Characterization of Polyketide Synthase Machinery from the pks Island Facilitates Isolation of a Candidate Precolibactin

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, productio...