Production of theobromine by N-demethylation of caffeine using metabolically engineered E. coli

This study reports bench-scale feasibility study for production and recovery of theobromine from caffeine using metabolically engineered E. coli. The N1-demethylase genes used in this study were originally discovered in Pseudomonas putida CBB5. Five strains of E. coli engineered with multiple combinations of the two N-demethylase genes were compared for growth and activity. Strain pAD1dDD containing one copy of ndmA and three copies of ndmD in two compatible plasmids, gave the best results (98.5% molar conversion of caffeine to theobromine). The reaction was scaled up to 2 L and theobromine was separated and recovered in nearly 100% pure form via preparative chromatography and drying. Final yield was 80% relative to caffeine. Purity of the product was identical to an authentic standard as judged by LC-MS and NMR. This is the first report of theobromine production via a metabolically engineered strain, with the highest yield and purity reported of any biological method. This method also eliminates the harsh reaction conditions and environmental issues associated with the chemical process.