Engineering a Silk Protein-Mediated Customizable Compartment for Modular Metabolic Synthesis.

Microbial cell factories provide a nontoxic, economical way for the synthesis of various chemicals and drugs, garnering significant attention from researchers. However, excessive dispersion of enzymes and accumulation of intermediate metabolites in the production process will weaken the reaction efficiency of the pathway enzyme. In this study, a cellular compartment was constructed to isolate the enzyme reaction space and optimize the modular metabolic synthesis. First, a special spider silk protein was designed and constructed to form protein condensates in microbial cells, and its synthetic microcompartment effects were investigated. Second, the interaction of short peptide pairs or direct fusion based on the silk protein was used to recruit a variety of enzymes to improve the efficiency of enzyme catalysis. Third, the 2'-fucosyllactose (2'-FL) de novo synthesis pathway and its modular optimization were carried out to verify the mode. Finally, a synthetic compartment was introduced into the pathway to directly aggregate the 2'-FL synthesis pathway, thus obtaining synthetic-compartment-mediated multienzyme aggregates. The experimental results showed that the titer of 2'-FL was significantly improved compared with those of wild-type and modular-optimized free enzymes. The utilization of this cell microcompartment offers a novel avenue for the aggregation of diverse enzymes, thereby offering an innovative approach for enhancing the efficiency of the microbial modular metabolic pathway.