Design and construction of a novel transcription factor-based glycine biosensor for directed enzyme evolution in...

Glycine is an essential metabolite and a valuable precursor for various industrially relevant compounds. However, the lack of efficient high-throughput detection methods has limited progress in metabolic engineering for glycine bioproduction. In this study, we developed a transcription factor-based glycine biosensor in Escherichia coli by reprogramming the native GcvA/GcvR regulatory complex. By fine-tuning the expression balance between GcvA and GcvR, the biosensor exhibited significantly reduced basal fluorescence while achieving strong glycine-dependent induction of up to 16.45-fold. The optimized system demonstrated rapid response kinetics, high sensitivity, and excellent specificity toward exogenously supplied glycine. The biosensor accurately reflected intracellular glycine concentrations in engineered glycine-producing strains, with fluorescence output correlating with production levels. Furthermore, its application enabled high-throughput screening of SerC (phosphoserine aminotransferase) mutants with enhanced glycine production. The best-performing SerC variant resulted in a 48.54-fold increase in fluorescence intensity and a 2.25-fold improvement in glycine accumulation, validating the biosensor as an effective tool for directed enzyme evolution. This work established a robust and tunable platform for glycine biosensing, offering broad potential for metabolic pathway optimization and enzyme engineering related to glycine metabolism.