Ding Qiang, Ji Mengqi, Yao Buhan, Wang Yongzhong
School of Life Sciences, Anhui University, Hefei 230601, China; Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes, Anhui University, Hefei 230601 Anhui, China; Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601 Anhui, China.
School of Life Sciences, Anhui University, Hefei 230601, China; Key Laboratory of Human Microenvironment and Precision Medicine of Anhui Higher Education Institutes, Anhui University, Hefei 230601 Anhui, China; Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601 Anhui, China.
Bioresour Technol. 2025 Mar;420:132109. doi: 10.1016/j.biortech.2025.132109. Epub 2025 Jan 24.
Microbial compartment provides a promising approach for achieving high-valued chemical biosynthesis from renewable feedstock. However, volatile precursor could be utilized by pathway enzyme, which may hinder and adverse the cascade catalysis within microbial cell factory. Here, a customizable compartment was developed for pathway sequestration using spatially assembled cascade catalysis reaction. Firstly, a phase separation protein was designed to form the intracellular protein condensates, facilitating the construction of a customizable compartment in Escherichia coli. Subsequently, modular assembly and recruitment of customizable compartment were achieved through using a short peptide interaction pair to cluster enzymes or fuse them directly. Finally, the 2'-fucosyllactose (2'-FL) salvage pathway was heterogeneously expressed in microorganisms as a stable targeted chemical and proof-of-concept model, the results showed that anchoring various enzymes required for the 2'-FL cascade catalysis pathway within the customizable compartment created a multiple enzyme condensate system, resulting an improvement of 2'-FL titer compared to both wild type and optimized free enzymes reaction. These findings illustrating an effectively cascade catalysis model that increasing titer and kick-starting metabolic flux control through co-localizing multiple enzymes condensate within microbial cell factories.
微生物区室为从可再生原料实现高价值化学品生物合成提供了一种有前景的方法。然而，挥发性前体可能会被途径酶利用，这可能会阻碍并对微生物细胞工厂内的级联催化产生不利影响。在此，利用空间组装的级联催化反应开发了一种用于途径隔离的可定制区室。首先，设计了一种相分离蛋白以形成细胞内蛋白凝聚物，便于在大肠杆菌中构建可定制区室。随后，通过使用短肽相互作用对将酶聚集或直接融合来实现可定制区室的模块化组装和招募。最后，将2'-岩藻糖基乳糖（2'-FL）补救途径作为一种稳定的目标化学品和概念验证模型在微生物中异源表达，结果表明，将2'-FL级联催化途径所需的各种酶锚定在可定制区室内创建了一个多酶凝聚物系统，与野生型和优化的游离酶反应相比，2'-FL滴度有所提高。这些发现说明了一种有效的级联催化模型，即通过在微生物细胞工厂内共定位多种酶凝聚物来提高滴度并启动代谢通量控制。
