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.
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.
Watanabe
I
I
Biomedical Research Laboratories, Sankyo Co. Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140, Japan.
Serizawa
N
N
D000595:Amino Acid Sequence; D000924:Anticholesteremic Agents / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000032:analysis; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008148:Lovastatin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology*; D017035:Pravastatin / Q000096:biosynthesis / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000187:drug effects*; D015854:Up-Regulation / Q000502:physiology
D000924:Anticholesteremic Agents; D000935:Antifungal Agents; D004268:DNA-Binding Proteins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D012333:RNA, Messenger; D012097:Repressor Proteins; C012258:mevastatin; D003577:Cytochrome P-450 Enzyme System; D008148:Lovastatin; D006899:Mixed Function Oxygenases; C069505:mevastatin hydroxylase; D017035:Pravastatin; D010634:Phenobarbital