School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, 637459, Singapore, Singapore.
Appl Microbiol Biotechnol. 2014 Aug;98(15):6739-50. doi: 10.1007/s00253-014-5758-8. Epub 2014 Apr 26.
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, flux toward the direct precursor fatty acids needs to be enhanced to approach high yields. Herein, fatty acyl-CoA metabolism in Saccharomyces cerevisiae was engineered to accumulate more free fatty acids (FFA). For this purpose, firstly, haploid S. cerevisiae double deletion strain △faa1△faa4 was constructed, in which the genes FAA1 and FAA4 encoding two acyl-CoA synthetases were deleted. Then the truncated version of acyl-CoA thioesterase ACOT5 (Acot5s) encoding Mus musculus peroxisomal acyl-CoA thioesterase 5 was expressed in the cytoplasm of the strain △faa1△faa4. The resulting strain △faa1△faa4 [Acot5s] accumulated more extracellular FFA with higher unsaturated fatty acid (UFA) ratio as compared to the wild-type strain and double deletion strain △faa1△faa4. The extracellular total fatty acids (TFA) in the strain △faa1△faa4 [Acot5s] increased to 6.43-fold as compared to the wild-type strain during the stationary phase. UFA accounted for 42 % of TFA in the strain △faa1△faa4 [Acot5s], while no UFA was detected in the wild-type strain. In addition, the expression of Acot5s in △faa1△faa4 restored the growth, which indicates that FFA may not be the reason for growth inhibition in the strain △faa1△faa4. RT-PCR results demonstrated that the de-repression of fatty acid synthesis genes led to the increase of extracellular fatty acids. 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, demonstrating great potential for exploitation in the platform of microbial fatty acid-derived biofuels.
近年来，由于微生物脂肪酸衍生生物燃料具有替代石油衍生燃料的潜力，因此引起了极大的关注。为了在成本上与当前的石油燃料竞争，需要增强流向直接前体脂肪酸的通量，以接近高产量。在此，通过工程改造酿酒酵母中的酰基辅酶 A 代谢以积累更多的游离脂肪酸 (FFA)。为此，首先构建了单倍体酿酒酵母双缺失菌株△faa1△faa4，其中缺失了编码两种酰基辅酶 A 合成酶的 FAA1 和 FAA4 基因。然后在菌株△faa1△faa4 的细胞质中表达了截短的肌型酰基辅酶 A 硫酯酶 ACOT5 (Acot5s)。与野生型菌株和双缺失菌株△faa1△faa4 相比，得到的菌株△faa1△faa4 [Acot5s]积累了更多的细胞外 FFA，且不饱和脂肪酸 (UFA) 比例更高。与野生型菌株相比，菌株△faa1△faa4 [Acot5s]在静止期时细胞外总脂肪酸 (TFA) 增加了 6.43 倍。UFA 占菌株△faa1△faa4 [Acot5s]中 TFA 的 42%，而在野生型菌株中则没有检测到 UFA。此外，在△faa1△faa4 中表达 Acot5s 恢复了生长，这表明 FFA 可能不是菌株△faa1△faa4 生长抑制的原因。RT-PCR 结果表明，通过删除酰基辅酶 A 合成酶和表达硫酯酶来解除脂肪酸合成基因的阻遏，可导致细胞外脂肪酸增加。本研究表明，通过控制酰基辅酶 A 代谢，通过删除酰基辅酶 A 合成酶和表达硫酯酶，可以在酿酒酵母中产生更多的 FFA，这表明在微生物脂肪酸衍生生物燃料的平台上具有很大的开发潜力。
