New paradigms for polyketide biosynthesis in the leinamycin family of potent antibiotics

Polyketides are a family of microbial natural products constructed from simple building blocks to generate a diverse range of often complex chemical structures cleanly and efficiently. They have been exploited in the production of many important high-value compounds including the antifungal strobilurins (Global Market Value (GMV) $3.8 billion) cholesterol-lowering, statins (HMG-CoA reductase inhibitors GMV ca $25 billion) and sulfur containing beta-lactam antibiotics, including penicillins ($11 billion) and cephalosporins ($20 billion). The leinamycin family of polyketides is a promising source of antitumor antibiotics, characterized by a sulfur-containing moiety essential for anticancer activity. While many aspects of the biosynthesis of these complex natural products are poorly understood, uncovering these processes will enable the rational design of sustainable pathways for producing novel compounds. Challenge. Complex polyketides are assembled from simple building blocks joined together on an acyl carrier protein (ACP) by large protein complexes called polyketide synthases. These function as modular nano-scale factories inside the microbe. In the leinamycin family, sulfur incorporation is introduced using the final module of the polyketide synthases, where the biosynthetic machinery combines ß-branching and thiocysteine incorporation to produce a diverse array of sulfur-based molecular scaffolds. Our Approach. The intricate biochemical steps involved in sulfur incorporation require in vitro analysis, however, bringing together the components is a complex, interdisciplinary challenge. The precise mechanistic steps leading to sulfur incorporation remain unresolved. To gain an understanding of these important transformations, we will build upon our experience of unravelling complex biosynthetic pathways by; The enantioselective synthesis of authentic biosynthetic intermediates that can be covalently tethered to ACPs in an elegant one-pot chemoenzymatic reaction. Using these functionalised ACPs as probes to understand chemoenzymatic reaction cascades that combine several novel enzymes to incorporate sulfur moieties and release the final product. These multi-enzyme transformations will be monitored via high resolution mass spectrometry. Gaining a structural and mechanistic description of these steps through the application of structural biology, computational modelling and AI approaches. This work will provide a detailed blueprint for sulfur incorporation in the leinamycin family and beyond, expanding our understanding of biosynthetic logic across the PKS field. This will enable the rational and selective incorporation of bespoke sulfur entities into other pathways to generate novel high-value bioactive hybrid molecules.