Fungal diseases are an escalating threat to human health, driven by rising antimicrobial resistance and a lack of new antifungal treatments1,2. Polyenes, a class of complex natural products, have been widely used as antifungal agents due to their broad-spectrum activity3. However, their clinical use is limited by severe toxicity and poor solubility, making safer and more effective alternatives urgently needed4. Previous efforts to generate improved polyenes have relied on costly, step-inefficient and atom-inefficient chemical syntheses5,6. Here we describe the discovery and characterization of pathways to previously undescribed polyenes, including unusual glycosyltransferase enzymes that introduce sugars onto polyene scaffolds. We also show how the reaction scope of an amidotransferase enzyme can be expanded to transform the detrimental carboxylate substituent of polyenes to alternative functionality. Introduction of a second sugar combined with carboxylate modifications leads to more potent polyene antifungals, with reduced toxicity, that are accessible by clean and efficient fermentation or enzymatic routes.
