Ursodeoxycholic acid (UDCA) is a bile acid of industrial interest as it is used as an agent for the treatment of primary sclerosing cholangitis and the medicamentous, non-surgical dissolution of gallstones. Currently, it is prepared industrially from cholic acid following a seven-step chemical procedure with an overall yield of <30%. In this study, we investigated the key enzymatic steps in the chemo-enzymatic preparation of UDCAthe two-step reduction of dehydrocholic acid (DHCA) to 12-keto-ursodeoxycholic acid using a mutant of 7 beta-hydroxysteroid dehydrogenase (7 beta-HSDH) from Collinsella aerofaciens and 3a-hydroxysteroid dehydrogenase (3a-HSDH) from Comamonas testosteroni. Three different one-pot reaction approaches were investigated using whole-cell biocatalysts in simple batch processes. We applied one-biocatalyst systems, where 3a-HSDH, 7 beta-HSDH, and either a mutant of formate dehydrogenase (FDH) from Mycobacterium vaccae N10 or a glucose dehydrogenase (GDH) from Bacillus subtilis were expressed in a Escherichia coli BL21(DE3) based host strain. We also investigated two-biocatalyst systems, where 3a-HSDH and 7 beta-HSDH were expressed separately together with FDH enzymes for cofactor regeneration in two distinct E. coli hosts that were simultaneously applied in the one-pot reaction. The best result was achieved by the one-biocatalyst system with GDH for cofactor regeneration, which was able to completely convert 100?mM DHCA to >99.5?mM 12-keto-UDCA within 4.5?h in a simple batch process on a liter scale. Biotechnol. Bioeng. 2013; 110: 6877. (C) 2012 Wiley Periodicals, Inc.