title: Decaffeination and measurement of caffeine content by addicted Escherichia coli with a refactored N-demethylation operon from Pseudomonas putida CBB5.
abstract: The widespread use of caffeine (1,3,7-trimethylxanthine) and other methylxanthines in beverages and pharmaceuticals has led to significant environmental pollution. We have developed a portable caffeine degradation operon by refactoring the alkylxanthine degradation (Alx) gene cluster from Pseudomonas putida CBB5 to function in Escherichia coli. In the process, we discovered that adding a glutathione S-transferase from Janthinobacterium sp. Marseille was necessary to achieve N 7 -demethylation activity. E. coli cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in de novo guanine biosynthesis that contain the refactored operon are ″addicted″ to caffeine: their growth density is limited by the availability of caffeine or other xanthines. We show that the addicted strain can be used as a biosensor to measure the caffeine content of common beverages. The synthetic N-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs.
authors: Center for Systems and Synthetic Biology, Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, The University of Texas at Austin , Austin, Texas 78712, United States.
authors: Erik M
authors: EM
authors: Quandt
authors: Michael J
authors: MJ
authors: Hammerling
authors: Ryan M
authors: RM
authors: Summers
authors: Peter B
authors: PB
authors: Otoupal
authors: Ben
authors: B
authors: Slater
authors: Razan N
authors: RN
authors: Alnahhas
authors: Aurko
authors: A
authors: Dasgupta
authors: James L
authors: JL
authors: Bachman
authors: Mani V
authors: MV
authors: Subramanian
authors: Jeffrey E
authors: JE
authors: Barrick
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001628:Beverages / Q000032:analysis; D015374:Biosensing Techniques; D002110:Caffeine / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D016680:Genome, Bacterial; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006147:Guanine / Q000096:biosynthesis; D008745:Methylation; D005810:Multigene Family; D009876:Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016958:Pseudomonas putida / Q000235:genetics*; D019820:Xanthine / Q000737:chemistry / Q000378:metabolism; D014970:Xanthines / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014970:Xanthines; D019820:Xanthine; C008514:methylxanthine; D002110:Caffeine; D006147:Guanine; D005982:Glutathione Transferase