Engineering the Saccharomyces cerevisiae β-oxidation pathway to increase medium chain fatty acid production as potential biofuel.
Fatty acid-derived biofuels and biochemicals can be produced in microbes using β-oxidation pathway engineering. In this study, the β-oxidation pathway of Saccharomyces 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 Δpox1 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 Δpox1 strain. The resulting Δpox1 [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 Δpox1 [pox2+] strain to transport the accumulated medium chain acyl-coAs out of the peroxisomes. The obtained Δpox1 [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 Δpox1 [pox2+] and Δpox1 [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 Δpox1 [pox2+] and Δpox1 [pox2+, crot+] were increased 29.5% and 15.6%, respectively. The intracellular level of MCFAs in Δpox1 [pox2+] and Δpox1 [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 accumulate 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.
School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.
Liwei
L
Chen
School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.
Jianhua
J
Zhang
School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.
Wei Ning
WN
Chen
D000214:Acyl Coenzyme A / Q000378:metabolism*; D042962:Acyl-CoA Oxidase / Q000172:deficiency / Q000235:genetics; D056804:Biofuels; D002333:Carnitine Acyltransferases / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D005227:Fatty Acids / Q000096:biosynthesis*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D010084:Oxidation-Reduction; D020675:Peroxisomes / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D019076:Transgenes; D025062:Yarrowia / Q000737:chemistry / Q000201:enzymology
D000214:Acyl Coenzyme A; D056804:Biofuels; D005227:Fatty Acids; D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D042962:Acyl-CoA Oxidase; C467991:POX1 protein, S cerevisiae; D002333:Carnitine Acyltransferases; C030162:carnitine octanoyltransferase