Overproduction of Cucurbitadienol through Modular Metabolic Engineering and Fermentation Optimization in <i>Saccharomyces cerevisiae</i>

Cucurbitadienol is a key intermediate in the biosynthesis of cucurbitane-type compounds and serves as a precursor for mogrosides, cucurbitacins, and other valuable natural products of potential biological and food importance. However, microbial fermentation for cucurbitadienol production remains inefficient, limiting its potential for further industrial application. This study achieved the efficient synthesis of cucurbitadienol through a multimodular strategy. First, an N-degron tag was used to direct metabolic flux toward cucurbitadienol synthesis without compromising cell growth. Second, enzyme engineering strategies were employed to improve the utilization efficiency of intermediate metabolites. Finally, to increase precursor availability, the transcription factor <i>UPC2-1</i> was introduced, which upregulated the expression of <i>ERGs</i> in the pre-squalene pathway. After eliminating nitrogen supplementation and optimizing fermentation conditions, cucurbitadienol accumulation in the 5 L bioreactor increased to 6.1 g/L, representing the highest titer reported to date. These findings provide a solid foundation for the industrial-scale production of cucurbitadienol and its derivatives.