Enzymatic features and biocatalytic applications of modular polyketide synthase domains
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Polyketides are a class of secondary metabolites that are notable for their chemical diversity and therapeutic relevance. They are biosynthesized by polyketide synthases (PKSs) megasynthase enzymes in an assembly-line fashion. Though the molecular architectures of polyketides are complex, their biological precursors are chemically simple. Thus, understanding this powerful biosynthetic machinery is of interest for synthetic biology and biocatalytic applications. This dissertation presents three projects that decipher underlying mechanistic features and explore biocatalytic applications of PKSs. In modular PKSs, one module corresponds to one round of keto-elongation followed by modification through the action of β-carbon processing domains. The first project employs a system wherein a single module is used in vitro to generate small, chiral PKS products (triketide lactones). Although triketide lactones are a common output for PKS enzymology assays, usually they are only observed in trace quantities. In this study, we performed a number of strategies to scale up the production of triketide lactones to facilitate their use as chiral building blocks for chemical synthesis. In this process, we also gained new insights regarding the interacting kinetics and selectivities of the domains in an in vitro environment. ix The second project focused on the ketoreductase (KR) domain, which sets the majority of the stereogenic centers within a polyketide, and thus has obvious potential for biocatalytic applications. This project employs a structure-activity relationship (SAR)- type approach to dissecting stereocontrol. The SAR results, in concert with crystallographic data inspired two rational mutations that were sufficient to reverse the stereoselectivity of a representative KR. Thus, we were able to employ a rational approach to engineering stereocontrol. The final project also focuses on the KR domain, however from a subclass of PKSs termed trans-acyltranferase (AT) PKSs. In contrast to the canonical cis-AT PKSs, the trans-AT PKSs have more varied modular organizations and architectures. One of these peculiar organizations one termed a “split” bimodule, wherein domains within a module are present on different polypeptides. Structural characterization of a KR from a split bimodule revealed features that may correspond to interpeptide interactions that afford communication between the two polypeptides of the split bimodule. Additionally, bioinformatic analysis of KRs from split bimodules reveals a number of diagnostic sequence motifs.