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

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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 Δ1-dehydrogenation of the steroid ring structure. Characterization of a 2.9-kb DNA fragment ofRhodococcus erythropolis SQ1 revealed an open reading frame (kstD) showing similarity with known 3-ketosteroid Δ1-dehydrogenase genes. Heterologous expression ofkstD yielded 3-ketosteroid Δ1-dehydrogenase (KSTD) activity under the control of the lac promoter inEscherichia coli. Targeted disruption of thekstD 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 9α-hydroxy-4-androstene-3,17-dione was not abolished by thekstD gene disruption. Bioconversion of phytosterols was also not blocked at the level of Δ1-dehydrogenation in the kstD mutant strain, since no accumulation of steroid pathway intermediates was observed. Thus, inactivation ofkstD is not sufficient for inactivation of the Δ1-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed thatR. erythropolis SQ1 in fact harbors two activity bands, one of which is absent in the kstD mutant strain.

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