Synthetic metabolic channel by functional membrane microdomains for compartmentalized flux control

• Published in: Metabolic engineering 2020-05, Vol.59, p.106-118
• Main Authors: Lv, Xueqin, Wu, Yaokang, Tian, Rongzhen, Gu, Yang, Liu, Yanfeng, Li, Jianghua, Du, Guocheng, Ledesma-Amaro, Rodrigo, Liu, Long
• Format: Article
• Language: English
• Subjects: Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Functional membrane microdomains; GlcNAc synthesis; Membrane Microdomains - genetics; Membrane Microdomains - metabolism; Metabolic channel; Metabolic Engineering; Metabolic Networks and Pathways; Multienzyme complex system
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2020.02.003

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. •A synthetic multi-enzyme complex system was successfully created based on an endogenous spatial scaffold named FMMs.•The existence of a synthetic substrate channel is verified by combinatorial metabolic model and kinetics analysis.•The synthetic multi-enzyme complex system can effectively alleviate the cell lysis.