Combining multiple metabolic strategies for efficient production of longifolene in Saccharomyces cerevisiae

Longifolene is a sesquiterpene commonly found in the heavy turpentine oil of pine plants, with various applications ranging from pest control and fragrance production to synthetic biofuels. While <i>S. cerevisiae</i> cell factories can effectively accumulate longifolene, further optimization and refinement of the metabolic modifications are still needed to improve the yield and conversion efficiency of longifolene production. In this study, we first explored enzyme fusion technology to enhance the efficiency of longifolene synthase catalysis, and improved acetyl-CoA availability by adjusting the pyruvate bypass pathway and introducing the synthetic chimeric citrate lyase pathway. The introduction of the formate dehydrogenase module was also used to supplement reducing power. By combining these strategies, the yield of longifolene reached 78.637 mg/L in shake flasks and 2063.7 mg/L in a 5 L bioreactor through fed-batch cultivation. This is the highest reported yield of longifolene to date. This study has important fundamental significance for the construction of biosynthetic factories for longifolene and other terpenes.