Metabolic Engineering of Limonene Overproduction in the Oleaginous Yeast Yarrowia Lipolytica

 Limonene is known for its use as an important precursor of many flavoring, pharmaceutical and biodiesel products. Here, we have reported the production of limonene in the non-limonene producing oleaginous yeast Yarrowia lipolytica and the improvement of strain productivity through the increasing of the endogenous supply of precursor metabolites to enhance pathway activity. The strain Y.lipolytica PO1f-LN-000, with the overexpression of two heterologous limonene synthesis genes encoding both neryl diphosphate synthase 1 (NDPS1) and limonene synthase (LS), was used as initial limonene production strain. This initial strain yielded limonene production of 6 μg/g DCW. To maximize limonene production, several genes in the MVA pathway were overexpressed, sometimes in different copies of the same gene and sometimes in combination. We found that overexpressing the HMG1 gene alone raised limonene content to 0.11±0.01 mg/g DCW, an 18-fold improvement. After the ERG12 gene was co-expressed with the HMG1 gene, the yield of limonene was further improved to 0.67±0.04 mg/g DCW, a 112-fold increase from the initial yield. Finally, the optimization of additive amounts of pyruvic acid and dodecane helps the limonene content reach 1.36 mg/g DCW which was 226-fold higher than the yield of the initial strain. The strategy using in this study lay a foundation for further producing value-added limonene derivatives, even other monoterpenes in oleaginous yeast.