Molecular approaches for production of pravastatin, a HMG-CoA reductase inhibitor: transcriptional regulation of the cytochrome P450sca gene from Streptomyces carbophilus by ML-236B sodium salt and phenobarbital

Abstract

We have characterized the transcriptional regulation of ML-236B.Na and phenobarbital-inducible cytochrome P450sca-2 (CytP450sca-2) from Streptomyces carbophilus, an industrial pravastatin-producing strain. ML-236B.Na and phenobarbital enhanced the expression of the cytP450sca-2 gene in S. carbophilus. The cytP450sca-2 gene was also ML-236B.Na-inductive in S. lividans. Analysis of various deletion and mutation of the 5'-flanking region of the cytP450sca-2 gene revealed that the 1-kb region was required for ML-236B.Na-dependent CytP450sca-2 induction. We have found a putative ORF in the 5'-flanking region that encodes a protein of 174 amino acid residues containing a helix-turn-helix DNA-binding motif. A gel mobility shift assay showed that the protein was bound by an imperfect palindromic sequence between -46bp and -24bp in the 5'-flanking region, and ML-236B.Na was found to inhibit its binding. These findings suggest that induction of cytP450sca-2 is negatively regulated at the transcriptional level and that the protein encoded by the putative ORF is possibly functional as a repressor of the cytP450sca-2 gene.