Abstract
Abstract 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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
