(437c) De-Orphanizing the Nocardiosis-Associated Polyketide Synthase

Large multimodular polyketide synthases (PKSs) and their associated tailoring enzymes are a major source of small molecules with important medicinal and agricultural capabilities such as antibacterial, anticancer, fungicidal, herbicidal, immunosuppressant and insecticidal activities. The recent surge in genomic sequencing has exposed many “orphan” PKSs and provides a vast resource for natural products research. We are particularly interested in the NOCardiosis-Associated Polyketide (NOCAP) synthase; it is an orphan PKS expressed in 12 clinical strains of the Actinomycete Nocardia isolated from nocardiosis-affected patients: N. abscessus, N. amamiensis, N. araoensis, N. arthritidis, N. asiatica, N. beijingensis, N. exalbida, N. gamkensis, N. niwae, N. paucivorans, N. pneumoniae and N. puris. A major barrier to identifying and elucidating the NOCAP synthase’s biosynthetic repertoire is its exceptionally large size: its homodimeric mass approaches 3 MDa. Despite this difficulty, we developed and optimized E. coli expression and purification protocols for the entire NOCAP synthase: modules X, 1-2, 3, 4-5’s ketosynthase, 5’s dehydratase-6-7-8-thioester reductase and trans-acyltransferases-thioesterase. Using stable isotope labeling with malonyl extender units, high-resolution mass-spectrometry and NMR spectroscopy, we identified an unprecedented set of pyrone- and resorcylaldehyde-containing polyketides produced by the NOCAP synthase. This accomplishment represents the first example of total decoding of an orphan assembly line PKS through direct in vitro reconstitution from purified proteins. As there are several examples of PKS-derived natural products being important fitness factors for disease-causing microorganisms, orphan PKSs like the NOCAP synthase may also provide a valuable resource for revealing paths to pathogenicity in microorganisms.