Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

中文翻译：

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工程大肠杆菌厌氧乙酸乙酯生产的多级优化


乙酸乙酯是一种广泛使用的工业溶剂，目前通过化石资源的化学转化生产。一些酵母种类能够在有氧条件下将糖转化为乙酸乙酯。然而，厌氧进行乙酸乙酯合成可能会提高生产效率，使该过程在经济上更加可行。我们设计了一种大肠杆菌菌株，能够在厌氧条件下将葡萄糖转化为乙酸乙酯作为主要发酵产物。该途径的关键酶是醇乙酰转移酶 (AAT)，它催化乙酰辅酶 A 和乙醇形成乙酸乙酯。为了选择合适的 AAT，比较了来自酿酒酵母的 Atf1、来自马克斯克鲁维酵母的 Eat1 和来自异常威克哈姆酵母的 Eat1 的乙酸乙酯形成能力。在诱导型 LacI/T7 和 XylS/Pm 启动子的控制下，AAT 编码基因的异源表达可以优化其表达水平。在蛋白质和发酵水平上的工程努力导致大肠杆菌菌株能够以前所未有的效率从葡萄糖厌氧生产 42.8 mM (3.8 g/L) 乙酸乙酯，即 0.48 C-mol/C-mol 或最大途径的 72%屈服。