7 beta-Hydroxysteroid dehydrogenase (7 beta-HSDH) is a key enzyme for the efficient biosynthesis of ursodeoxycholic acid (UDCA), an effective pharmaceutical for primary biliary cirrhosis and human cholesterol gallstones. In this work, a new 7 beta-HSDH from Ruminococcus torques ATCC 35915, designated as 7 beta-HSDHRt, was identified and heterologously overexpressed in Escherichia coli for the enzymatic synthesis of ursodeoxycholic acid from chenodeoxycholic acid (COCA). 7 beta-HSDHRt was firstly employed in one-pot mode together with 7 alpha-HSDHCa, another NADPH-dependent 7 alpha-HSDH from Clostridium absonum, to convert CDCA into UDCA without additional coenzyme regeneration. However, the final yield was limited to merely 73%, probably due to chemical equilibrium. Therefore, to enhance the UDCA yield, we alternatively adopted a two-step reaction strategy where the enzymes involved in the first reaction were simply heat-inactivated between the 1st-step reaction (dehydrogenation) and the 2nd-step reaction (hydrogenation), in order to prevent the undesired bioreduction of 7-oxo-LCA into CDCA in the 2nd step. Consequently, the analytic yield of UDCA was significantly improved up to above 98% at a substrate load of 10 mM (Co. 4 g L-1), without any detectable intermediate (7-oxo-LCA) as observed in the case of one-pot reaction. (C) 2015 Elsevier Ltd. All rights reserved.