Targeted disruption of the kstD gene encoding a 3-ketosteroid Delta(1)-dehydrogenase isoenzyme of Rhodococcus erythropolis strain SQ1

Microbial phytosterol degradation is accompanied by the formation of steroi d pathway intermediates, which are potential precursors in the synthesis of bioactive steroids. Degradation of these steroid intermediates is initiate d by Delta(1)-dehydrogenation of the steroid ring structure. Characterizati on of a 2.9-kb DNA fragment of Rhodococcus erythropolis SQ1 revealed an ope n reading frame (kstD) showing similarity with known 3-ketosteroid Delta(1) -dehydrogenase genes. Heterologous expression of kstD yielded 3-ketosteroid Delta(1)-dehydrogenase (KSTD) activity under the control of the lac promot er in Escherichia coli. Targeted disruption of the kstD gene in R. erythrop olis SQ1 was achieved, resulting in loss of more than 99% of the KSTD activ ity, However, growth on the steroid substrate 4-androstene-3,17-dione or 9 alpha-hydroxy-4-androstene-3,17-dione was not abolished by the kstD gene di sruption. Bioconversion of phytosterols was also not blocked at the level o f Delta(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 Delta(1)-dehydrogenase activity. Native polyacrylamide gel electrophoresis of cell extracts stained for KSTD activity showed that R. erythropolis SQ1 in fact harbors two activity band s, one of which is absent in the kstD mutant strain.