基于内在无序蛋白质域的相分离合成细胞器用于大肠杆菌中的代谢途径组装,ACS Nano

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基于内在无序蛋白质域的相分离合成细胞器用于大肠杆菌中的代谢途径组装
ACS Nano ( IF 15.8 ) Pub Date : 2023-05-16 , DOI: 10.1021/acsnano.3c02333
Li Wan ₁ , Yingying Zhu ₁ , Wenli Zhang ₁ , Wanmeng Mu _{1,

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Affiliation

广泛的研究工作集中在受限细胞环境中生物催化级联或催化网络的空间组织。受自然代谢系统的启发，该系统通过隔离到亚细胞区室来空间调节通路，通过在宿主菌株中表达本质上无序的蛋白质来形成人工无膜细胞器已被证明是一种可行的策略。在这里，我们报告了一个合成无膜细胞器平台的工程，该平台可用于扩展区室化和空间组织途径顺序酶。我们展示了来自大肠杆菌中无序 P 颗粒蛋白 LAF-1 的 RGG 结构域的异源过表达菌株可以通过液-液相分离形成细胞内蛋白质浓缩物。我们进一步证明，可以通过直接与 RGG 结构域融合或与不同的蛋白质相互作用基序合作，将不同的客户招募到合成隔间。使用 2'-岩藻糖基乳糖从头生物合成途径作为模型系统，我们表明，与具有自由漂浮途径酶的菌株相比，将顺序酶聚集到合成区室中可以有效地提高目标产物的滴度和产量。这里构建的合成无膜细胞器系统为微生物细胞工厂的发展提供了一种有前途的方法，其中它可用于通路酶的区室化以简化代谢通量。

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Phase-Separated Synthetic Organelles Based on Intrinsically Disordered Protein Domain for Metabolic Pathway Assembly in Escherichia coli

Extensive research efforts have been focused on spatial organization of biocatalytic cascades or catalytic networks in confined cellular environments. Inspired by the natural metabolic systems that spatially regulate pathways via sequestration into subcellular compartments, formation of artificial membraneless organelles through expressing intrinsically disordered proteins in host strains has been proven to be a feasible strategy. Here we report the engineering of a synthetic membraneless organelle platform, which can be used to extend compartmentalization and spatially organize pathway sequential enzymes. We show that heterologous overexpression of the RGG domain derived from the disordered P granule protein LAF-1 in an Escherichia coli strain can form intracellular protein condensates via liquid–liquid phase separation. We further demonstrate that different clients can be recruited to the synthetic compartments via directly fusing with the RGG domain or cooperating with different protein interaction motifs. Using the 2′-fucosyllactose de novo biosynthesis pathway as a model system, we show that clustering sequential enzymes into synthetic compartments can effectively increase the titer and yield of the target product compared to strains with free-floating pathway enzymes. The synthetic membraneless organelle system constructed here gives a promising approach in the development of microbial cell factories, wherein it could be used for the compartmentalization of pathway enzymes to streamline metabolic flux.

更新日期：2023-05-16

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