Abstract
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.
Highlights
The renewable synthesis of fatty acid-derived biofuels and chemicals has gained considerable attention in recent years
The resulting plasmid pVTU260-POX2-carnitine octanoyltransferase (CROT) was transformed into strain Dpox1, creating strain Dpox1 [pox2+, crot+]
Our study showed that when oleic acid was used as a cosubstrate of glucose, the total fatty acids were increased in engineered strains compared to the WT strain
Summary
The renewable synthesis of fatty acid-derived biofuels and chemicals has gained considerable attention in recent years. Fatty acids are overproduced in microbes and later converted to fuels or chemicals through chemical conversion [1] or bioconversion [2,3]. Other studies directly utilize fatty acid-rich feedstock as a carbon source to produce biofuels and chemicals through the complete oxidation of fatty acids [4] or using the intermediates of b-oxidation [5,6]. Fatty acids have a product yield advantage over sugars when used as a carbon source due to the efficient b-oxidation metabolism process [4], which has 100% carbon recovery. Fatty acids can be exploited as an alternative feedstock to lignocellulosic sugars for microbial bioconversion platforms
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