Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica

•We report a proof-of-concept pathway for pentane production in Yarrowia lipolytica.•A soybean lipoxygenase enzyme cleaves linoleic acid substrate into pentane and a byproduct.•Strong pathway overexpression and increased fatty acid substrate supply increased yields threefold to 4.98mg/L.•This demonstrates that short chain n-alkane synthesis is conceivable in model cellular hosts. The complete biosynthetic replacement of petroleum transportation fuels requires a metabolic pathway capable of producing short chain n-alkanes. Here, we report and characterize a proof-of-concept pathway that enables microbial production of the C5n-alkane, pentane. This pathway utilizes a soybean lipoxygenase enzyme to cleave linoleic acid to pentane and a tridecadienoic acid byproduct. Initial expression of the soybean lipoxygenase enzyme within a Yarrowia lipolytica host yielded 1.56mg/L pentane. Efforts to improve pentane yield by increasing substrate availability and strongly overexpressing the lipoxygenase enzyme successfully increased pentane production three-fold to 4.98mg/L. This work represents the first-ever microbial production of pentane and demonstrates that short chain n-alkane synthesis is conceivable in model cellular hosts. In this regard, we demonstrate the potential pliability of Y. lipolytica toward the biosynthetic production of value-added molecules from its generous fatty acid reserves.