ORGANIZATION OF THE BIOSYNTHETIC GENE-CLUSTER FOR RAPAMYCIN IN STREPTOMYCES-HYGROSCOPICUS - ANALYSIS OF GENES FLANKING THE POLYKETIDE SYNTHASE

Analysis of the gene cluster from Streptomyces hygroscopicus that gove rns the biosynthesis of the polyketide immunosuppressant rapamycin (Rp ) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorpor ating enzyme that probably also catalyzes closure of the macrolide rin g. On either side of these very large genes are ranged a total of 22 f urther ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Se veral of these ORFs appear to encode enzymes that would be required fo r Rp biosynthesis. These include two cytochrome P-450 monooxygenases ( P450s), designated RapI and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in 'late' modif ication of the macrocycle, The cluster also contains a novel gene (rap t) whose product is proposed to catalyze the formation of the Rp precu rsor, L-pipecolate, through the cyclodeamination of L-lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usag e of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster.