Engineering Temperature-Powered Synthetic Multilayer Molecular Bioswitch for High-Level Pyruvate Derivative Production in Escherichia coli

Microbial engineering and genetically encoded bioswitches provide an excellent platform for the production of high-value biochemicals. However, the synthesis of biobased compounds associated with central pathways or multiple bypasses is often restricted by robust central metabolism, incompatible metabolic homeostasis, and limited genetic tools. Here, a programmable and bifunctional temperature- and pyruvate-responsive biosensor was designed to establish an efficient biosynthesis platform for pyruvate derivatives. Especially, the novel variant L185P of regulator CI⁸⁵⁷ exhibited a 37.08% improvement in thermostability by forming a ring oscillation and a more rigid structure. Subsequently, the tool TPLC, based on thermosensor- and pyruvate biosensor-based layered circuit, was created. Using iso-butylamine synthesis as a proof-of-concept, a 3.31-fold increase in titer was achieved by precisely fine-tuning cell metabolism and bioproduct synthesis, and its titer further reached 46.24 g/L in a fermenter. This work highlights powerful toolkits for regulating multigene pathways, enabling the efficient production of high-value bioproducts of industrial interest of industry.