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 × 10