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Menthol, a high-value commodity monoterpenoid chemical, holds an important market share commercially because of its distinct functions. The menthol on the market mainly originates from plant extraction, which is facing challenges such as the seasonal fluctuations and long growth cycle of plants. Therefore, this study attempted to realize the de novo synthesis of menthol through microbial fermentation. First, through heterologous expression and subcellular localization observation, a synthetic route from glucose to (-)-menthol was successfully designed and constructed in Saccharomyces cerevisiae . Then, the mevalonate (MVA) pathway was enhanced, and the expression of farnesyl diphosphate synthase ( ERG20 ) was dynamically regulated to improve the synthesis of D-limonene, a key precursor of (-)-menthol. Shake flask fermentation results showed that the D-limonene titer of the recombinant strain reached 459.59 mg/L. Next, the synthesis pathway from D-limonene to (-)-menthol was strengthened, and the fermentation medium was optimized. The (-)-menthol titer of 6.28 mg/L was obtained, implying that the de novo synthesis of menthol was successfully realized for the first time. This study provides a good foundation for the synthesis of menthol through microbial fermentation.

Engineered Saccharomyces cerevisiae for the De Novo Biosynthesis of (−)-Menthol