Investigating structural and biochemical factors guiding concanamycin A biosynthesis

Proposal Summary Bacteria produce a wide array of diverse natural products through secondary metabolism. Polyketides stand out in this group of metabolites owing to their remarkable chemical complexity and varied bioactivities. This fascinating group of natural products includes FDA-approved antibiotics, antifungals, immunosuppressants, and many other therapeutic agents. Concanamycin A (CMA) is one such polyketide natural product that was recently found to inhibit the activity of Nef, a Human Immunodeficiency Virus (HIV) protein that blocks normal immune signaling in infected cells. Following treatment with CMA, cells with active HIV infections can properly relay immune response signals. Because of its therapeutic potential in combating HIV infections, a thorough understanding of the systems by which CMA is produced and assembled is needed, as it will help us develop analogs with improved pharmaceutical properties. CMA is produced by several members of the Streptomyces genus of bacteria, where the bulk of its biosynthesis is undertaken by a Type I assembly-line polyketide synthase (PKS) complex comprising 14 functional modules that build the initial CMA precursor polyketide through the stepwise addition of acyl units. Components of Type I PKSs, from individual enzymatic domains to entire functional modules, have been structurally characterized in the past. However, the native higher-order arrangement and interactome of PKS complexes within bacteria remains to be discovered. Similarly, how nascent CMA precursor molecules are tailored and stored following their biosynthesis remains unknown. To address the aforementioned knowledge gaps, I propose (1) elucidating the architecture of the concanamycin- producing PKS complex directly within cells (i.e., in situ) using cryo-electron tomography, cross-linking mass spectrometry, and integrative modeling and (2) determining key players in CMA storage, and their impact on CMA tailoring, by pulldown of CMA-associated proteins using CMA-based molecular probes and subsequent quantitative proteomics, coupled with liquid chromatography-mass spectrometry (LC-MS) analysis of gradient fractions of CMA-producing cell lysate and high-resolution infrared nanospectroscopy analysis of CMA- producing cells. The proposed work will provide the first high-resolution structural insights into the PKS machinery in situ and an improved understanding of CMA biosynthesis, with downstream applications in the engineering of PKS systems towards producing novel polyketides. I also expect this research to broadly apply to understanding the biosynthesis of other pharmaceutically relevant polyketides. Finally, understanding polyketide tailoring and storage will reveal critical insights for improving CMA production, which is necessary for derivatization to produce other analogs and downstream applications.