Decaffeination and Measurement of Caffeine Content by Addicted <i>Escherichia coli</i> with a Refactored <i>N</i>‑Demethylation Operon from <i>Pseudomonas putida</i> 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 <i>Pseudomonas putida</i> CBB5 to function in <i>Escherichia coli</i>. In the process, we discovered that adding a glutathione <i>S</i>-transferase from <i>Janthinobacterium</i> sp. Marseille was necessary to achieve <i>N</i>₇-demethylation activity. <i>E. coli</i> cells with the synthetic operon degrade caffeine to the guanine precursor, xanthine. Cells deficient in <i>de novo</i> 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 <i>N</i>-demethylation operon could be useful for reclaiming nutrient-rich byproducts of coffee bean processing and for the cost-effective bioproduction of methylxanthine drugs