Heterologous biosynthesis of (+)-nootkatone in unconventional yeast Yarrowia lipolytica

Yarrowia lipolytica is an unconventional yeast that is regarded as safe. It is the potential platform for terpenoids production because it possesses the endogenous MVA pathway which can provide precursors for terpenoids synthesis. Herein, we constructed (+)-nootkatone, a sesquiterpenoid which is highly demanded in cosmetics and fragrance industries, synthetic pathway in Yarrowia lipolytica ATCC 201249. Heterologous production of (+)-nootkatone was achieved by co-expressing (+)-valencene synthase CnVS, codon-optimized (+)-nootkatone synthase opCYP706M1 and codon-optimized NADPH-cytochrome P450 reductase opAtCPR1. The initial (+)-nootkatone production was 45.6 μg/L. Fusion of opCYP706M1 and opt46AtCPR1 (opAtCPR1 with 46 amino acids truncated at N-terminal) increased (+)-valencene conversion efficiency to (+)-nootkatone and (+)-nootkatone production increased to 312.2 μg/L, nearly six times higher than the initial. Overexpression of the MVA pathway rate limiting enzymes 3-hydroxy-3-methylglutaryl-coenzyme A reductase tHMG1 and FPP synthase ERG20 improved the (+)-nootkatone production furtherly. The final engineered strain achieved a (+)-nootkatone titer of 978.2 μg/L, which was a 20.5-fold increase compared to those simply coexpressed CnVS, opCYP706M1 and opAtCPR1. This is the first report of heterologous biosynthesis of (+)-nootkatone in Y. lipolytica, which will provide a favorable reference for studies on heterologous production of other sesquiterpenoids and high-efficiency expression of P450 enzymes in Y. lipolytica.