초록
<P>The gene encoding the putative reductase component (KshB) of 3-ketosteroid 9α-hydroxylase was cloned from <I>Rhodococcus equi</I> USA-18, a cholesterol oxidase-producing strain formerly named <I>Arthrobacter simplex</I> USA-18, by PCR according to consensus amino acid motifs of several bacterial KshB subunits. Deletion of the gene in <I>R. equi</I> USA-18 by a PCR-targeted gene disruption method resulted in a mutant strain that could accumulate up to 0.58 mg/ml 1,4-androstadiene-3,17-dione (ADD) in the culture medium when 0.2% cholesterol was used as the carbon source, indicating the involvement of the deleted enzyme in 9α-hydroxylation of steroids. In addition, this mutant also accumulated 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid (Δ<SUP>1,4</SUP>-BNC). Because both ADD and Δ<SUP>1,4</SUP>-BNC are important intermediates for the synthesis of steroid drugs, this mutant derived from <I>R. equi</I> USA-18 may deserve further investigation for its application potential.</P>