Pseudomonas putida CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential N-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine N-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske non-heme iron monooxygenases with N1- and N3-specific N-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one FMN per molecule. In addition, two more genes, ndmC and ndmE, along with ndmD are involved in 7-N-demethylation. All ndm genes are located in a 13.2-kb genomic DNA fragment, which also contains a formaldehyde dehydrogenase. Several E. coli strains have been metabolically engineered with various combinations and copy numbers of ndmA, B and D. Some of these strains are able to convert caffeine, theophylline, and theobromine to high value methylxanthines like 1-, 2-, 7-methylxanthines, and 1,7-dimethylxanthines. These strains also have the potential to: (a) decaffeinate spent coffee and tea extracts so that the left over solids can be used as feedstock for ethanol or as animal feed. (b) The coffee and tea extracts can also be specifically decaffeinated to produce high value methylxanthines. This is the first report of complete genetic characterization and utility of bacterial N-demethylase genes involved in caffeine, theophylline and theobromine utilization. NdmA, B, and D represent a new class of Rieske oxygenases. NdmE is a glutathione transferase homolog that is specifically involved in 7-N-demethylation, with Ndm C and D. This is also the first report of absolute requirement of glutathione transferase-like protein in an oxygenase reaction.

New genetic insights to consider coffee and tea waste as “biomass” for fuels and chemicals

245th National Meeting (2013)

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