De novo biosynthesis of mogroside V by multiplexed engineered yeasts.

High sugar intake has become a global health concern due to its association with various diseases. Mogroside V (MG-V), a zero-calorie sweetener with multiple medical properties, is emerging as a promising sugar substitute. However, its application is hindered by low natural abundance and the inefficiency of conventional plant extraction methods. In this study, two glycosyltransferases were introduced into an engineered mogrol-producing Saccharomyces cerevisiae strain to enable the first de novo MG-V biosynthesis. Then, MG-V titer increased by 2.3 × 10⁴-fold through a series of efficient metabolic engineering strategies, including the enhancement of precursors, inhibition of the competitive pathway, and prevention of MG-V degradation. The challenges of enzyme spatial separation and high protein folding stress were addressed through lipid droplet (LD) compartmentalization and endoplasmic reticulum expansion, respectively. The ty1 transposon was employed to increase the copies of LD-targeted fusion protein AtCPR2-CYP87D18, which possessed higher CYP450 catalytic efficiency, resulting in an MG-V titer of 10.25 mg/L in shake flasks and 28.62 mg/L in a 5-L bioreactor. Overall, this study realized de novo MG-V synthesis in S. cerevisiae for the first time and provided a valuable reference for constructing microbial factories for triterpenoid saponin synthesis.