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With increasing global consumption of caffeine-rich products, such as coffee, tea, and energy drinks, there is also an increase in urban and processing waste full of residual caffeine with limited disposal options. This waste caffeine has been found to leach into the surrounding environment where it poses a threat to microorganisms, insects, small animals, and entire ecosystems. Growing interest in harnessing this environmental contaminant has led to the discovery of 79 bacterial strains, eight yeast strains, and 32 fungal strains capable of metabolizing caffeine by N-demethylation and/or C-8 oxidation. Recently observed promiscuity of caffeine-degrading enzymes in vivo has opened up the possibility of engineering bacterial strains capable of producing a wide variety of caffeine derivatives from a renewable resource. These engineered strains can be used to reduce the negative environmental impact of leached caffeine-rich waste through bioremediation efforts supplemented by our increasing understanding of new techniques such as cell immobilization. Here, we compile all of the known caffeine-degrading microbial strains, discuss their metabolism and related enzymology, and investigate their potential application in bioremediation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

7-Methylxanthine, a derivative of caffeine (1,3,7-trimethylxanthine), is a high-value compound that has multiple medical applications, particularly with respect to eye health. Here, we demonstrate the biocatalytic production of 7-methylxanthine from caffeine using Escherichia coli strain MBM019, which was constructed for production of paraxanthine (1,7-dimethylxanthine). The mutant N-demethylase NdmA4, which was previously shown to catalyze N 3 -demethylation of caffeine to produce paraxanthine, also retains N 1 -demethylation activity toward paraxanthine. This study demonstrates that whole cell biocatalysts containing NdmA4 are more active toward paraxanthine than caffeine. We used four serial resting cell assays, with spent cells exchanged for fresh cells between each round, to produce 2,120 μM 7-methylxanthine and 552 μM paraxanthine from 4,331 μM caffeine. The purified 7-methylxanthine and paraxanthine were then isolated via preparatory-scale HPLC, resulting in 177.3 mg 7-methylxanthine and 48.1 mg paraxanthine at high purity. This is the first reported strain genetically optimized for the biosynthetic production of 7-methylxanthine from caffeine., (© 2022 Wiley Periodicals LLC.)

Caffeine, found in many foods, beverages, and pharmaceuticals, is the most used chemical compound for mental alertness. It is originally a natural product of plants and exists widely in environmental soil. Some bacteria, such as Pseudomonas putida CBB5, utilize caffeine as a sole carbon and nitrogen source by degrading it through sequential N-demethylation catalyzed by five enzymes (NdmA, NdmB, NdmC, NdmD, and NdmE). The environmentally friendly enzymatic reaction products, methylxanthines, are high-value biochemicals that are used in the pharmaceutical and cosmetic industries. However, the structures and biochemical properties of bacterial N-demethylases remain largely unknown. Here, we report the structures of NdmA and NdmB, the initial N 1 - and N 3 -specific demethylases, respectively. Reverse-oriented substrate bindings were observed in the substrate-complexed structures, offering methyl position specificity for proper N-demethylation. For efficient sequential degradation of caffeine, these enzymes form a unique heterocomplex with 3:3 stoichiometry, which was confirmed by enzymatic assays, fluorescent labeling, and small-angle x-ray scattering. The binary structure of NdmA with the ferredoxin domain of NdmD, which is the first structural information for the plant-type ferredoxin domain in a complex state, was also determined to better understand electron transport during N-demethylation. These findings broaden our understanding of the caffeine degradation mechanism by bacterial enzymes and will enable their use for industrial applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N-demethylation and C-8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N-demethylation and C-8 oxidation. All of the genes for the N-demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2-kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C-8 oxidation of caffeine have been located on a 25.2-kb genomic DNA fragment of Pseudomonas sp. CBB1. The C-8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio-decaffeination, remediation of caffeine-contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated., (© 2015 The Author. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Understanding the genes and enzymes involved in caffeine metabolism can lead to applications such as production of methylxanthines and environmental waste remediation. Pseudomonas sp. CES may provide insights into these applications, since this bacterium degrades caffeine and thrives in concentrations of caffeine that are three times higher (9.0 g L(-1)) than the maximum tolerable levels of other reported bacteria. We took a novel approach toward identifying the enzymatic pathways in Pseudomonas sp. CES that metabolize caffeine, which largely circumvented the need for exhaustive isolation of enzymes and the stepwise reconstitution of their activities. Here we describe an optimized, rapid alternative strategy based on multiplexed LC-MS/MS assays and show its application by discovering caffeine-degrading enzymes in the CES strain based on quantitative comparison of proteomes from bacteria grown in the absence and presence of caffeine, the latter condition of which was found to have a highly induced capacity for caffeine degradation. Comparisons were made using stable isotope dimethyl labeling, differences in the abundance of particular proteins were substantiated by reciprocal labeling experiments, and the role of the identified proteins in caffeine degradation was independently verified by genetic sequencing. Overall, multiple new components of a N-demethylase system were identified that resulted in rapid pathway validation and gene isolation using this new approach.

Caffeine and other N-methylated xanthines are natural products found in many foods, beverages, and pharmaceuticals. Therefore, it is not surprising that bacteria have evolved to live on caffeine as a sole carbon and nitrogen source. The caffeine degradation pathway of Pseudomonas putida CBB5 utilizes an unprecedented glutathione-S-transferase-dependent Rieske oxygenase for demethylation of 7-methylxanthine to xanthine, the final step in caffeine N-demethylation. The gene coding this function is unusual, in that the iron-sulfur and non-heme iron domains that compose the normally functional Rieske oxygenase (RO) are encoded by separate proteins. The non-heme iron domain is located in the monooxygenase, ndmC, while the Rieske [2Fe-2S] domain is fused to the RO reductase gene, ndmD. This fusion, however, does not interfere with the interaction of the reductase with N1- and N3-demethylase RO oxygenases, which are involved in the initial reactions of caffeine degradation. We demonstrate that the N7-demethylation reaction absolutely requires a unique, tightly bound protein complex composed of NdmC, NdmD, and NdmE, a novel glutathione-S-transferase (GST). NdmE is proposed to function as a noncatalytic subunit that serves a structural role in the complexation of the oxygenase (NdmC) and Rieske domains (NdmD). Genome analyses found this gene organization of a split RO and GST gene cluster to occur more broadly, implying a larger function for RO-GST protein partners.

The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown. 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 nonheme iron monooxygenases with N(1)- and N(3)-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 flavin mononucleotide (FMN) per enzyme. All ndm genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase. ndmA, ndmB, and ndmD were cloned as His(6) fusion genes, expressed in Escherichia coli, and purified using a Ni-NTA column. NdmA-His(6) plus His(6)-NdmD catalyzed N(1)-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His(6) plus His(6)-NdmD catalyzed N(3)-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an N(7)-specific N-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial N-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.

The coffee berry borer, the most economically important insect pest of coffee worldwide, is the only insect capable of feeding and reproducing solely on the coffee seed, a food source containing the purine alkaloid caffeine. Twenty-one bacterial species associated with coffee berry borers from Hawai'i, Mexico, or a laboratory colony in Maryland (Acinetobacter sp. S40, S54, S55, Bacillus aryabhattai , Delftia lacustris , Erwinia sp. S38, S43, S63, Klebsiella oxytoca , Ochrobactrum sp. S45, S46, Pantoea sp. S61, Pseudomonas aeruginosa , P. parafulva , and Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75) were found to have at least one of five caffeine N-demethylation genes (ndmA , ndmB , ndmC , ndmD , ndmE), with Pseudomonas spp. S31, S32, S37, S60 and P. parafulva having the full complement of these genes. Some of the bacteria carrying the ndm genes were detected in eggs, suggesting possible vertical transmission, while presence of caffeine-degrading bacteria in frass, e.g., P. parafulva (ndmABCDE) and Bacillus aryabhattai (ndmA) could result in horizontal transmission to all insect life stages. Thirty-five bacterial species associated with the insect (Acinetobacter sp. S40, S54, S55, B. aryabhattai , B. cereus group, Bacillus sp. S29, S70, S71, S72, S73, D. lacustris , Erwinia sp. S38, S43, S59, S63, K. oxytoca , Kosakonia cowanii , Ochrobactrum sp. S45, S46, Paenibacillus sp. S28, Pantoea sp. S61, S62, P. aeruginosa , P. parafulva , Pseudomonas sp. S30, S31, S32, S37, S44, S60, S75, Stenotrophomonas sp. S39, S41, S48, S49) might contribute to caffeine breakdown using the C-8 oxidation pathway, based on presence of genes required for this pathway. It is possible that caffeine-degrading bacteria associated with the coffee berry borer originated as epiphytes and endophytes in the coffee plant microbiota. [ABSTRACT FROM AUTHOR]

This study reports bench-scale feasibility study for production and recovery of theobromine from caffeine using metabolically engineered E. coli . The N 1 -demethylase genes used in this study were originally discovered in Pseudomonas putida CBB5. Five strains of E. coli engineered with multiple combinations of the two N -demethylase genes were compared for growth and activity. Strain pAD1dDD containing one copy of ndmA and three copies of ndmD in two compatible plasmids, gave the best results (98.5% molar conversion of caffeine to theobromine). The reaction was scaled up to 2 L and theobromine was separated and recovered in nearly 100% pure form via preparative chromatography and drying. Final yield was 80% relative to caffeine. Purity of the product was identical to an authentic standard as judged by LC-MS and NMR. This is the first report of theobromine production via a metabolically engineered strain, with the highest yield and purity reported of any biological method. This method also eliminates the harsh reaction conditions and environmental issues associated with the chemical process. [ABSTRACT FROM AUTHOR]

1-Methylxanthine is a high-value derivative of caffeine of limited natural availability with many potential pharmaceutical applications. Unfortunately, production of 1-methylxanthine through purely chemical methods of synthesis are unfavorable due to lengthy chemical processes and the requirement of hazardous chemicals, ultimately resulting in low yields. Here, we describe a novel biosynthetic process for the production of 1-methylxanthine from theophylline using engineered Escherichia coli whole-cell biocatalysts and reaction optimization. When scaled-up to 1590 mL, the simple biocatalytic reaction produced approximately 1188 mg 1-methylxanthine from 1444 mg theophylline, constituting gram-scale production of 1-methylxanthine in as little as 3 hours. Following HPLC purification and solvent evaporation, 1163 mg of dried 1-methylxanthine powder was collected, resulting in a 97.9 wt% product recovery at a purity of 97.8%. This is the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 1-methylxanthine from theophylline. This process is also the most robust methylxanthine N -demethylation process featuring engineered E. coli to date, capable of gram-scale production. • Novel, biosynthetic pathway for production of 1-methylxanthine at gram scale. • Engineered E. coli for high-value biochemical production. • High performance liquid chromatography (HPLC) separation for high purity. • Co-factor (NADH) recycle through by-product (formaldehyde) degradation. [ABSTRACT FROM AUTHOR]