Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae

The production of fuels and chemicals from biorenewable resources is important to alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Fatty acids are attractive biomolecules due to the flexibility of their iterative biosynthetic pathway, high energy content, and suitability for conversion into other secondary chemicals. Free fatty acids (FFAs) that can be secreted from the cell are particularly appealing due to their lower harvest costs and straightforward conversion into a broad range of biofuel and biochemical products. Saccharomyces cerevisiae was engineered to overproduce extracellular FFAs by targeting three native intracellular processes, p-oxidation was disrupted by gene knockouts in FAA2, PXA1 and POX1, increasing intracellular fatty acids levels up to 55%. Disruptions in the acyl-CoA synthetase genes FAA1, FAA4 and FAT1 allowed the extracellular detection of free fatty acids up to 490 mg/L. Combining these two disrupted pathways, a sextuple mutant (Delta faa1 Delta faa4 Delta fat1 Delta faa2 Delta pxa1 Delta pox1) was able to produce 1.3 g/L extracellular free fatty acids. Further diversion of carbon flux into neutral lipid droplet formation was investigated by the overexpression of DGA1 or ARE1 and by the co-overexpression of a compatible lipase, TGL1,TGL3 or TGL5. The sextuple mutant overexpressing the diacylglycerol acyltransferase, DEAL and the triacylglycerol lipase, TGL3, yielded 2.2 g/L extracellular free fatty acids. This novel combination of pathway interventions led to 4.2-fold higher extracellular free fatty acid levels than previously reported for S. cerevisiae. (C) 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.