Engineering a Temperature-Programmable Biosensor Toolkit for Recombinant Protein Production in Corynebacterium glutamicum

Dynamic metabolic regulation is crucial for optimizing microbial cell factories. To address the limitations of chemical inducers, this study developed a temperature-responsive synthetic biology toolkit for Corynebacterium glutamicum . A high-performance, heat-inducible biosensor was engineered by optimizing the CI ⁸⁵⁷ repressor and its cognate promoter, yielding a variant (CI ⁸⁵⁷ -M3/H1) with a 107-fold dynamic range and minimal background leakage. Additionally, a cold-inducible RNA thermometer was implemented using the Escherichia coli csapA 5'UTR. These components were integrated into a dual-functional genetic circuit enabling bidirectional metabolic control. Finally, the optimized heat-inducible sensor was applied to the production of three secretory proteins with distinct characteristics (AmyE, XylA, and VHH), and the scale-up cultivation of AmyE was successfully achieved in 1-L shake-flasks. This work provides an efficient, inducer-free strategy for precise metabolic regulation, offering a scalable and cost-effective tool for advanced biomanufacturing.