Mechanisms and Evolution of Assembly-Line Polyketide Synthases

PROJECT DESCRIPTION Assembly-line polyketide synthases (PKSs) are enzyme machines that catalyze vectorial biosynthesis of a growing polyketide chain through a uniquely defined sequence of acyl carrier protein and ketosynthase domains involving alternating chain translocation and elongation reactions. Notwithstanding the discovery of >3000 naturally occurring assembly-line PKSs, we do not understand how they blend catalytic specificity with evolutionary flexibility. Our lab is motivated by the goal of understanding the enzymology and evolution of assembly-line PKSs while enhancing our ability to engineer known PKSs and decode “orphan” ones. Our Goals for the next five years are to: 1) Understand the chemical logic of vectorial biosynthesis by an assembly-line PKS: We will study: (i) the structural dynamics of individual PKS modules; (ii) how different conformations of a module enable its elementary reactions; and (iii) the extent to which transitions between successive reactions are coordinated across an assembly line. Our proposed mechanistic investigations will exploit: (i) our ability to functionally reconstitute PKSs in vitro; (ii) epitope-specific monoclonal antibodies to trap individual PKS modules in specific conformational or catalytic states; and (iii) advances in X-ray, SAXS, and cryoEM analysis of PKSs. 2) De-orphanize the nocardiosis-associated NOCAP synthase: We have de-orphanized the nonamodular NOCAP synthase found in isolates of Nocardia associated with nocardiosis. Now, we propose to solve the structure of the fully tailored natural product, and to elucidate its biological role in nocardiosis. This will require us to: (i) characterize a putatively doubly glycosylated polyketide product; (ii) establish a phenotypic assay for its bioactivity in a macrophage-like human cell line; and (ii) harness genome-wide CRISPR knockout and shRNA knockdown screens to gain insight into its mode of action. 3) Decipher the role of GRINS in the evolution of assembly-line PKSs: We have discovered a new genetic element, named GRINS (genetic repeats of intense nucleotide skews), that is widespread in assembly-line PKS genes. We hypothesize that GRINS play a major role in diversifying assembly-line PKSs. To test this hypothesis, we will: (i) identify candidate genes in Streptomyces that are involved in introducing nucleotide skews or enabling gene conversion; (ii) identify a bacterial host in which gene conversion is enabled by GRINS under laboratory conditions; and (iii) develop an experimental model for GRINS-based PKS engineering. The significance of our proposal is two-fold. On one hand, it offers the opportunity to break new ground in our understanding of the structure, mechanism, and evolution of assembly-line PKSs. On the other hand, it tests the extent to which our understanding of these remarkable megasynthases can be harnessed to discover novel bioactive polyketides from “orphan” assembly-line PKSs.