Elevating Limonene Production in Oleaginous Yeast Yarrowia lipolytica via Genetic Engineering of Limonene Biosynthesis Pathway and Optimization of Medium Composition

Abstract

As an alternative terpenoid producer, non-conventional oleaginous yeast Yarrowia lipolytica was chosen for limonene production. Y. lipolytica can give high biomass yield and metabolize a broad range of substrates such as glycerol, alkanes, fatty acid, fats, and oils. As previously reported, optimization of limonene synthesis pathway and mevalonate (MVA) pathway leads to the accumulation of 112-fold higher limonene as compared to an initial strain. In this study, we introduced an additional copy of limonene synthesis gene (LS), which resulted in an increase of limonene production. This engineered strain was used to carry out further optimization study. Amongst all the carbon sources tested, the highest level of limonene production was obtained from glycerol, and citrate was selected as an auxiliary carbon source. In fed-batch fermentation with an optimized medium, the engineered strain was found to produce 165.3 mg/L limonene, which corresponds to the highest yield till date for the production of limonene in Y. lipolytica.