Overproduction\nof Cucurbitadienol through Modular\nMetabolic Engineering and Fermentation Optimization in Saccharomyces cerevisiae

Cucurbitadienol is a key intermediate in the biosynthesis\nof cucurbitane-type\ncompounds and serves as a precursor for mogrosides, cucurbitacins,\nand other valuable natural products of potential biological and food\nimportance. However, microbial fermentation for cucurbitadienol production\nremains inefficient, limiting its potential for further industrial\napplication. This study achieved the efficient synthesis of cucurbitadienol\nthrough a multimodular strategy. First, an N-degron tag was used to\ndirect metabolic flux toward cucurbitadienol synthesis without compromising\ncell growth. Second, enzyme engineering strategies were employed to\nimprove the utilization efficiency of intermediate metabolites. Finally,\nto increase precursor availability, the transcription factor <i>UPC2–1</i> was introduced, which upregulated the expression\nof <i>ERGs</i> in the pre-squalene pathway. After eliminating\nnitrogen supplementation and optimizing fermentation conditions, cucurbitadienol\naccumulation in the 5 L bioreactor increased to 6.1 g/L, representing\nthe highest titer reported to date. These findings provide a solid\nfoundation for the industrial-scale production of cucurbitadienol\nand its derivatives.