Molecular and functional characterization of the kstD2 gene of Rhodococcus erythropolis SQ1 encoding a second 3-ketosteroid Δ1-dehydrogenase isoenzyme b bThe GenBank accession number for the sequence reported in this paper is AY078169

Abstract

Previously, Rhodococcus erythropolis SQ1 kstD, encoding ketosteroid ∆ 1 -dehydrogenase (KSTD1) was characterized. Surprisingly, a kstD gene deletion mutant (strain RG1) grew normally on steroids. UV mutagenesis of strain RG1 allowed isolation of strains (e.g. strain RG1-UV29) unable to perform the ∆ 1 -dehydrogenation of 4-androstene-3,17-dione (AD) and 9α-hydroxy-4-androstene-3,17-dione (9OHAD). Functional complementation of strain RG1-UV29 with total genomic DNA of strain RG1 resulted in identification of a 1698 nt ORF (kstD2) showing clear similarity (35 % identity at amino acid sequence level) with KSTD1. Expression of kstD2 in Escherichia coli resulted in high KSTD2 activity levels. Single gene deletion mutants of either kstD (strain RG1) or kstD2 (strain RG7) appeared unaffected in growth on the steroid substrates AD, 1,4-androstadiene-3,17-dione and 9OHAD. Strain RG7, but not strain RG1, showed temporary accumulation of 9OHAD during AD conversion. A kstD kstD2 double deletion mutant (strain RG8) was constructed. Strain RG8 was unable to grow on steroid substrates, had undetectable steroid ∆ 1 -dehydrogenation activity and efficiently converted AD into 9OHAD. Strain SQ1 thus employs two KSTD isoenzymes in steroid catabolism. Analysis of two null mutants in KSTD2 showed that the semi-conserved Ser325 and the highly conserved Thr503 play a role in KSTD enzyme activity.

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

“…However, it is also true that other reductase enzymes present in the cell could replace their function. Nevertheless, we also decided to delete kshB1 because the effect of the deletion of the kshB homologous gene has already been analysed in Rhodococcus (van der Geize et al ., 2002a,b) and we would like to compare the performance of M. smegmatis having the same gene deletion.…”

“…To explain this performance, it was proposed that the KshB reductase from R. erythropolis was not only involved in the production of 9OH‐AD, but also in sterol side‐chain removal (van der Geize et al ., 2002b). In M. smegmatis , the absence of KshB1 does not hinder the complete degradation of the sterol side‐chain (see Discussion).…”

“…This observation contrasts with other cholesterol‐degrading bacteria, where several KstD dehydrogenases are involved in the metabolism of sterols (van der Geize et al ., 2000, 2002b; Fernández de las Heras et al ., 2012; Yao et al ., 2014). …”

“…the best characterized cholesterol‐degrading organism) to produce AD, ADD and 9α‐hydroxy‐4‐androstene‐3,17‐dione (9OH‐AD) from natural sterols (e.g. cholesterol or phytosterols), but these mutants have not been used at industrial scale yet (van der Geize et al ., 2000, 2001a,b, 2002a,b, 2008; Wilbrink et al ., 2011; Yeh et al ., 2014). …”

“…Two KstD enzymes were reported in Rhodococcus erythropolis SQ1 (van der Geize et al ., 2000) and accordingly, targeted disruption of only one of the kstD genes did not result on the accumulation of intermediates, but the strain lacking both dehydrogenase activities was able to convert AD into 9OH‐AD stoichiometrically (van der Geize et al ., 2002b). Similarly, Fernández de las Heras et al .…”