The anchoring of metabolic pathway enzymes to spatial scaffolds can significantly improve their reaction efficiency. Here, we successfully constructed a multi-enzyme complex assembly system able to enhance bioproduction in bacteria by using the endogenous spatial scaffolds─functional membrane microdomains (FMMs). First, using VA-TIRFM and SPT analysis, we reveal that FMMs possess high temporal and spatial stability at the plasma membrane and can be used as endogenous spatial scaffolds to organize enzyme pathways. Then, taking the synthesis of N-acetylglucosamine (GlcNAc) in Bacillus subtilis as a proof-of-concept demonstration, we found that anchoring of various enzymes required for GlcNAc synthesis onto FMMs to obtain the FMMs-multi-enzyme complex system resulted in a significant increase in GlcNAc titer and an effectively alleviate in cell lysis at the later stage of fermentation compared to that in control strains expressing the related enzymes in the cytoplasm. Combining with metabolic model and kinetics analysis, the existence of a constructed substrate channel that maximizes the reaction efficiency is verified. In summary, we propose a novel metabolic pathway assembly model which allowed improved titers and compartmentalized flux control with high spatial resolution in bacterial metabolism.

代谢途径酶在空间支架上的锚定可以显着提高其反应效率。在这里，我们成功地构建了一种多酶复合物组装系统，该系统可以通过使用内源性空间支架功能膜微域（FMM）来增强细菌的生物生产。首先，使用VA-TIRFM和SPT分析，我们发现FMMs在质膜上具有较高的时空稳定性，可以用作组织酶途径的内源性空间支架。然后，在枯草芽孢杆菌中合成N-乙酰氨基葡萄糖（GlcNAc）作为概念验证的证明，我们发现将GlcNAc合成所需的各种酶锚固在FMM上以获得FMMs-多酶复合物系统，导致GlcNAc滴度显着增加，并在以后有效缓解了细胞裂解相比于在细胞质中表达相关酶的对照菌株而言 结合代谢模型和动力学分析，验证了存在的底物通道的存在，该通道可使反应效率最大化。总之，我们提出了一种新的代谢途径装配模型，该模型允许在细菌代谢中以更高的空间分辨率改善滴度和分区流量控制。