Engineering combinatorial biosynthesis of polyketide synthase-pepide synthetase products using novel fungal genetic tools

Fungal polyketides are a complex class of natural products with diverse scaffolds. The biosynthesis of these molecules involves the iterative condensation of acetate units using a minimal set of domains on a single polypeptide. The different levels of reduction at each iterative step generates the structural diversity observed via the stuttering action of these domains. For some polyketides, further structural complexity is introduced via amidation by a nonribosomal peptide synthetase (NRPS) module fused to the polyketide synthase (PKS). The NRPS contains a condensation domain (C) that catalyzes the coupling of the polyketide to a specific amino acid. Studies by others have suggested that PKS function is independent of NRPS activity and thus, this system is amenable to combinatorial biosynthesis to generate analogues with different polyketide chains and amino acids by performing module swaps. Forging intermodular interactions and understanding C domain selectivity for substrates is key to successful engineering of these analogues. Herein, I investigate the impact of these factors on the ability to synthesize unnatural products by this route. Studying these components required the overexpression of chimeric gene constructs, which was anticipated to result in low compound yields. Therefore, a novel platform to express fungal genes with yields exceeding 1 g per kg media was developed and validated through the characterization of a silent pathway. Application to the PKS-NRPS combinatorial biosynthesis problem led to the discovery that C domains are highly selective for closely related substrates in the presence of favorable PKS/NRPS interactions.