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Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a β-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/β-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and β-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.

Analyses of microbial genome sequences have revealed numerous examples of 'cryptic' or 'orphan' biosynthetic gene clusters, with the potential to direct the production of novel, structurally complex natural products. This article summarizes the various methods that have been developed for discovering the products of cryptic biosynthetic gene clusters in microbes and gives an account of my group's discovery of the products of two such gene clusters in the model actinomycete Streptomyces coelicolor M145. These discoveries hint at new mechanisms, roles and specificities for natural product biosynthetic enzymes. Our efforts to elucidate these are described. The identification of new secondary metabolites of S. coelicolor raises the question: what is their biological function? Progress towards answering this question is also summarized.

MbtH-like proteins are a family of small proteins encoded by genes found in many, but not all, non-ribosomal peptide synthetase-encoding gene clusters that direct the biosynthesis of peptide antibiotics and siderophores. Studies published to date have not elucidated the function of MbtH-like proteins, nor have they clarified whether they are required for metabolite biosynthesis. Here it is shown that only one of two genes (cdaX or cchK) encoding MbtH-like proteins in Streptomyces coelicolor is required for biosynthesis of the peptide siderophore coelichelin and the calcium-dependent peptide antibiotic (CDA). The cdaX and cchK genes can functionally complement each other in trans, suggesting that CdaX and CchK can cross-talk with the coelichelin and CDA biosynthetic pathways, respectively. Transcriptional analyses of wild-type S. coelicolor and a double cchK/cdaX replacement mutant indicate that CchK and CdaX may not be involved in transcriptional regulation of coelichelin and CDA biosynthetic gene clusters.

Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because it contributes to virulence. In contrast, siderophore-mediated iron acquisition by saprophytic bacteria has received relatively little attention. The independent identification of the des and cch gene clusters that direct production of the tris-hydroxamate ferric iron- chelators desferrioxamine E and coelichelin, respectively, which could potentially act as siderophores in the saprophyte Streptomyces coelicolor A3(2), has recently been reported. Here it is shown that the des cluster also directs production of desferrioxamine B in S. coelicolor and that very similar des and cch clusters direct production of desferrioxamines E and B, and coelichelin, respectively, in Streptomyces ambofaciens ATCC 23877. Sequence analyses of the des and cch clusters suggest that components of ferric-siderophore uptake systems are also encoded within each cluster. The construction and analysis of a series of mutants of S. coelicolor lacking just biosynthetic genes or both the biosynthetic and siderophore uptake genes from the des and cch clusters demonstrated that coelichelin and desferrioxamines E and B all function as siderophores in this organism and that at least one of these metabolites is required for growth under defined conditions even in the presence of significant quantities of ferric iron. These experiments also demonstrated that a third siderophore uptake system must be present in S. coelicolor, in addition to the two encoded within the cch and des clusters, which show selectivity for coelichelin and desferrioxamine E, respectively. The ability of the S. coelicolor mutants to utilize a range of exogenous xenosiderophores for iron acquisition was also examined, showing that the third siderophore-iron transport system has broad specificity for tris-hydroxamate-containing siderophores. Together, these results define a complex system of multiple biosynthetic and uptake pathways for siderophore-mediated iron acquisition in S. coelicolor and S. ambofaciens.