Fatty acids as biofuel precursors accumulation : an investigation by metabolic engineering approach
Date of Issue2015
School of Chemical and Biomedical Engineering
Production of biofuels derived from microbial fatty acids has attracted great attention in recent years owing to their potential to replace petroleum-derived fuels. To be cost competitive with current petroleum fuel, production of the direct precursor fatty acids need to be enhanced to approach high yields. Herein, metabolic engineering approach was adopted to engineer Saccharomyces cerevisiae to accumulate more free fatty acids (FFA). In the third chapter, the β-oxidation pathway of S. cerevisiae was engineered to accumulate a higher ratio of medium chain fatty acids (MCFAs) when cells were grown on fatty acid-rich feedstock. For this purpose, the haploid deletion strain △pox1 was obtained, in which the sole acyl-CoA oxidase encoded by POX1 was deleted. Next, the POX2 gene from Yarrowia lipolytica, which encodes an acyl-CoA oxidase with a preference for long chain acyl-CoAs, was expressed in the △pox1 strain. The resulting △pox1 [pox2+] strain exhibited a growth defect because the β-oxidation pathway was blocked in peroxisomes. To unblock the β-oxidation pathway, the gene CROT, which encodes carnitine O-octanoyltransferase, was expressed in the △pox1 [pox2+] strain to transport the accumulated medium chain acyl-CoAs out of the peroxisomes. The obtained △pox1 [pox2+, crot+] strain grew at a normal rate. The effect of these genetic modifications on fatty acid accumulation and profile was investigated when the strains were grown on oleic acids-containing medium. It was determined that the engineered strains △pox1 [pox2+] and △pox1 [pox2+, crot+] had increased fatty acid accumulation and an increased ratio of MCFAs. Compared to the wild-type (WT) strain, the total fatty acid production of the strains △pox1 [pox2+] and △pox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in △pox1 [pox2+] and △pox1 [pox2+, crot+] increased 2.26- and 1.87-fold compared to the WT strain, respectively. In addition, MCFAs in the culture medium increased 3.29-fold and 3.34-fold compared to the WT strain. These results suggested that fatty acids with an increased MCFAs ratio had accumulated in the engineered strains with a modified β-oxidation pathway. Our approach exhibits great potential for transforming low value fatty acid-rich feedstock into high value fatty acid-derived products. In the fourth chapter, fatty acyl-CoA metabolism in S. cerevisiae was engineered to accumulate more FFA. For this purpose, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed first, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of Mus musculus peroxisomal acyl-CoA thioesterase Acot5 (Acot5s) was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acids (UFA) ratio as compared to the WT strain and double deletion strain △faa1△faa4. The extracellular total FFA in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the WT strain during the stationary phase. A high level of FFA about 500 μg/ml was achieved under shake-flask condition. UFA accounted for 42% of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the WT strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth. The study presented here showed that through control of the acyl-CoA metabolism by deleting acyl-CoA synthetase and expressing thioesterase, more FFA could be produced in S. cerevisiae. In the fifth chapter, comparative proteomic analysis were performed to get a global overview of metabolic regulation in the strain △faa1△faa4 [Acot5s]. Over 500 proteins were identified, and 82 of those proteins were found to change signiﬁcantly in the engineered strains. Proteins involved in glycolysis, acetate metabolism, fatty acid synthesis, TCA cycle, glyoxylate cycle, pentose phosphate pathway, respiration, transportation and stress response were found to be up-regulated in △faa1△faa4 [Acot5s] as compared to the WT strain. On the other hand, proteins involved in glycerol, ethanol, ergosterol and cell wall synthesis were down-regulated. Taken together with our metabolite analysis, our results showed that the disruption of Faa1 and Faa4 and expression of Acot5s in the engineered strain △faa1△faa4 [Acot5s] not only relieved the feedback inhibition of fatty acyl-CoAs on fatty acid synthesis, but also caused a major metabolic rearrangement. The rearrangement redirected carbon flux towards the pathways which generate the essential substrates and cofactors for fatty acid synthesis, such as acetyl-CoA, ATP and NADPH. Therefore, our results help shed light on the mechanism for the increased production of fatty acids in the engineered strains, which is useful in providing information for future studies in biofuel production. In short, our study demonstrated the great potential in improving metabolic efficiency of FFA production in S. cerevisiae, and implied that it could serve as a good platform for producing microbial fatty acid-derived biofuels. In addition, it added another excellent tool in guiding future biofuel production studies.