(620cd) Isolation of Caffeine-Degrading Bacteria from Soil

Caffeine (1,3,7-trimethylxanthine) is a natural compound frequently found in many common food and pharmaceutical products.  Consumption of caffeine in low to moderate levels is not toxic to humans, but is lethal for many bacteria and insects.  Caffeine infiltrates the environment through many methods, including human waste and plant decomposition.  As a result, some bacteria have evolved to use caffeine as a nutrient source. Some of the metabolic intermediates formed by the bacterial degradation of caffeine are high-value chemicals that can be used in the food, cosmetics, and pharmaceutical industries.

 Two pathways exist for the degradation of caffeine: N-demethylation and C-8 oxidation.  During C-8 oxidation, caffeine is oxidized to 1,3,7-trimethyluric acid, which is further degraded to CO₂ and NH₃.  In the N-demethylation pathway, caffeine is degraded through a series of N-demethylation reactions to form theobromine (3,7-dimethylxanthine), 7-methylxanthine, and xanthine.  Xanthine then enters the normal purine catabolic pathway.  In some bacteria, paraxanthine (1,7-dimethylxanthine) is also transiently formed during the first metabolic step by N₃-demethylation of caffeine, with subsequent degradation through 7-methylxanthine and xanthine. 

 Paraxanthine is a high-value compound that provides the stimulatory effects of caffeine with fewer side effects.  In order to isolate a paraxanthine-producing strain of bacteria, we isolated several bacterial colonies from soil samples using an enrichment procedure.  Soil samples were incubated in a modified liquid M9 media with 2.5% caffeine as the sole source of carbon and nitrogen.  After three subcultures, bacteria were isolated on solid M9 + caffeine media.  The isolates were characterized phylogenetically by 16S ribosomal RNA sequencing.  Metabolism of each strain was determined by analyzing metabolites in growth media and resting cell assays by HPLC.  Paraxanthine-producing strains were identified and selected for use in future studies.