Colibactin represents an as-yet uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886 that uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block in its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal cancer-linked microbial chemical.

Colibactin is a potent genotoxin that induces DNA double-strand breaks; it is produced by Escherichia coli strains harboring a pks+ island. However, the structure of this compound remains elusive. Here, using transformation-associated recombination (TAR) cloning to perform heterologous expression, we took advantage of the significantly increased yield of colibactin pathway-related compounds to determine and isolate a series of vital (pre)colibactin intermediates. The chemical structures of compounds 8, 10 and 11 were identified by NMR and MS(n) analyses. The new 1H-pyrrolo[3,4-c]pyridine-3,6(2H,5H)-dione- and thiazole-containing compound 10 provides new insights regarding the biosynthetic pathway to (pre)colibactin and establishes foundations for future investigation of the intriguing (pre)colibactin structures and its modes of action.