Clean enzymatic production of ursodeoxycholic acid enabled by a newly identified NADH-dependent 7β-hydroxysteroid dehydrogenase

7β-Hydroxysteroid dehydrogenases (7β-HSDHs) play an important role in the enzymatic synthesis of ursodeoxycholic acid (UDCA), which is a value-added compound with a range of pharmacological activities. Since the cofactor NADH is much cheaper than NADPH, the production of UDCA using an NADH-dependent 7β-HSDH has better prospects than that using an NADPH-dependent 7β-HSDH. However, a major bottleneck in UDCA biosynthesis is the poor catalytic activity of the current NADH-dependent 7β-HSDHs. In this work, a new and natively NADH-dependent Rs 7β-HSDH from Roseococcus sp. was identified, which showed a high specific activity of 63.3 U mg −1 protein toward 7-oxo-lithocholic acid, with a catalytic efficiency ( k cat / K M ) of 515 mM –1 s –1 . In a preparative biotransformation (100-mL scale) using Rs 7β-HSDH and an NAD + -dependent 7α-HSDH, with O 2 /NOX and HCOO − /FDH systems for the regeneration of cofactors (NAD + and NADH), 25 mM chenodeoxycholic acid was completely converted to UDCA in one-pot two-step cascade, with an 80% isolated yield and a total turnover number (TTN) of 6586 for Rs 7β-HSDH. The environmental factor ( E -factor) of this process was 8.37 when water was excluded, much lower than those obtained from other processes reported so far, indicating a great potential of this Rs 7β-HSDH for more sustainable and cleaner enzymatic production of UDCA.