Cytochrome P450 125 (CYP125) catalyses C26-hydroxylation to initiate sterol side-chain degradation in Rhodococcus jostii RHA1

The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3-hydroxysterols such as cholesterol, but not on 3-oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side-chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side-chain. The RG32Ωcyp125 mutant failed to transform the side-chain of cholesterol, but degraded that of 5-cholestene-26-oic acid-3β-ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125RHA1 was < 10% in the low-spin state, cholesterol (KDapp = 0.20 ± 0.08 µM), 5α-cholestanol (KDapp = 0.15 ± 0.03 µM) and 4-cholestene-3-one (KDapp = 0.20 ± 0.03 µM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26-carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.

Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme

In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the steroid with >95 % regioselectivity to form 16-β-OH norandrostenedione, as confirmed by NMR analysis. Whole cells of R. erythropolis RG9 expressing P450 BM3 M02 enzyme also converted norandrostenedione into the 16-β-hydroxylated product, resulting in the formation of about 0.35 g/L. Whole cells of strain RG9 itself did not convert norandrostenedione, indicating that metabolite formation is P450 BM3 M02 enzyme mediated. This study shows that R. erythropolis is a novel and interesting host for the heterologous expression of highly selective and active P450 BM3 M02 enzyme variants able to perform steroid bioconversions