Advancing the formation of artificial membraneless compartments with organizational complexity and diverse functionality remains a challenge. Typically, synthetic compartments or membraneless organelles are made up of intrinsically disordered proteins featuring low-complexity sequences or polypeptides with repeated distinctive short linear motifs. In order to expand the repertoire of tools available for the formation of synthetic membraneless compartments, here, a range of DIshevelled and aXin (DIX) or DIX-like domains undergoing head-to-tail polymerization were demonstrated to self-assemble into aggregates and generate synthetic compartments within E. coli cells. Then, synthetic complex compartments with diverse intracellular morphologies were generated by coexpressing different DIX domains. Further, we genetically incorporated a pair of interacting motifs, comprising a homo-dimeric domain and its anchoring peptide, into the DIX domain and cargo proteins, respectively, resulting in the alteration of both material properties and client recruitment of synthetic compartments. As a proof-of-concept, several human milk oligosaccharide biosynthesis pathways were chosen as model systems. The findings indicated that the recruitment of pathway sequential enzymes into synthetic compartments formed by DIX–DIX heterotypic interactions or by DIX domains embedded with specific interacting motifs efficiently boosted metabolic pathway flux and improved the production of desired chemicals. We propose that these synthetic compartment systems present a potent and adaptable toolkit for controlling metabolic flux and facilitating cellular engineering.

中文翻译：

---

将通路序列酶区室化为合成蛋白区室，以优化大肠杆菌的代谢通量


推进具有组织复杂性和多样化功能的人工无膜隔室的形成仍然是一项挑战。通常，合成区室或无膜细胞器由具有低复杂性序列的固有无序蛋白质或具有重复独特短线性基序的多肽组成。为了扩大可用于形成合成无膜区室的工具库，这里证明了一系列经历头到尾聚合的 DIshevelled 和 aXin （DIX） 或 DIX 样结构域自组装成聚集体并在大肠杆菌细胞内产生合成区室。然后，通过共表达不同的 DIX 结构域生成具有不同细胞内形态的合成复合区室。此外，我们在基因上将一对相互作用的基序（包括同型二聚体结构域及其锚定肽）分别整合到 DIX 结构域和转运蛋白中，导致材料特性的改变和合成区室的客户端募集。作为概念验证，选择了几种母乳低聚糖生物合成途径作为模型系统。研究结果表明，将通路顺序酶募集到由 DIX-DIX 异型相互作用或嵌入特定相互作用基序的 DIX 结构域形成的合成区室中，有效地提高了代谢途径通量并改善了所需化学物质的产生。我们建议这些合成隔室系统提供了一种有效且适应性强的工具包，用于控制代谢通量和促进细胞工程。