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-incorporating enzyme (PIE) has been identified in the gene cluster fur the biosynthesis of rapamycin in Streptomyces hygroscopicus. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation,,and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetases. Disruption of rapP by phage 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 expressed in both Escherichia coli and Streptomyces coelicolor. and recombinant PIE was purified to homogeneity from both hosts, Although low-level incorporation of [C-14]beta-alanine into recombinant PIE isolated from E. coli was detected. formation of the covalent acylenzyme intermediate could only be shown with the PIE from S. coelicolor. suggesting that while the recombinant PIE from S. coelicolor was phosphopantetheinylated, only a minor proportion of the recombinant enzyme from E. coli was post-translationally modified.