Engineered Regulon to Enable Autonomous Azide Ion Biosensing, Recombinant Protein Production, and in Vivo Glycoengineering

Detection of azide-tagged biomolecules ( e.g. , azido sugars) inside living cells using "click" chemistry has been revolutionary to the field of chemical biology. However, we currently still lack suitable synthetic biology tools to autonomously and rapidly detect azide ions. Here, we have developed an engineered synthetic promoter system called cyn regulon, and complementary Escherichia coli engineered strains, to selectively detect azide ions and autonomously induce downstream expression of reporter genes. The engineered cyn azide operon allowed highly tunable reporter green fluorescent protein (GFP) expression over three orders of inducer azide ion concentrations (0.01-5 mM) and rapidly induced GFP expression by over 600-fold compared to the uninduced control. Next, we showcase the superior performance of this engineered cyn -operon over the classical lac -operon for recombinant protein production. Finally, we highlight how this synthetic biology toolkit can enable glycoengineering-based applications by facilitating in vivo activity screening of mutant carbohydrate-active enzymes (CAZymes), called glycosynthases, using azido sugars as donor substrates.