Modular metabolic flux control for kick-starting cascade catalysis through engineering customizable compartment.

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.