydroxysteroid dehydrogenases for the asymmetric reduction of dehydrocholic acid.
abstract: Ursodeoxycholic acid is an important pharmaceutical so far chemically synthesized from cholic acid. Various biocatalytic alternatives have already been discussed with hydroxysteroid dehydrogenases (HSDH) playing a crucial role. Several whole-cell biocatalysts based on a 7α-HSDH-knockout strain of Escherichia coli overexpressing a recently identified 7β-HSDH from Collinsella aerofaciens and a NAD(P)-bispecific formate dehydrogenase mutant from Mycobacterium vaccae for internal cofactor regeneration were designed and characterized. A strong pH dependence of the whole-cell bioreduction of dehydrocholic acid to 3,12-diketo-ursodeoxycholic acid was observed with the selected recombinant E. coli strain. In the optimal, slightly acidic pH range dehydrocholic acid is partly undissolved and forms a suspension in the aqueous solution. The batch process was optimized making use of a second-order polynomial to estimate conversion as function of initial pH, initial dehydrocholic acid concentration, and initial formate concentration. Complete conversion of 72 mM dehydrocholic acid was thus made possible at pH 6.4 in a whole-cell batch process within a process time of 1 h without cofactor addition. Finally, a NADH-dependent 3α-HSDH from Comamonas testosteroni was expressed additionally in the E. coli production strain overexpressing the 7β-HSDH and the NAD(P)-bispecific formate dehydrogenase mutant. It was shown that this novel whole-cell biocatalyst was able to convert 50 mM dehydrocholic acid directly to 12-keto-ursodeoxycholic acid with the formation of only small amounts of intermediate products. This approach may be an efficient process alternative which avoids the costly chemical epimerization at C-7 in the production of ursodeoxycholic acid.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Braun
authors.forename: Boqiao
authors.initials: B
authors.lastname: Sun
authors.forename: Bernd
authors.initials: B
authors.lastname: Anselment
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
mesh_terms: D039903:Actinobacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D003685:Dehydrocholic Acid / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods*; D007700:Kinetics; D015394:Molecular Structure; D009161:Mycobacterium / Q000201:enzymology; D010084:Oxidation-Reduction; D014580:Ursodeoxycholic Acid / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014580:Ursodeoxycholic Acid; D006913:Hydroxysteroid Dehydrogenases; D005560:Formate Dehydrogenases; D003685:Dehydrocholic Acid