Lin Jyun-Liang, Zhu Jie, Wheeldon Ian
Department of Chemical and Environmental Engineering, University of California , Riverside, California 92521, United States.
ACS Synth Biol. 2017 Aug 18;6(8):1534-1544. doi: 10.1021/acssynbio.7b00041. Epub 2017 May 23.
Eukaryotic biochemistry is organized throughout the cell in and on membrane-bound organelles. When engineering metabolic pathways this organization is often lost, resulting in flux imbalance and a loss of kinetic advantages from enzyme colocalization and substrate channeling. Here, we develop a protein-based scaffold for colocalizing multienzyme pathways on the membranes of intracellular lipid droplets. Scaffolds based on the plant lipid droplet protein oleosin and cohesin-dockerin interaction pairs recruited upstream enzymes in yeast ester biosynthesis to the native localization of the terminal reaction step, alcohol-O-acetyltransferase (Atf1). The native localization is necessary for high activity and pathway assembly in close proximity to Atf1 increased pathway flux. Screening a library of scaffold variants further showed that pathway structure can alter catalysis and revealed an optimized scaffold and pathway expression levels that produced ethyl acetate at a rate nearly 2-fold greater than unstructured pathways. This strategy should prove useful in spatially organizing other metabolic pathways with key lipid droplet-localized and membrane-bound reaction steps.
真核生物化学在整个细胞内的膜结合细胞器内部和表面进行组织。在构建代谢途径时，这种组织性常常丧失，导致通量失衡以及酶共定位和底物通道化带来的动力学优势丧失。在此，我们开发了一种基于蛋白质的支架，用于在细胞内脂质滴的膜上使多酶途径共定位。基于植物脂质滴蛋白油质蛋白和黏连蛋白 - 对接蛋白相互作用对构建的支架，将酵母酯生物合成中的上游酶招募到终端反应步骤乙醇 - O - 乙酰转移酶（Atf1）的天然定位处。这种天然定位对于高活性是必需的，并且在Atf1附近的途径组装增加了途径通量。对支架变体文库的筛选进一步表明，途径结构可以改变催化作用，并揭示了一种优化的支架和途径表达水平，其产生乙酸乙酯的速率比无结构途径快近2倍。该策略在空间组织其他具有关键脂质滴定位和膜结合反应步骤的代谢途径方面应会被证明是有用的。
