Enhanced limonene production by metabolically engineered Yarrowia lipolytica from cheap carbon sources

Limonene is a valuable monoterpene widely used in the food and pharmaceutical industries. Previously, we successfully engineered Yarrowia lipolytica to produce limonenes. In this study, we focused on improving the titers of limonenes in Y. lipolytica by optimizing the metabolic flux of the limonene biosynthetic pathway and the medium composition. First, we adopted a combinatorial gene (over)expression strategy to improve the production of limonenes, obtaining the highest titer production strains. Subsequently, the medium composition and fed-batch fermentation were optimized to improve limonene biosynthesis, and it was confirmed that waste cooking oil (WCO) is the superior substrate to produce limonenes in Y. lipolytica. Under optimal fermentation conditions, the titers of D-limonene and L-limonene were improved to 91.24 mg/L and 83.06 mg/L from WCO. These findings provide valuable insights into the engineering of Y. lipolytica for a higher-level production of limonene and its utilization in converting WCO into other industrial products.