Complete Reconstitution and Deorphanization of the 3 MDa NOCAP (NOCardiosis-Associated Polyketide) Synthase

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly-line polyketide synthase (PKS) that presumably synthesizes an unknown natural product. Here, we report the discovery and structure elucidation of the NOCAP (NOCardiosis-Associated Polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly-line PKS either in vitro or in E. coli, and of using these approaches to deorphanize a complete assembly-line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.