Structural investigation of a type III polyketide synthase involved in cannabinoid biosynthesis

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PROJECT SUMMARY Phytocannabinoids from Cannabis sativa L. are natural plant products with therapeutic potential in humans. Rapidly changing Cannabis legislation has fostered a burgeoning pharmaceutical cannabinoid industry, which is projected to be worth USD 5.7 billion in 2027. To keep pace with rising demand, research groups are working to develop heterologous expression methods for cannabinoid production. Current strategies show promise but suffer from low product yields. In this biosynthetic pathway, a plant type III polyketide synthase referred to as tetraketide synthase (TKS) catalyzes a highly modular reaction to generate essential precursors in the biogenesis of more than 113 phytocannabinoids, such as cannabidiol and Δ9-tetrahydrocannabinol. The reaction is prone to forming premature hydrolysis by- products derailing phytocannabinoid biosynthesis. The objective of this application is to establish a structural and mechanistic basis for understanding the TKS reaction. Preliminary experiments employing X-ray protein crystallography, structure-guided mutagenesis, and high performance liquid chromatography provide critical insight into this highly modular reaction. A method to generate catalytically active TKS crystals was developed. The resulting crystal structures provide a mechanistically relevant view of the TKS substrate-binding pocket. This is the first example of such high-resolution structural information being obtained for TKS. The proposed research strategy builds upon these preliminary experiments to characterize the architecture of the TKS substrate-binding pocket to identify key residues regulating substrate discrimination and polyketide formation (Aim 1). Time-resolved X-ray protein crystallography experiments will be pursued to investigate the structural and conformational changes occurring within the TKS substrate-binding pocket during polyketide elongation (Aim 2). Completion of these proposed aims will provide much needed structural insight elucidating the mechanism of multiple substrate discrimination and production formation in the TKS-catalyzed reaction. The results will have broad implications for enabling biosynthetic approaches to cannabinoid production.