Project Summary. Polyketide natural products have played, and will continue to play, critical roles in cancer chemotherapy. Though they are potent and often tumor-selective agents, access to gram scale supplies of these agents complicates their clinical translation. Current solutions, including chemical synthesis and strain optimization, have provided modest success, but our lack in understanding the machines responsible for their production ultimately limits the ability to attain effective levels of production. Type I polyketide synthases (PKSs) are large multifunctional enzymes that are organized into modules of discrete enzymatic proteins, minimally containing a β-ketoacyl synthase, an acyltransferase, and an acyl carrier protein (ACP). While an ideal size for cryo-electron microscopy (cryo-EM), only a few Type I PKS structures currently exist, and of these many lack resolution of their ACP domain. Like many large proteins, they exist naturally in a highly dynamic state, wherein their activity operates through discrete, mechanical processes. In most Type I PKSs, biosynthesis arises through assembly of ketide units, which are shuttled between the enzymatic domains by means of the ACP. Here, the complex movement between domains in each module results in synchronic capture of multiple states of the same protein, therein complicating structural evaluation. In this program, our team explores the pathways to enable the selective evaluation of state trapped Type I PKSs. Through two specific aims, we offer a unique solution towards isolating PKS megasynthases from host cells or producing them through recombinant technologies for cryo-EM studies. Overall, two key fundamental discoveries will arise from this program: an approach to isolating PKS enzymes for cryo-EM studies by directly challenging the current methods; and a primary understanding of the selectivity and mechanics involved in PKS catalysis. We anticipate that this program will offer not only a basis for structural resolution of large PKS enzymes, but also offer insight into modular and intermodular dynamics within these complex multimodular synthases.