Targeted Disruption of the kstD Gene Encoding a 3-Ketosteroid Δ ¹ -Dehydrogenase Isoenzyme of Rhodococcus erythropolis Strain SQ1

Abstract

Microbial phytosterol degradation is accompanied by the formation of steroid pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiated by ⌬ 1 -dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid ⌬ 1 -dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid ⌬ 1 -dehydrogenase (KSTD) activity under the control of the lac promoter in Escherichia coli. Targeted disruption of the kstD gene in R. erythropolis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activity. However, growth on the steroid substrate 4-androstene-3,17-dione or 9␣-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of ⌬ 1 -dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation of kstD is not sufficient for inactivation of the ⌬ 1 -dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.Rhodococcus species are well known for their catabolic potential (5, 40). Several Rhodococcus species degrade natural phytosterols. Microbial phytosterol degradation proceeds via the formation of steroids as pathway intermediates (16,21,22), i.e., 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione, and 9␣-hydroxy-4-androstene-3,17-dione (Fig.

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

“…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). …”

“…Nevertheless, in this mutant, we have observed some enzyme redundancy because we were able to detected small amounts of ADD when high concentrations of phytosterols are transformed. Therefore, we can conclude that at least one of the other five putative KstD enzymes identified in M. smegmatis mc 2 155 (Table 1) can also replace this function, but this alternative KstD activity is very low and does not appear to fulfil the relevant function observed in other cholesterol‐degrading strains (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 .…”

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

“…r et al, 1995 ;Morii et al, 1998 ;Plesiat et al, 1991 ;Sih & Bennet, 1962 ;Wagner et al, 1992 ;Wovcha et al, 1979). Functional studies of the steroid ∆"-dehydrogenation system in the micro-organism itself and analysis of the metabolic effects of their inactivation, however, are limited (van der Geize et al, 2000(van der Geize et al, , 2001, probably due to the paucity of genetic tools for actinomycetes able to perform the KSTD reaction (e.g. Rhodococcus, Arthrobacter, Nocardia, Mycobacterium).…”