INTRODUCTION: Research on the human microbiome has revealed extensive correlations between bacterial populations and host physiology and disease states. However, moving past correlations to understanding causal relationships between the bacteria in our bodies and our health remains a challenge. A well-studied human-bacteria relationship is that of certain gut Escherichia coli strains whose presence correlates with colorectal cancer in humans. These E. coli damage host DNA and cause tumor formation in animal models, and this genotoxic phenotype is thought to derive from a secondary metabolite—known as colibactin—that is synthesized by the bacteria. Because colibactin’s biosynthetic pathway is only partially resolved, the complete structure of colibactin has remained unknown for more than a decade. Similarly, because colibactin is unstable and is produced in vanishingly small quantities, it has yet to be isolated and characterized by means of standard spectroscopic methods. RATIONALE: Determining colibactin’s chemical structure and related biological activity will allow researchers to determine whether the metabolite is the causal agent underlying many colorectal cancers. To that end, we used an interdisciplinary approach to overcome the challenges that have impeded determination of colibactin’s structure. Inspired by an earlier study that showed that colibactin-producing bacteria cross-link DNA, we used DNA as a probe to isolate colibactin from bacterial cultures. Using a combination of isotope labeling and tandem mass spectrometry analysis, we deduced the structure of the colibactin residue when bound to two nucleobases. This information allowed us to then identify and characterize colibactin in bacterial extracts and to identify plausible biosynthetic precolibactin precursors. Last, we developed a method to recreate colibactin in the laboratory and thereby confirm these structure-function relationships. RESULTS: Colibactin is formed through the union of two complex biosynthetic intermediates. This coupling generates a nearly symmetrical structure that contains two electrophilic cyclopropane warheads. We found that each of these residues undergoes ring-opening through nucleotide addition, a determination that is consistent with earlier studies of truncated colibactin derivatives and the observation that colibactin-producing bacteria cross-link DNA. Using genome editing techniques, we were able to show that the production of colibactin’s precursor, precolibactin 1489, requires every biosynthetic gene in the colibactin gene cluster, implicating it as being derived from the long-elusive and now completed biosynthetic pathway. Because natural colibactin remains non-isolable, the chemical synthetic route to colibactin we developed will allow researchers to probe for causal relationships between the metabolite and inflammation-associated colorectal cancer. CONCLUSION: These studies reveal the structure of colibactin, which accounts for the entire gene cluster encoding its biosynthesis, a goal that has remained b...

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The clb gene cluster encodes the biosynthesis of metabolites known as precolibactins and colibactins. The clb pathway is found in gut commensal E. coli, and clb metabolites are thought to initiate colorectal cancer via DNA cross-linking. Here we report confirmation of the structural assignment of the complex clb product precolibactin 886 via a biomimetic synthetic pathway. We show that a α-ketoimine linear precursor undergoes spontaneous cyclization to precolibactin 886 upon HPLC purification. Studies of this α-ketoimine and the related α-dicarbonyl revealed that these compounds are unexpectedly susceptible to nucleophilic cleavage under mildly basic conditions. This cleavage pathway forms other known clb metabolites or biosynthetic intermediates and explains the difficulties in isolating fully mature biosynthetic products. This cleavage also accounts for a recently identified colibactin-adenine adduct. The colibactin peptidase ClbP deacylates synthetic precolibactin 886 to form a non-genotoxic pyridone, suggesting precolibactin 886 lies off-path of the major biosynthetic route.

Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3′ phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.

This review recounts the chemical journey from 2015 to 2020 that culminated in the structure elucidation of the elusive bacterial metabolite, colibactin.