Efficient Synthesis of 12‐Oxochenodeoxycholic Acid Using a 12α‐Hydroxysteroid Dehydrogenase from Rhodococcus ruber

Abstract

Abstract12α‐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 (Rr12α‐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. Rr12α‐HSDH was heterologously overexpressed in Escherichia coli BL21 (DE3), purified and characterized. The purified Rr12α‐HSDH showed a high oxidative activity of 290 U mg−1protein toward CA, with a catalytic efficiency (kcat/KM) of 5.10×103 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, Rr12α‐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 Rr12α‐HSDH as a promising biocatalyst for the synthesis of various therapeutic bile acids.magnified image

Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

“…Thanks to these interesting features, HSDHs have been widely studied during the last years for their exploitation in the biocatalyzed synthesis of key intermediates of the drug ursodeoxycholic acid (UDCA), a largely applied therapeutic agent used for the dissolution of cholesterol gallstones and for the treatment of different hepatic diseases. These efforts have led to the characterization of different enzymes showing either 7α‐, 7β‐ or 12α‐HSDH activity, as well as to the development of biocatalyzed processes for the preparation of UDCA intermediates …”

Discovery and Engineering of a Novel Baeyer‐Villiger Monooxygenase with High Normal Regioselectivity

“…BVMO with higher activity and regioselectivity will significantly reduce the formation of by‐products, and simplify downstream separation and purification steps. The protein sequence of Pp BVMO has been employed as a probe to perform pBLAST in the NCBI database [34,35] . Pp BVMO has shown the highest catalytic activity among the four BVMOs reported, with normal selectivity toward 10‐ketostearic acid, and has been widely used in the biotransformation of unsaturated fatty acids.…”

Recent trends in biocatalysis

“…Industrially relevant steroid modifications are Baeyer-Villiger oxidations, 354,355 hydrogenation and dehydrogenation of CQC and C-C bonds, respectively, 337,[356][357][358] and alcohol/carbonyl group interconversions. [359][360][361][362] Several wild-type organisms are used at industrial scales to perform these functionalisation reactions. 333,335,363 Of particular interest are direct hydroxylations of inert C-H bonds as carried out, for example, by P450s, a superfamily of heme-containing enzymes.…”

“…The responsible enzyme, a 12a-HSDH from Rhodococcus ruber, was identified using a structure-guided genome mining approach and is applied as lyophilised E. coli whole-cell powder during the process, yielding 12-oxo-CDCA. 362,409 The second example is the exploitation of a metabolically engineered S. cerevisiae strain harboring an artificial biosynthetic pathway consisting of four mammalian P450s. The heterologous cascade reaction yields cortisol from glucose by mimicking human steroid biosynthesis (Scheme 8).…”

“…410,411 The lack of steroid-modifying enzymes as bottleneck has been overcome with the continuous discovery of new P450s from microbial but also eukaryotic sources. 362,[412][413][414][415][416][417] Recently, Szaleniec et al reviewed P450s for the degradation of cholesterol (CYP125 and CYP142 family) and steroid hydroxylations (CYP106A, CYP109, CYP154, and CYP260), as well as Riesketype monooxygenases, 3-ketosteroid 9a-hydroxylases, and molybdenum-containing steroid C25-dehydrogenases as alternative (bacterial) steroid hydroxylases. 344 The impressive successful engineering of CYPs complements the steroid hydroxylations found in nature.…”