Identification of a cytochrome P450 hydroxylase gene involved in rifamycin biosynthesis by Amycolatopsis mediterranei S699

Abstract

In analyzing the region of the Amycolatopsis mediterranei S699 chromosome responsible for the biosynthesis of the ansamycin antibiotic rifamycin, we identified a gene, designated orf0, which is located immediately upstream of the rifamycin polyketide synthase (PKS). Orf0 encodes a protein, on the basis of sequence-comparative analysis, that is similar to several cytochrome P450 monooxygenases from different sources. The rifamycin producer, A. mediterranei, predominantly produces rifamycin B from its macrocyclic intermediate, proansamycin X, through dehydrogenation and hydroxylation steps. However, an A. mediterranei strain, deleted in orf0 by gene replacement, no longer produced rifamycin B. Furthermore, a versatile replicative vector in A. mediterranei was constructed and rifamycin B production was restored in a complementation experiment of orf0 using this novel vector. These consecutive results verified that the Orf0 protein, which is a P450 hydroxylase, is required for the production of rifamycin B in A. mediterranei.