α-Pinene, an important biologically active natural monoterpene, has been widely used in fragrances, medicines, and fine chemicals, especially, in high-density renewable fuels such as jet fuel. The development of an α-pinene production platform in a highly modifiable microbe from renewable substitute feedstocks could lead to a green, economical avenue, and sustainable biotechnological process for the biosynthesis of α-pinene. Here, we report engineering of an orthogonal biosynthetic pathway for efficient production of α-pinene in oleaginous yeast Yarrowia lipolytica that resulted in an α-pinene titer of 19.6 mg/L when using glucose as the sole carbon source, a significant 218-fold improvement than the initial titer. In addition, the potential of using waste cooking oil and lignocellulosic hydrolysate as carbon sources for α-pinene production from the engineered Y. lipolytica strains was analyzed. The results indicated that α-pinene titers of 33.8 and 36.1 mg/L were successfully obtained in waste cooking oil and lignocellulosic hydrolysate medium, thereby representing the highest titer reported to date in yeast. To our knowledge, this is also the first report related to microbial production of α-pinene from waste cooking oil and lignocellulosic hydrolysate.

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