Oleochemicals have diverse applications in health, fuel, and chemical markets with growing product demands. Traditionally, these compounds are generated from relatively unsustainable petroleum processes or natural oils extractions (e.g. plant, fish, etc.). While these natural oil sources are commonly thought of as renewable, these processes require significant environmental inputs of land, water, and time and can be rather unsustainable. Alternatively, oleochemicals can be generated by microbes and often utilize waste products as the input nutrient feedstock. The yeast Yarrowia lipolytica is uniquely positioned as a host for this oleochemical production due to its innate oleaginous nature. Additionally, Y. lipolytica has native tolerance to a variety of usually inhibitory molecules, thus allowing for growth in a variety of waste sources. In this work, the oleochemical palette of this host was expanded to include the polyunsaturated, ω-3 fatty acid α-linolenic acid and fatty alcohols along with significant progress toward rewiring for phospholipid-linked modified fatty acids. On the feedstock side, waste valorization and chemical co-production was evaluated in Hydrothermal Liquefaction Aqueous Phase (HTL-AP) leading to enhanced tolerance and productivity.
In brief, this work allowed for high content (maximum titer of 1.4 g/L and lipid content of 27%) of α-linolenic acid production through expression of a heterologous Δ12-15 desaturase along with a low temperature fermentation. In a subsequent approach, fatty alcohols were produced using a fatty acyl-CoA reductase along with dodecane extractive fermentation leading to 75% product secretion and 5.8 g/L fatty alcohols in bioreactor fermentation. To access a third class of additional oleochemicals with unique modifications, progress was made toward understanding and improving phospholipid availability in Y. lipolytica. A three-pronged approach addressed co-factor availability, pathway biosynthesis, and lipid shuffling to identify limiting factors that could lead to a 5-fold improvement in phospholipid titer. To complement these production phenotypes, Y. lipolytica was evaluated for its potential to both detoxify and valorize HTL-AP. Bioreactor fermentation demonstrated chemical production exceeding 20 g/L in a mixture of lignocellulosic hydrolysate and HTL-AP while adaptation isolated new clones capable of growth in 27% HTL-AP. Collectively, this work in teaching Y. lipolytica new tricks demonstrates significant steps toward sustainable biochemical production and waste utilization.