Modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) comprise giant multidomain enzymes responsible for the “assembly line” biosynthesis of many genetically encoded small molecules. Site-directed mutagenesis, protein biochemical, and structural studies have focused on elucidating the catalytic mechanisms of individual multidomain proteins and protein domains within these megasynthases. However, probing their functions at the cellular level typically has invoked the complete deletion (or overexpression) of multidomain-encoding genes or combinations of genes and comparing those mutants with a control pathway. Here we describe a “domain-targeted” metabolomic strategy that combines genome editing with pathway analysis to probe the functions of individual PKS and NRPS catalytic domains at the cellular metabolic level. We apply the approach to the bacterial colibactin pathway, a genotoxic NRPS–PKS hybrid pathway found in certain Escherichia coli. The pathway produces precolibactins, which are converted to colibactins by a dedicated peptidase, ClbP. Domain-targeted metabolomics enabled the characterization of “multidomain signatures”, or functional readouts of NRPS–PKS domain contributions to the pathway-dependent metabolome. These multidomain signatures provided experimental support for individual domain contributions to colibactin biosynthesis and delineated the assembly line timing events of colibactin heterocycle formation. The analysis also led to the structural characterization of two reactive precolibactin metabolites. We demonstrate the fate of these reactive intermediates in the presence and absence of ClbP, which dictates the formation of distinct product groups resulting from alternative cyclization cascades. In the presence of the peptidase, the reactive intermediates are converted to a known genotoxic scaffold, providing metabolic support of our mechanistic model for colibactin-induced genotoxicity. Domain-targeted metabolomics could be more widely used to characterize NRPS–PKS pathways with unprecedented genetic and metabolic precision.

Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey’s analysis. Compounds 1–4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9 μm.

Lumiquinone A (1), an unusual aminobenzoquinone member within the phenylpropanoid class of natural products, together with the known compound 3,5-dihydroxy-4-isopropyl-trans-stilbene (2), was isolated from the entomopathogenic bacterium Photorhabdus luminescens TT01. On the basis of the analysis of extensive 2D NMR and high-resolution ESI-QTOF-MS spectral data, the structure of 1 was determined to be a 2-amino-5-hydroxy-1,4-benzoquinone substituted with (E)-2-phenylvinyl and isopropyl functional groups. Free α-aminomalonate medium supplementation significantly enhanced production of 1 relative to 2 in a dose-dependent manner, suggesting that promiscuous polyketide synthase processing of malonate- versus α-aminomalonate-derived substrates represents a competitive route for polyketide structural diversification. Metabolites 1 and 2 were active against Bacillus subtilis and Saccharomyces cerevisiae.

The gene cluster responsible for synthesis of the unknown molecule “colibactin” has been identified in mutualistic and pathogenic Escherichia coli. The pathway endows its producer with a long-term persistence phenotype in the human bowel, a probiotic activity used in the treatment of ulcerative colitis, and a carcinogenic activity under host inflammatory conditions. To date, functional small molecules from this pathway have not been reported. Here we implemented a comparative metabolomics and targeted structural network analyses approach to identify a catalog of small molecules dependent on the colibactin pathway from the meningitis isolate E. coli IHE3034 and the probiotic E. coli Nissle 1917. The structures of 10 pathway-dependent small molecules are proposed based on structural characterizations and network relationships. The network will provide a roadmap for the structural and functional elucidation of a variety of other small molecules encoded by the pathway. From the characterized small molecule set, in vitro bacterial growth inhibitory and mammalian CNS receptor antagonist activities are presented.

•An unusual orphan carbohydrate-NRPS pathway was identified by genome synteny analysis•Five oligosaccharides with a rare 1,6-anhydro-β-D-Glc-NAc moiety were characterized•Genetic and biochemical studies elucidated oligosaccharide biosynthesis and function•A hypothetical protein was discovered to be a 1,6-anhydropyranose synthaseMicrobial genome sequencing platforms have produced a deluge of orphan biosynthetic pathways suspected of biosynthesizing new small molecules with pharmacological relevance. Genome synteny analysis provides an assessment of genomic island content, which is enriched in natural product gene clusters. Here we identified an atypical orphan carbohydrate-nonribosomal peptide synthetase genomic island in Photorhabdus luminescens using genome synteny analysis. Heterologous expression of the pathway led to the characterization of five oligosaccharide metabolites with lysozyme inhibitory activities. The oligosaccharides harbor a 1,6-anhydro-β-D-N-acetyl-glucosamine moiety, a rare structural feature for natural products. Gene deletion analysis and biochemical reconstruction of oligosaccharide production led to the discovery that a hypothetical protein in the pathway is a lytic transglycosylase responsible for bicyclic sugar formation. The example presented here supports the notion that targeting select genomic islands with reduced reliance on known protein homologies could enhance the discovery of new metabolic chemistry and biology.Figure optionsDownload full-size imageDownload high-quality image (287 K)Download as PowerPoint slide

The integration of chemical ecology and bacterial genome mining can enhance the discovery of structurally diverse natural products in functional contexts. By examining bacterial secondary metabolism in the framework of its ecological niche, insights into the upregulation of orphan biosynthetic pathways and the enhancement of the enzyme substrate supply can be obtained, leading to the discovery of new secondary metabolic pathways that would otherwise be silent or undetected under typical laboratory cultivation conditions. Access to these new natural products (i.e., the chemotypes) facilitates experimental genotype-to-phenotype linkages. Here, we describe certain functional natural products produced by Xenorhabdus and Photorhabdus bacteria with experimentally linked biosynthetic gene clusters as illustrative examples of the synergy between chemical ecology and bacterial genome mining in connecting genotypes to phenotypes through chemotype characterization. These Gammaproteobacteria share a mutualistic relationship with nematodes and a pathogenic relationship with insects and, in select cases, humans. The natural products encoded by these bacteria distinguish their interactions with their animal hosts and other microorganisms in their multipartite symbiotic lifestyles. Though both genera have similar lifestyles, their genetic, chemical, and physiological attributes are distinct. Both undergo phenotypic variation and produce a profuse number of bioactive secondary metabolites. We provide further detail in the context of regulation, production, processing, and function for these genetically encoded small molecules with respect to their roles in mutualism and pathogenicity. These collective insights more widely promote the discovery of atypical orphan biosynthetic pathways encoding novel small molecules in symbiotic systems, which could open up new avenues for investigating and exploiting microbial chemical signaling in host–bacteria interactions.