Creating natural product diversity via the fabrication of a hybrid fatty acid-nonribosomal peptide synthetase

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Abstract: Natural products and derivatives/mimics of natural products make up the majority of modern therapeutic drugs due to their unique complex organic scaffolds that contribute to their wide range of bioactivities. These natural products are synthesized by the fatty acid synthase (FAS), the nonribosomal peptide synthetase (NRPS) or the polyketide synthase (PKS). This program studies the role of protein•protein interactions in the modular synthases responsible for the formation of clinically relevant fatty acids and nonribosomal peptides for the long-term goal of re-designing and completely controlling the biosynthetic assembly lines. An example of a natural product therapeutic in the clinic is the antimicrobial daptomycin, which is a lipopeptide synthesized from a hybrid fatty acid synthase (FAS) and nonribosomal peptide synthetase (NRPS) that is used to treat methicillin- resistant Staphylococcus aureus. Thus, the hybrid FAS/NRPS are valuable systems to study and control so we may diversify natural products with new or enhanced bioactivities. Over the last three decades, true control over the biosynthetic assembly lines has remained elusive; the lack of a fundamental understanding on how the mega- synthase’s protein•protein interactions govern natural product biosynthesis has hindered this promising advance. In order to control and develop a hybrid FAS/NRPS biosynthetic pathway, first we must understand the fundamentals that guides the related biosynthetic machinery. Both FASs and NRPSs are dependent on the carrier protein (CP); the CP shuttles the growing substrate between multiple partner proteins (PP) for functionalization and incorporation into the natural product. The modular nature of fatty acid and nonribosomal peptide biosynthesis is conserved among organisms, however, the mechanism of CP•PP recognition in each pathway is distinct. Thus, to re-design these biosynthetic assembly lines, this project focuses on studying the molecular details of the CP•PP interface. We aim to 1) Characterize NRPS CP•PP interactions via X-ray crystallography, 2) establish a computational methodology to design a hybrid NRPS/FAS interaction, and 3) explore the cross-pathway productivity of the hybrid NRPS/FAS interaction. The results from these studies will be integral to the future bioengineering of these modular synthases.