Summary: The biosynthetic pathway of the red‐pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2‐methyl‐3‐n‐amyl‐pyrrole (MAP) and the bipyrrole, 4‐methoxy‐2,2′‐bipyrrole‐5‐carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in‐frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC‐MS, cross‐feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3‐acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).

The prodigiosin biosynthesis gene cluster (pigcluster) from two strains ofSerratia(S. marcescensATCC 274 andSerratiasp. ATCC 39006) has been cloned, sequenced and expressed in heterologous hosts. Sequence analysis of the respectivepigclusters revealed 14 ORFs inS. marcescensATCC 274 and 15 ORFs inSerratiasp. ATCC 39006. In eachSerratiaspecies, predicted gene products showed similarity to polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs) and the Red proteins ofStreptomyces coelicolorA3(2). Comparisons between the twoSerratia pigclusters and theredcluster fromStr. coelicolorA3(2) revealed some important differences. A modified scheme for the biosynthesis of prodigiosin, based on the pathway recently suggested for the synthesis of undecylprodigiosin, is proposed. The distribution of thepigcluster within severalSerratiasp. isolates is demonstrated and the presence of cryptic clusters in some strains shown. Thepigcluster ofSerratia marcescensATCC 274 is flanked bycueRandcopAhomologues and this configuration is demonstrated in severalS. marcescensstrains, whilst these genes are contiguous in strains lacking thepigcluster.