School of Chemical and Biomedical Engineering, College of Engineering, Nanyang Technological University, Singapore, Singapore.
PLoS One. 2014 Jan 21;9(1):e84853. doi: 10.1371/journal.pone.0084853. eCollection 2014.
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.
脂肪酸衍生的生物燃料和生物化学物质可以通过β-氧化途径工程在微生物中生产。在这项研究中，通过工程改造酿酒酵母的β-氧化途径，使细胞在富含脂肪酸的饲料上生长时积累更高比例的中链脂肪酸（MCFAs）。为此，获得了单倍体缺失菌株Δpox1，其中仅由 POX1 编码的酰基辅酶 A 氧化酶被删除。接下来，在Δpox1 菌株中表达了来自解脂耶氏酵母的 POX2 基因，该基因编码一种优先作用于长链酰基辅酶 A 的酰基辅酶 A 氧化酶。结果，Δpox1[pox2+] 菌株表现出生长缺陷，因为过氧化物酶体中的β-氧化途径被阻断。为了解除β-氧化途径的阻断，在Δpox1[pox2+] 菌株中表达了编码肉碱 O-辛酰基转移酶的基因 CROT，以将积累的中链酰基辅酶 A 从过氧化物酶体中转运出去。获得的Δpox1[pox2+, crot+] 菌株以正常速度生长。当菌株在含有油酸的培养基中生长时，研究了这些遗传修饰对脂肪酸积累和谱的影响。结果表明，工程菌株Δpox1[pox2+]和Δpox1[pox2+, crot+]增加了脂肪酸积累和 MCFAs 的比例。与野生型（WT）菌株相比，菌株Δpox1[pox2+]和Δpox1[pox2+, crot+]的总脂肪酸产量分别增加了 29.5%和 15.6%。Δpox1[pox2+]和Δpox1[pox2+, crot+]菌株的细胞内 MCFAs 水平分别比 WT 菌株增加了 2.26 倍和 1.87 倍。此外，与 WT 菌株相比，培养基中 MCFAs 的水平分别增加了 3.29 倍和 3.34 倍。这些结果表明，在修饰的β-氧化途径的工程菌株中积累了具有增加 MCFAs 比例的脂肪酸。我们的方法为将低价值富含脂肪酸的饲料转化为高价值脂肪酸衍生产品提供了巨大潜力。
