THE PIPECOLATE-INCORPORATING ENZYME FOR THE BIOSYNTHESIS OF THE IMMUNOSUPPRESSANT RAPAMYCIN - NUCLEOTIDE-SEQUENCE ANALYSIS, DISRUPTION AND HETEROLOGOUS EXPRESSION OF RAPP FROM STREPTOMYCES-HYGROSCOPICUS

An open reading frame (rapP) encoding the putative pipecolate-incorpor ating enzyme (PIE) has been identified in the gene cluster fur the bio synthesis of rapamycin in Streptomyces hygroscopicus. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate format ion,,and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetases. Disruption of rapP by p hage insertion abolished rapamycin production in S, hygroscopicus, and the production of the antibiotic was specifically restored upon loss of the inserted phage by a second recombination event. rapP was expres sed in both Escherichia coli and Streptomyces coelicolor. and recombin ant PIE was purified to homogeneity from both hosts, Although low-leve l incorporation of [C-14]beta-alanine into recombinant PIE isolated fr om E. coli was detected. formation of the covalent acylenzyme intermed iate could only be shown with the PIE from S. coelicolor. suggesting t hat while the recombinant PIE from S. coelicolor was phosphopantethein ylated, only a minor proportion of the recombinant enzyme from E. coli was post-translationally modified.