Engineering Organisms and Processes for Cost-Effective Lipid Production

Lipids are an important product that can be used for the production of biofuels such as biodiesel. The latter is presently produced from vegetables and seed oils, however, its long-term cost-effective production should utilize carbohydrate feedstocks for optimal land use. To this end, we have engineered an oleaginous yeast for the high yield conversion of carbohydrates to lipids and accumulation at high titers. The engineering strategy followed a push-pull approach whereby the fatty acid synthesis pathway was enhanced through the overexpression of Acetyl-CoA-Carboxylase (ACC1) along with the downstream lipid storage pathway for lipid sequestration in lipid bodies. We found that both pathways are important for optimal conversion yield and productivity, which is further obtained by minimizing citrate byproduct formation and maximizing pathway throughput. Pathway throughput is maximized by reducing or eliminating regulation of fatty acid synthesis, a goal that was achieved through the modulation of various target desaturase-expressing genes. This strategy yielded sharp increases in overall growth rate and lipid synthesis in small-scale flasks and bioreactor runs. We also examined lipid synthesis from acetate and other volatile fatty acid (VFA) substrates that can be obtained from inexpensive feedstocks such as synthesis gas or municipal solid waste by anaerobic digestion. We show that, using our engineered strains, VFAs can be converted efficiently to lipids at high yields. Additionally, using efficient cell recycle systems, dilute VFA streams can be converted to lipids that, as intracellular products, accumulate in the yeast cells at high titers. Our analysis shows that efficient lipid and biofuel production processes depend critically on feedstock cost and process yield, however, our results, taken together, suggest that these products are possible to produce in a cost-effective manner and be used at scale for biofuel production.