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.

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.

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 nonisolable, the chemical synthetic route to colibactin we developed will allow researchers to probe for causal relationships between the metabolite and inflammation-associated colorectal cancer.

These studies reveal the structure of colibactin, which accounts for the entire gene cluster encoding its biosynthesis, a goal that has remained beyond reach for more than a decade. The complete identity of colibactin has been a missing link in determining whether and how often colibactin is the causal agent underlying colorectal cancers. The interdisciplinary approach we used—marrying chemical synthesis, metabolomics, and probe-mediated natural product capture—may be applicable toward other spectroscopically intractable metabolites that are implicated in disease phenotypes but are currently undetected in the enormous chemical space encoded by the microbiome. Our studies represent a substantial advance toward our understanding of causative …