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 72mM dehydrocholic acid was thus made possible at pH6.4 in a whole-cell batch process within a process time of 1h 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 50mM 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.