Recent advances in the production of biofuels by microbes have attracted attention due to increasingly limited fossil fuels. Biodiesels, especially fatty acid ethyl esters (FAEEs), are considered a potentially fully sustainable fuel in the near future due to similarities with petrodiesels and compatibility with existing infrastructure. However, biosynthesis of FAEEs is limited by the supply of precursor lipids and acetyl-CoA. In the present study, we explored the production potential of an engineered biosynthetic pathway coupled to the addition of ethanol in the oleaginous yeast Yarrowia lipolytica. This type of yeast is able to supply a greater amount of precursor lipids than species typically used. To construct the FAEEs synthesis pathway, WS genes that encode wax ester synthases (WSs) from different species were codon-optimized and heterologously expressed in Y. lipolytica. The most productive engineered strain was found to express a WS gene from Marinobacter hydrocarbonoclasticus strain DSM 8798. To stepwisely increase FAEEs production, we optimized the promoter of WS overexpression, eliminated β-oxidation by deleting the PEX10 gene in our engineered strains, and redirected metabolic flux toward acetyl-CoA. The new engineered strain, coupled with an optimized ethanol concentration, led to an approximate 5.5-fold increase in extracellular FAEEs levels compared to the wild-type strain and a maximum FAEEs titer of 1.18 g/L in shake flask cultures. In summary, the present study demonstrated that an engineered Y. lipolytica strain possessed a high capacity for FAEEs production and may serve as a platform for more efficient biodiesel production in the future.

中文翻译：

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产油酵母酵母解脂耶氏酵母通过代谢工程和工艺优化生产脂肪酸乙酯

由于化石燃料的日益有限，微生物在生物燃料生产中的最新进展引起了人们的关注。由于与石油柴油的相似性以及与现有基础设施的兼容性，生物柴油，尤其是脂肪酸乙酯（FAEE），在不久的将来被认为是潜在的完全可持续的燃料。但是，FAEE的生物合成受到前体脂质和乙酰辅酶A的供应的限制。在本研究中，我们探索了在油性酵母解脂耶氏酵母中添加乙醇的工程化生物合成途径的生产潜力。这种类型的酵母能够提供比通常使用的种类更多的前体脂质。为了构建FAEEs合成途径，WS编码不同物种蜡酯合酶（WSs）的基因经过密码子优化，并在解脂耶氏酵母中异源表达。已发现生产效率最高的工程菌株可从马氏杆菌碳氢破损菌株DSM 8798中表达WS基因。为了逐步提高FAEEs的产量，我们优化了WS过表达的启动子，通过删除PEX10消除了β-氧化基因在我们的工程菌株中，并且将代谢通量重定向到乙酰辅酶A。与野生型菌株相比，新的工程菌株加上优化的乙醇浓度可导致细胞外FAEEs水平增加约5.5倍，摇瓶培养中的最大FAEEs效价为1.18 g / L。总而言之，本研究表明，工程化解脂耶氏酵母菌株具有高的FAEE生产能力，并且可以作为将来更有效地生产生物柴油的平台。