479159 Production of Theobromine by N-demethylation of Caffeine Using Metabolically Engineered E. coli

Wednesday, November 16, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Khalid Algharrawi1, Ryan M. Summers2 and Mani Subramanian1, (1)Chemical and Biochemical Engineering, University of Iowa, Coralville, IA, (2)Chemical and Biological Engineering, The University of Alabama, Tuscaloosa, AL

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 caffeine). The reaction was scaled up to 1.95 L and theobromine was 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.

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See more of this Session: Poster Session: Process Development
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