Characterization and Engineering of Fungal Megasynthases

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DESCRIPTION (provided by applicant): Polyketide synthases (PKSs) of fungal origin are among the most enigmatic enzyme machineries known in microbial secondary metabolism. These enzymes are associated with the biosynthesis of very important fungal natural products, both beneficial, such as the cholesterol lowering blockbuster drug lovastatin from Aspergillus terreus; and deleterious, such as the carcinogenic mycotoxin aflatoxin from different aspergilli. The highly reducing fungal PKSs (HRPKS) are architecturally and mechanistically distinct from the well-characterized bacterial PKSs, and therefore produce compounds of different structural diversity and biological activities. In contrast to bacterial Type I PKSs or the structurally relatd mammalian FASs, our knowledge of the programming rules of fungal HRPKSs remains very limited, especially with regard to how a single set of domains are iteratively used and precisely orchestrated in the synthesis of the final product, often exceeding a combined 30 catalytic steps. Therefore, understanding fungal HRPKS function will enhance our knowledge of natural product biosynthesis and enzymology of multidomain, processive systems. In this proposal, we will comprehensively investigate the mechanisms of HRPKSs using several model systems and compounds. Our overarching goal is to understand, and to be able to predict, the relationship between HRPKS sequence and product structure. Four aims will be pursued in parallel during the proposal period: 1) Classifying and mining fungal HRPKS based on function and product structure; 2) Fingerprinting the specificity of HRPKS tailoring domains; 3) Understanding protein-protein interactions in HRPKS off-loading mechanisms; and 4) Elucidating the enzymes responsible for post- PKS intramolecular cyclization reactions. Each aim is targeted at one unique aspect of HRPKS function that is vital to structural diversity generation. Fundamental enzymology questions will also be answered in addressing these aims. Our work tackles the least understood, the most difficult and perhaps the last frontier of polyketide biosynthesis.