Restoring a synthetic methylxanthine degradation operon to its native genes

Caffeinated Coli are Escherichia coli strains engineered to degrade methylxanthines, providing an accessible bioassay for determining methylxanthine concentrations, with applications in food safety, bioremediation, medicine, and STEM education. The Caffeinated Coli decaffeination operon encodes four N-demethylation enzymes, including three N-demethylases, from Pseudomonas putida CBB5. A fifth P. putida protein essential for N ₇ -demethylation, NdmE, was substituted with the homolog Gst9, which may have unfavorable interactions with the P. putida enzymes, compromising N ₇ -demethylation in Caffeinated Coli. Here, we restored ndmE to the decaffeination operon and found most of the new N ₇ -demethylating strains can grow at slightly higher temperatures than their gst9 -containing counterparts.