Proteomic methodologies for polyketide biosynthesis(RMI)

University Of California, San Diego, La Jolla CA

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Abstract

DESCRIPTION (provided by applicant): This project develops new tools for the identification, isolation, and characterization of polyketide synthases from producer organisms. We focus on anticancer polyketides from marine dinoflagellates (dinophyceae), a class of single-cellular marine eukaryotes that synthesize an extraordinary variety of bioactive polyketides, including okadaic acid, brevetoxin and amphidinolides. The biosynthesis of these polyketides has been difficult to characterize genetically due to the fact that the large nuclear genomes of dinoflagellates contain high gene duplication with multiple intron content. These complications are intensified by the fact that dinoflagellates often harbor bacterial endo- and exo-symbionts. We propose the application of a novel suite of protein methods for the general study of polyketide biosynthesis in dinoflagellates using a model system, the biosynthesis of amphidinolides in Amphidinium sp. The amphidinolides are a large class of macro ides, of which several molecules demonstrate promising anti-cancer activity. This suite of methods has been engineered to visualize, isolate, and manipulate polyketide synthases through manipulation of their carrier protein (CP) domains. Using these techniques synthase enzymes in Amphidinium lysates will be tagged with fluorescent or affinity tags, identified, and purified. Methods such as photocleavable reporter linkages, enzymatic 4'-phosphopantetheine cleavage, and phosphopantetheinyltransferase inhibitors will be examined as a means to provide pure synthases in their natural apo- and holo- state. A variety of downstream assays will be conducted on the purified synthases to identify the individual loading domains and modular organization. The methodologies introduced here constitute a new platform of proteomic tools to investigate natural product biosynthetic enzymes from modular synthases, including polyketide and non-ribosomal peptide synthases and their hybrids. These tools will allow further understanding of natural product pathways and guide metabolic engineering to produce therapeutically important molecules.