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.

Bacterial prodiginines are a family of red-pigmented, tripyrrolic compounds that display numerous biological activities, including antibacterial, antifungal, antiprotozoal, antimalarial, immunosuppressive and anticancer properties. Recently, significant progress has been made in understanding the biosynthesis and regulation of bacterial prodiginines. An understanding of the biosynthesis of prodiginines will allow engineering of bacterial strains capable of synthesizing novel prodiginines through rational design and mutasynthesis experiments. Bacterial prodiginines and synthetic derivatives are effective proapoptotic agents with multiple cellular targets, and they are active against numerous cancer cell lines, including multidrug-resistant cells, with little or no toxicity towards normal cell lines. A synthetic derivative, GX15-070 (Obatoclax), developed through structure-activity relationship studies of the pyrrolic ring A of GX15, is in multiple Phase I and II clinical trials in both single and dual-agent studies to treat different types of cancer. Therefore, prodiginines have real therapeutic potential in the clinic.

We show here that isonitriles can perform click reactions with tetrazines in aqueous media, making them promising candidates for ligation reactions in chemical biology and polymer chemistry. This is the first time that a [4+1] cycloaddition has been used as a biocompatible ligation reaction.