EXPRESSION AND PURIFICATION OF POLYKETIDE SYNTHASE DOMAINS TO INVESTIGATE PROTEIN-PROTEIN INTERACTIONS IN POLYKETIDE BIOSYNTHETIC PATHWAYS

Polyketide synthases (PKSs) are multienzyme proteins catalyzing the production of the polyketide class of natural products with vast structural diversity. Type II PKSs catalyze polyketide biosynthesis using discrete (standalone) enzymes (domains), while type I PKSs use covalently linked enzymes arranged in a module to catalyze polyketide production. It is hypothesized that type II PKSs are the precursors to type I PKSs, proposing that the genes encoding individual discrete enzymes in type II PKSs fused to give the multienzyme modules of type I PKSs. We reasoned that the intramolecular-like reactions occurring in type I PKS modules might be a driving force for this evolution. This thesis reports the heterologous expression and purification of acyl carrier proteins (ACPs) and ketoreductase (KR) domains from the 6-deoxyerythronolide B synthase’s (DEBS) first module and the amphotericin B synthase’s second module as model enzymes to investigate this hypothesis. These enzymes were expressed as didomains (KR-ACP) and standalone domains in E. coli strains and purified by fast protein liquid chromatography (FPLC). The domain boundaries of the ACP domains were established to express these enzymes as soluble standalone domains. Chimeric didomains were also produced to evaluate the effect of protein-protein interactions in rate acceleration.