Some strains of commensal and pathogenic Escherichia coli have a gene cluster, named the pks island, that encodes for the biosynthesis of a small-molecule genotoxin named colibactin. To date, there has been no report of a successful isolation of colibactin, and the structure of colibactin is unknown. Colibactin biosynthesis is hypothesized to involve a conserved prodrug resistance strategy that is predicted to be operative in otherwise unrelated non-ribosomal peptide biosynthetic gene clusters across microbial genera. The prodrug strategy is predicted to involve the synthesis of an N-terminal signal sequence by an unusual non-ribosomal peptide synthetase (NRPS) module and cleavage of the signal sequence by a periplasmic D-amino peptidase. Here we report the in vitro characterization of the activities of ClbN, the NRPS module, and ClbP, the D-amino peptidase, that together constitute the prodrug machinery from the colibactin biosynthetic pathway. We have also shown that the hybrid NRPS/ polyketide synthase (PKS) enzyme ClbB, which we propose to act immediately downstream of ClbN in colibactin biosynthesis, extends the N-terminal signal sequence by incorporating the first amino acid of colibactin. Our work has revealed the molecular basis of the prodrug strategy in colibactin biosynthesis and marks the first biosynthetic study of colibactin, an important small-molecule in human microbiome related disease.

Characterization of the prodrug resistance mechanism of colibactin biosynthesis

245th National Meeting (2013)

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