High-oleate yeast oil without polyunsaturated fatty acids.

BACKGROUND: Oleate-enriched triacylglycerides are well-suited for lubricant applications that require high oxidative stability. Fatty acid carbon chain length and degree of desaturation are key determinants of triacylglyceride properties and the ability to manipulate fatty acid composition in living organisms is critical to developing a source of bio-based oil tailored to meet specific application requirements. RESULTS: We sought to engineer the oleaginous yeast Yarrowia lipolytica for production of high-oleate triacylglyceride oil. We studied the effect of deletions and overexpressions in the fatty acid and triacylglyceride synthesis pathways to identify modifications that increase oleate levels. Oleic acid accumulation in triacylglycerides was promoted by exchanging the native ∆9 fatty acid desaturase and glycerol-3-phosphate acyltransferase with heterologous enzymes, as well as deletion of the Δ12 fatty acid desaturase and expression of a fatty acid elongase. By combining these engineering steps, we eliminated polyunsaturated fatty acids and created a Y. lipolytica strain that accumulates triglycerides with > 90% oleate content. CONCLUSIONS: High-oleate content and lack of polyunsaturates distinguish this triacylglyceride oil from plant and algal derived oils. Its composition renders the oil suitable for applications that require high oxidative stability and further demonstrates the potential of Y. lipolytica as a producer of tailored lipid profiles.

Metabolic engineering of microbial competitive advantage for industrial fermentation processes.

Microbial contamination is an obstacle to widespread production of advanced biofuels and chemicals. Current practices such as process sterilization or antibiotic dosage carry excess costs or encourage the development of antibiotic resistance. We engineered Escherichia coli to assimilate melamine, a xenobiotic compound containing nitrogen. After adaptive laboratory evolution to improve pathway efficiency, the engineered strain rapidly outcompeted a control strain when melamine was supplied as the nitrogen source. We additionally engineered the yeasts Saccharomyces cerevisiae and Yarrowia lipolytica to assimilate nitrogen from cyanamide and phosphorus from potassium phosphite, and they outcompeted contaminating strains in several low-cost feedstocks. Supplying essential growth nutrients through xenobiotic or ecologically rare chemicals provides microbial competitive advantage with minimal external risks, given that engineered biocatalysts only have improved fitness within the customized fermentation environment.

Engineering of a high lipid producing Yarrowia lipolytica strain.

BACKGROUND: Microbial lipids are produced by many oleaginous organisms including the well-characterized yeast Yarrowia lipolytica, which can be engineered for increased lipid yield by up-regulation of the lipid biosynthetic pathway and down-regulation or deletion of competing pathways. RESULTS: We describe a strain engineering strategy centered on diacylglycerol acyltransferase (DGA) gene overexpression that applied combinatorial screening of overexpression and deletion genetic targets to construct a high lipid producing yeast biocatalyst. The resulting strain, NS432, combines overexpression of a heterologous DGA1 enzyme from Rhodosporidium toruloides, a heterlogous DGA2 enzyme from Claviceps purpurea, and deletion of the native TGL3 lipase regulator. These three genetic modifications, selected for their effect on lipid production, enabled a 77 % lipid content and 0.21 g lipid per g glucose yield in batch fermentation. In fed-batch glucose fermentation NS432 produced 85 g/L lipid at a productivity of 0.73 g/L/h. CONCLUSIONS: The yields, productivities, and titers reported in this study may further support the applied goal of cost-effective, large -scale microbial lipid production for use as biofuels and biochemicals.