The low enzymatic capability of terpene synthases and the limited availability of precursors often hinder the productivity of terpenes in microbial hosts. Herein, a systematic approach combining protein engineering and pathway compartmentation was exploited in Yarrowia lipolytica for the high-efficient production of trans-nerolidol, a sesquiterpene with various commercial applications. Through the single-gene overexpression, the reaction catalyzed by nerolidol synthase (FaNES1) was identified as another rate-limiting step. An optimized FaNES1^G498Q was then designed by rational protein engineering using homology modeling and docking studies. Additionally, further improvement of trans-nerolidol production was observed as enhancing the expression of an endogenous carnitine acetyltransferase (CAT2) putatively responsible for acetyl-CoA shuttling between peroxisome and cytosol. To harness the peroxisomal acetyl-CoA pool, a parallel peroxisomal pathway starting with acetyl-CoA to trans-nerolidol was engineered. Finally, the highest reported titer of 11.1 g/L trans-nerolidol in the Y. lipolytica platform was achieved in 5 L fed-batch fermentation with the carbon restriction approach.

中文翻译：

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通过改善前体供应和优化橙花醇合酶活性提高解脂耶氏酵母中的反式橙花醇生产力

萜烯合酶的低酶促能力和前体的有限可用性通常阻碍微生物宿主中萜烯的生产力。在此，在解脂耶氏酵母中开发了一种结合蛋白质工程和通路区室的系统方法，用于高效生产反式橙花醇，一种具有多种商业应用的倍半萜烯。通过单基因过表达，橙花醇合酶 ( Fa NES1) 催化的反应被确定为另一个限速步骤。然后使用同源建模和对接研究通过合理的蛋白质工程设计优化的Fa NES1 ^{G498Q 。}此外，进一步改进反式-nerolidol 的产生被观察为增强内源性肉碱乙酰转移酶 (CAT2) 的表达，该酶推测负责乙酰辅酶 A 在过氧化物酶体和胞质溶胶之间穿梭。为了利用过氧化物酶体乙酰辅酶 A 池，设计了从乙酰辅酶 A 到反式橙花醇的平行过氧化物酶体途径。最后，解脂耶氏酵母平台中报道的最高滴度为 11.1 g/L反式橙花醇是在采用碳限制方法的 5 L 补料分批发酵中实现的。