Efficient Synthesis of 12‐Oxochenodeoxycholic Acid Using a 12α‐Hydroxysteroid Dehydrogenase from <i>Rhodococcus ruber</i>

Abstract

Abstract 12α‐Hydroxysteroid dehydrogenase (12α‐HSDH) has the potential to convert cheap and readily available cholic acid (CA) to 12‐oxochenodeoxycholic acid (12‐oxo‐CDCA), a key precursor for chemoenzymatic synthesis of the therapeutic bile acid ursodeoxycholic acid (UDCA). In this work, a native nicotinamide adenine dinucleotide (NAD + )‐dependent 12α‐hydroxysteroid dehydrogenase ( Rr 12α‐HSDH) from Rhodococcus ruber was identified using a structure‐guided genome mining (SSGM) approach, which is based on the structure of cofactor pocket and the conserved nicotinamide cofactor binding motif alignment. Rr 12α‐HSDH was heterologously overexpressed in Escherichia coli BL21 (DE3), purified and characterized. The purified Rr 12α‐HSDH showed a high oxidative activity of 290 U mg −1 protein toward CA, with a catalytic efficiency ( k cat / K M ) of 5.10×10 3 mM −1 s −1 . In a preparative biotransformation (100 mL), CA (200 mM, 80 g L −1 ) was efficiently converted to 12‐oxo‐CDCA in 1 h, with a 85% isolated yield and a space‐time yield (STY) of up to 1632 g L −1 d −1 . Furthermore, Rr 12α‐HSDH was shown to be able to catalyze the oxidation of other 12α‐hydroxysteroids at high substrate loads (up to 200 mM), giving the corresponding 12‐oxo‐hydroxysteroids in 71%–85% yields, indicating the great potential of Rr 12α‐HSDH as a promising biocatalyst for the synthesis of various therapeutic bile acids. magnified image