Zhu Jie, Schwartz Cory, Wheeldon Ian
Biochemistry, University of California Riverside, Riverside, CA 92521, USA.
Chemical and Environmental Engineering, University of California Riverside, Riverside, CA 92521 USA.
Metab Eng Commun. 2018 Dec 26;8:e00085. doi: 10.1016/j.mec.2018.e00085. eCollection 2019 Jun.
In metabolic engineering, most available pathway engineering strategies aim to control enzyme expression by making changes at the transcriptional level with an underlying assumption that translation and functional expression follow suit. In this work, we engineer expression of a key reaction step in medium chain ester biosynthesis that does not follow this common assumption. The native alcohol acyltransferses Eeb1 and Eht1 condense acyl-CoAs with ethanol to produce the corresponding ester, a reaction that is rate limiting in engineering ester biosynthesis pathways. By changing the N- and C-termini of Eeb1 to those of Eht1, Eeb1 localization is changed from the mitochondria to lipid droplets. The change has no significant effect on transcription, but increases protein expression by 23-fold thus enabling a 3-fold increase in enzyme activity. This system demonstrates one example of the impact of protein trafficking on functional pathway expression, and will guide future metabolic engineering of ester biosynthesis and, potentially, other pathways with critical membrane-bound enzymes.
在代谢工程中，大多数现有的途径工程策略旨在通过在转录水平上进行改变来控制酶的表达，其潜在假设是翻译和功能表达也会随之发生。在这项工作中，我们对中链酯生物合成中一个关键反应步骤的表达进行了工程改造，该步骤并不遵循这一普遍假设。天然的醇酰基转移酶Eeb1和Eht1将酰基辅酶A与乙醇缩合以产生相应的酯，该反应在工程化酯生物合成途径中是限速反应。通过将Eeb1的N端和C端换成Eht1的N端和C端，Eeb1的定位从线粒体改变为脂滴。这种变化对转录没有显著影响，但使蛋白质表达增加了23倍，从而使酶活性提高了3倍。该系统展示了蛋白质转运对功能性途径表达影响的一个例子，并将指导未来酯生物合成的代谢工程，以及潜在地指导其他具有关键膜结合酶的途径的代谢工程。
