Colibactin is a human gut bacterial genotoxin of unknown structure that has been linked to colon cancer. The biosynthesis of this elusive metabolite is directed by the pks gene cluster, which encodes a hybrid nonribosomal peptide synthetase-polyketide synthase (NRPS-PKS) assembly line that is hypothesized to use the unusual polyketide building block aminomalonate. This biosynthetic pathway is thought to initially produce an inactive intermediate (precolibactin) that is processed to the active toxin. Here, we report the first in vitro biochemical characterization of the PKS components of the pks enzymatic assembly line. We evaluate PKS extender unit utilization and show that ClbG, a freestanding acyltransferase (AT) from the pks gene cluster, recognizes aminomalonyl-acyl carrier protein (AM-ACP) and transfers this building block to multiple PKS modules, including a cis-AT PKS ClbI. We also use genetics to explore the in vivo role of ClbG in colibactin and precolibactin biosynthesis. Unexpectedly, production of previously identified pks-associated metabolites is dramatically increased in a ΔclbP/ΔclbG mutant strain, enabling the first structure elucidation of a bithiazole-containing candidate precolibactin. This work provides new insights into the unusual biosynthetic capabilities of the pks gene cluster, offers further support for the hypothesis that colibactin directly damages DNA, and suggests that additional, uncharacterized pks-derived metabolites containing aminomalonate play critical roles in genotoxicity.