Development and characterization of a glycine biosensor system for fine-tuned metabolic regulation in Escherichia...

In vivo biosensors have a wide range of applications, ranging from the detection of metabolites to the regulation of metabolic networks, providing versatile tools for synthetic biology and metabolic engineering. However, in view of the vast array of metabolite molecules, the existing number and performance of biosensors is far from sufficient, limiting their potential applications in metabolic engineering. Therefore, we developed the synthetic glycine-ON and -OFF riboswitches for metabolic regulation and directed evolution of enzyme in Escherichia coli.

The results showed that a synthetic glycine-OFF riboswitch (glyOFF6) and an increased-detection-range synthetic glycine-ON riboswitch (glyON14) were successfully screened from a library based on the Bacillus subtilis glycine riboswitch using fluorescence-activated cell sorting (FACS) and tetA-based dual genetic selection. The two synthetic glycine riboswitches were successfully used in tunable regulation of lactate synthesis, dynamic regulation of serine synthesis and directed evolution of alanine-glyoxylate aminotransferase in Escherichia coli, respectively. Mutants AGXT22 and AGXT26 of alanine-glyoxylate aminotransferase with an increase of 58% and 73% enzyme activity were obtained by using a high-throughput screening platform based on the synthetic glycine-OFF riboswitch, and successfully used to increase the 5-aminolevulinic acid yield of engineered Escherichia coli.

A synthetic glycine-OFF riboswitch and an increased-detection-range synthetic glycine-ON riboswitch were successfully designed and screened. The developed riboswitches showed broad application in tunable regulation, dynamic regulation and directed evolution of enzyme in E. coli.