De Novo High-Titer Biosynthesis of (-)-α-Bisabolol in Engineered Candida tropicalis via Green Metabolic Engineered Strategies.

(-)-α-bisabolol, a valuable sesquiterpene with anti-inflammatory and antibacterial properties, was sustainably produced via de novo high-titer biosynthesis in Candida tropicalis using engineered cell factories. To minimize resource-intensive extraction methods, we employed green metabolic engineering strategies, including an optimized Scaffold-Mediated Assembly Regulation Tool for coexpressing MrBBS and ERG20 genes, enhancing (-)-α-bisabolol titer 3.24-fold over the control strain. A novel Linker- and Scaffold-Mediated Assembly Regulation Tool further improved pathway efficiency by coordinating eight key acetyl-CoA/mevalonate pathway genes, achieving a 5.23-fold increase. Sustainable strain optimization included knockout of the competing gene DPP1 (yielding 261.2 mg•L⁻¹) and the peroxisomal surface display of Cat2 and ERG10 (290.1 mg•L⁻¹). Additional deletions of genes ADH1 and GPD1 reduced metabolic waste, boosting the titer 2.46-fold. A low-energy two-stage temperature control strategy further improved the titer to 1108.2 mg•L⁻¹, while fed-batch fermentation in a 5 L fermenter─optimized for minimal energy and substrate input─achieved a record titer of 36.9 g•L⁻¹. This study demonstrated a green, scalable biosynthesis platform for (-)-α-bisabolol, reducing reliance on petrochemical or plant-extraction routes, and provided a sustainable blueprint for terpenoid production in nonconventional yeasts.