(4dr) Microbial Caffeine Junkies: Growth of Pseudomonas Putida CBB5 On Caffeine and Metabolic Engineering of E. Coli for Production of High-Value Methylxanthines From Caffeine

Alkylxanthines are naturally occurring compounds that are used in pharmaceutical preparations as diuretics, asthma treatments, and cardiac, pulmonary, and neurological stimulants.  The most common of these compounds, caffeine (1,3,7-trimethylxanthine), is also found in many common food and beverage products such as coffee, tea, soft drinks, and chocolates.  Because of their wide use, caffeine and related alkylxanthines enter the environment through human waste water and both solid and liquid wastes from coffee and tea processing facilities.  Not surprisingly, bacteria capable of growing on caffeine have been isolated previously.  However, the mechanism of bacterial caffeine degradation has not been elucidated until now.

Metabolism of caffeine by new enzymes in Pseudomonas putida CBB5.

We have discovered a novel caffeine-degrading bacterium, Pseudomonas putida CBB5, from soil at the University of Iowa by enrichment on caffeine as sole carbon and nitrogen source.  CBB5 utilizes five novel enzymes belonging to the Rieske oxygenase (RO) family, NdmABCDE, to metabolize caffeine by sequential N-demethylation to xanthine, which enters normal purine catabolism.  NdmA, NdmB, and NdmC are positional-specific N-demethylases that remove the methyl groups from caffeine as formaldehyde.  All three enzymes require an unusually large redox-dense reductase protein, NdmD, which transfers electrons from NADH to the non-heme iron at the catalytic site of NdmA, NdmB, and NdmC.  NdmA and NdmB, which catalyze the first two steps in the caffeine degradation pathway, contain the basic RO structure with both Rieske [2Fe-2S] and non-heme iron domains.  NdmC, however, contains only a non-heme iron domain.  Curiously, the Rieske [2Fe-2S] domain for NdmC is fused to the N-terminus of NdmD, which is the only characterized RO reductase that contains an extra Rieske [2Fe-2S] cluster.  NdmE is a novel protein belonging to the glutathione-S-transferase (GST) superfamily that may serve a structural role in bridging the Rieske [2Fe-2S] from NdmD and non-heme iron from NdmC.  Together, NdmCDE form a large protein complex that carries out the N₇-demethylation of 7-methylxanthine to xanthine, the last step in the caffeine N-demethylation pathway.  This is the first report of a GST-dependent oxygenase.

Metabolic engineering of E. coli to produce high-value methylxanthines.

Many of the methylxanthine metabolites produced in the caffeine N-demethylation pathway, such as paraxanthine (1,7-dimethylxanthine), 1-methylxanthine, 3-methylxanthine, and 7-methylxanthine, are high-value compounds used in the pharmaceutical industry.  We have engineered over 40 strains of E. coli with various combinations of ndmABD to create these compounds from caffeine and theophylline (1,3-dimethylxanthine), a 200- to 5,000-fold increase in value.  We are currently performing protein engineering experiments to increase production of paraxanthine from caffeine.  The caffeine-degrading genes from P. putida CBB5 also provide many more potential applications, including (i) environmental remediation of coffee and tea waste and by-products, (ii) bio-decaffeination of caffeine, (iii) biosynthesis of pharmaceuticals, (iv) decaffeination of coffee and tea waste prior to fermentation, (v) development of caffeine biosensors, and (vi) regulation of synthetic gene networks using caffeine- or theophylline-responsive RNA switches.