Background Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.

中文翻译：

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从 Eat 到 trEat：改造线粒体 Eat1 酶以增强大肠杆菌中乙酸乙酯的产生。

背景 微生物的基因工程已成为建立微生物细胞工厂以生产多种化合物的常见做法。乙酸乙酯是一种工业溶剂，有多种用途，主要用作低毒的可生物降解有机溶剂。虽然乙酸乙酯是由几种天然酵母菌种生产的，但其主要生产机制仍然难以捉摸，直到在异常 Wickerhamomyces anomalus 中发现 Eat1。与其他酵母酒精乙酰转移酶 (AAT) 不同，Eat1 位于酵母线粒体中，表明编码序列包含线粒体前序列。对于在大肠杆菌等原核宿主中的表达，具有真核信号序列的异源蛋白的表达可能不是最佳的。结果已在体外比较了马克斯克鲁维酵母和异常柳杆菌的未加工和合成截断的eat1变体的酶活性和稳定性。虽然比活性不受影响，但几个截断变体的半衰期有所改善。当在大肠杆菌中表达时，相同的变体在乙酸乙酯生产方面表现出更好的性能。结论通过分析预测不同Eat1蛋白的N端前序列并对其进行系统修剪，可以提高酶的体外稳定性，从而全面提高大肠杆菌体内乙酸乙酯的产量。因此，eat1 的截短变体可能有利于未来有效生产乙酸乙酯的工程方法。