Catabolic pathways and biotechnological applications of microbial caffeine degradation

Catabolism of caffeine (1,3,7-trimethylxanthine) in microorganisms commences via two possible mechanisms: demethylation and oxidation. Through the demethylation route, the major metabolite formed in fungi is theophylline (1,3-dimethylxanthine), whereas theobromine (3,7-dimethylxanthine) is the major metabolite in bacteria. In certain bacterial species, caffeine has also been oxidized directly to trimethyl uric acid in a single step. The conversion of caffeine to its metabolites is primarily brought about by N-demethylases (such as caffeine demethylase, theobromine demethylase and heteroxanthinedemethylase), caffeine oxidase and xanthine oxidase that are produced by several caffeine-degrading bacterial species such as Pseudomonas putida and species within the genera Alcaligenes, Rhodococcus and Klebsiella. Development of biodecaffeination techniques using these enzymes or using whole cells offers an attractive alternative to the present existing chemical and physical methods removal of caffeine, which are costly, toxic and non-specific to caffeine. This review mainly focuses on the biochemistry of microbial caffeine degradation, presenting recent advances and the potential biotechnological application of caffeine-degrading enzymes.     

Studies on the microbial production of theobromine and heteroxanthine from caffeine

Summary From soil a caffeine degrading bacterium was isolated which is able to grow on media containing up to 2% caffeine as the sole source of carbon and nitrogen. The organism was identified as Pseudomonas putida and referred to as Pseudomonas putida WS. Mutants of this strain converted caffeine and were shown to accumulate a mixture of theobromine and heteroxanthine during resting cells experiments.The highest yield in accumulation products was obtained with the mutant strain H8, however the production rate with resting cells was too small for commercial purposes. The yield was significantly increased by growth of the mutant on diluted complex media. With this technique a yield of 50% based on the amount of caffeine could be obtained for heteroxanthine. The concentration maximum is reached when caffeine is completely converted and only traces of theobromine are present.Dedicated to Professor G. Braunitzer on the occasion of his 65th birthday     

Genetic characterization of caffeine degradation by bacteria and its potential applications

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.     

Microbial Production of Theobromine from Caffeine

Production of theobromine from caffeine by caffeine-degrading bacteria was studied. We found that addition of metal ions such as Zn²⁺ to intact cells of a caffeine-degrading isolate from soil, Pseudomonas sp. No.6, resulted in a high theobromine accumulation from caffeine. We hypothesized that Zn²⁺ acts as a selective inhibitor of one of the theobromine-demethylating enzymes and further screened for theobromine-producing activities in the presence of Zn²⁺ among a number of caffeine-using microorganisms. A strain identified taxonomically as Pseudomonas putida No. 352 showed the best productivity among 973 microorganisms of stock cultures and soil isolates. Culture conditions for the production of theobromine from caffeine by P. putida No. 352 were studied. Under optimal conditions, nearly 20 g/liter of theobromine was produced from caffeine in a yield of 92%.     

Biodegradation of crystal violet by Pseudomonas putida

Crystal violet (CV), which has been extensively used as a biological stain and a commercial textile dye, is a recalcitrant molecule. A strain of Pseudomonas putida was isolated that effectively degraded CV: up to 80% of 60 μM CV as the sole carbon source, was degraded in liquid media within 1 week. Nine degradation products were isolated and identified. We propose that CV degradation occurs via a stepwise demethylation process to yield mono-, di-, tri-, tetra-, penta- and hexa-demethylated CV species.     

Removal of caffeine in sewage by Pseudomonas putida: Implications for water pollution index

A strain of Pseudomonas putida (biotype A) capable of growing on caffeine (1,3,7-trimethylxanthine) was isolated from a domestic wastewater processing operation. It used caffeine as the sole carbon source with a mean growth rate constant (k) of 0.049 h^-1 (approximately 20 h per generation), whereas k for glucose utilization under similar incubation conditions was 0.31 (3.3 h per generation). The isolate contained at least two plasmids, and the increased expression of a 40 kDa protein was attributable to growth on caffeine. Degradation byproducts of caffeine metabolism by the bacterial isolate included other xanthine derivatives. The slow bacterial catabolism of caffeine in sewage has implications for the effectiveness of wastewater purification, re-use and disposal.The author is with the Laboratory for Molecular Ecology, Department of Environmental Analysis and Design, University of California at Irvine, Irvine, CA 92717-5150 U.S.A.     

Caffeine-inducible enzyme activity in Pseudomonas putida ATCC 700097

Caffeine (1,3,7-trimethylxanthine), a ubiquitous component of human diet has been suggested as a chemical indicator of ecosystem impacts of sewage spills and treated effluent discharges because it is not sufficiently metabolized by wastewater microorganisms. This study identified enzymes responsible for caffeine metabolism in sewage bacteria. Pseudomonas putida biotype A (ATCC 700097) originally isolated as a rare caffeine-degrading organism in domestic wastewater exhibited diauxic growth on caffeine, concomitant with the expression of a P450-type cytochrome and peroxidase enzyme activities. Initial growth phase lasted 13.8 ± 1.4 h with a growth rate that was five times slower than the secondary growth phase that lasted 5.5 ± 1.2 h. Molecular and enzymatic characteristics of the cytochrome P450-type enzyme differ from the previously described cytochrome P450 (P450_cam) of P. putida (ATCC 17453) involved in camphor metabolism. The caffeine-inducible cytochrome P450-type enzyme exhibited a carbon monoxide difference spectrum peak at 450 nm, but does not allow growth on camphor. Caffeine induced production of haem-associated peroxidase activity was confirmed with 3,3, 5,5-tetramethylbenzidine–H₂O₂ reaction in polyacrylamide gels. Polymerase chain reaction (PCR) primers derived from the gene for cytochrome P450_cam (camC) of P. putida (ATCC 17453) did not yield an amplification product when DNA extracted from P. putida strain ATCC 700097 was used as template. The data demonstrate that caffeine is metabolized through a specific biphasic pathway driven by oxygen-demanding enzymes.     

Caffeine Junkie: an Unprecedented Glutathione S-Transferase-Dependent Oxygenase Required for Caffeine Degradation by Pseudomonas putida CBB5

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.     

Biosynthesis of zeaxanthin in recombinant Pseudomonas putida

Pseudomonas putida KT2440 strain was investigated for biosynthesis of the valuable xanthophyll zeaxanthin. A new plasmid was constructed harboring five carotenogenic genes from Pantoea ananatis and three genes from Escherichia coli under control of an l-rhamnose-inducible promoter. Pseudomonas putida KT2440 wild type hardly tolerated the plasmids for carotenoid production. Mating experiments with E. coli S17-1 strains revealed that the carotenoid products are toxic to the Pseudomonas putida cells. Several carotenoid-tolerant transposon mutants could be isolated, and different gene targets for relief of carotenoid toxicity were identified. After optimization of cultivation conditions and product processing, 51 mg/l zeaxanthin could be produced, corresponding to a product yield of 7 mg zeaxanthin per gram cell dry weight. The effect of various additives on production of hydrophobic zeaxanthin was investigated as well. Particularly, the addition of lecithin during cell cultivation increased volumetric productivity of Pseudomonas putida by a factor of 4.7 (51 mg/l vs. 239 mg/l).     

Kinetics of hydrogen sulfide oxidation by immobilized autotrophic and heterotrophic bacteria in bioreactors

Summary The kinetics of H₂S oxidation in bioreactors with separately packed autotrophic Thiobacillus thioparus CH11 and heterotrophic Pseudomonas putida CH11 were evaluated. The reaction rates were determined to be first-order below 20 ppm, zero-order above 60 ppm, and fractional-order in the intermediate concentration ranges for the Thiobacillus thioparus CH11 bioreactor, and first-order below 35 ppm, zero-order above 80 ppm, and fractional-order in the intermediate concentration ranges for the Pseudomonas putida CH11 bioreactor. The saturation constants for H₂S by Thiobacillus thioparus CH11 and Pseudomonas putida CH11 were calculated to be 30.3 ppm and 44.2 ppm, respectively.     

Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72?h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48?h. Even when the inoculation by P. putida was delayed for 24?h, P. fluorescens was eliminated from the capillary within 48?h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.     

Degradation of caffeine by immobilized cells of Pseudomonas putida strain C 3024

Summary A caffeine-resistant strain of Pseudomonas putida was isolated from soil and was grown with caffeine as the sole source of carbon, energy and nitrogen. Cells were immobilized in agar gel particles which were continuously supplied with a caffeine solution (0.52 g · l^–1, D=1.0 h^–1) in a homogeneously mixed aerated reaction vessel. In the presence of the ATPase inhibitor arsenate the caffeine was removed by the immobilized cells at an average rate of 0.25 mg caffeine · h^–1 · (mg cell carbon)^–1 during 6 days. Thereafter a rapid decline of activity was observed. From a similar system without arsenate supplied with a growth medium containing a limiting amount of caffeine (0.13 g · l^–1) the caffeine was almost completely oxidized by the immobilized cells. The concentration of the remaining caffeine was 1.4 mg · l^–1, which is much lower than the substrate constant for caffeine (9.7 mg · l^–1) observed with freshly harvested suspended resting cells.     

Electroporation of pKK1 silver-resistance plasmid fromPseudomonas stutzeri AG259 intoPseudomonas putida CYM318

Plasmid pKK1 (49.4 Mda), which encodes for Ag⁺ resistance, was isolated fromPseudomonas stutzeri AG259 (pKK1) and introduced intoPseudomonas putida CYM318 by high-voltage electroporation. Upon acquiring pKK1,P. putida CYM318 became resistant to AgNO₃. This demonstrated that electroporation is a useful method to introduce a nonconjugative metal-resistance plasmid into a bacterial strain and to conduct further comparative research on Ag accumulation.     

The Wsp intermembrane complex mediates metabolic control of the swim-attach decision of Pseudomonas putida

Pseudomonas putida is a microorganism of biotechnological interest that—similar to many other environmental bacteria—adheres to surfaces and forms biofilms. Although various mechanisms contributing to the swim-attach decision have been studied in this species, the role of a 7-gene operon homologous to the wsp cluster of Pseudomonas aeruginosa—which regulates cyclic di-GMP (cdGMP) levels upon surface contact—remained to be investigated. In this work, the function of the wsp operon of P. putida KT2440 has been characterized through inspection of single and multiple wsp deletion variants, complementation with Pseudomonas aeruginosa's homologues, combined with mutations of regulatory genes fleQ and fleN and removal of the flagellar regulator fglZ. The ability of the resulting strains to form biofilms at 6 and 24 h under three different carbon regimes (citrate, glucose and fructose) revealed that the Wsp complex delivers a similar function to both Pseudomonas species. In P. putida, the key components include WspR, a protein that harbours the domain for producing cdGMP, and WspF, which controls its activity. These results not only contribute to a deeper understanding of the network that regulates the sessile-planktonic decision of P. putida but also suggest strategies to exogenously control such a lifestyle switch.     

Tolerance of plastic-encapsulated Pseudomonas putida KT2440 to chemical stress

Pseudomonas putida dried in the presence of hydroxyectoine or trehalose can withstand exposure to organic solvents and therefore can be encapsulated inside plastics such as polystyrene. Here we show that P. putida in a plastic-encapsulated dried tablet exhibits remarkable tolerance to chemical stress, comparable to that of spores of Bacillus subtilis.     

Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5

Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria.Caffeine (1,3,7-trimethylxanthine) and related methylxanthines are widely distributed in many plant species. Caffeine is also a major human dietary ingredient that can be found in common beverages and food products, such as coffee, tea, and chocolates. In pharmaceuticals, caffeine is used generally as a cardiac, neurological, and respiratory stimulant, as well as a diuretic (3). Hence, caffeine and related methylxanthines enter soil and water easily through decomposed plant materials and other means, such as effluents from coffee- and tea-processing facilities. Therefore, it is not surprising that microorganisms capable of degrading caffeine have been isolated from various natural environments, with or without enrichment procedures (3, 10). Bacteria use oxidative and N-demethylating pathways for catabolism of caffeine. Oxidation of caffeine by a Rhodococcus sp.-Klebsiella sp. mixed-culture consortium at the C-8 position to form 1,3,7-trimethyluric acid (TMU) has been reported (8). An 85-kDa, flavin-containing caffeine oxidase was purified from this consortium (9). Also, Mohapatra et al. (12) purified a 65-kDa caffeine oxidase from Alcaligenes sp. strain CF8. Cells of a caffeine-degrading Pseudomonas putida strain (ATCC 700097) isolated from domestic wastewater (13) showed a fourfold increase in a cytochrome P450 absorption spectrum signal compared to cells grown on glucose. Recently, we reported a novel non-NAD(P)⁺-dependent heterotrimeric caffeine dehydrogenase from Pseudomonas sp. strain CBB1 (20). This enzyme oxidized caffeine to TMU stoichiometrically and hydrolytically, without producing hydrogen peroxide. Further metabolism of TMU has not been elucidated.Several caffeine-degrading bacteria metabolize caffeine via the N-demethylating pathway and produce theobromine (3,7-dimethylxanthine) or paraxanthine (1,7-dimethylxanthine) as the initial product. Theophylline (1,3-dimethylxanthine) has not been reported to be a metabolite in bacterial degradation of caffeine. Subsequent N demethylation of theobromine or paraxanthine to xanthine is via 7-methyxanthine. Xanthine is further oxidized to uric acid by xanthine dehydrogenase/oxidase (3, 10). Although the identities of metabolites and the sequence of metabolite formation for caffeine N demethylation are well established, there is very little information on the number and nature of N-demethylases involved in this pathway.The lack of adequate information on the metabolism and enzymology of theophylline, caffeine, and related methylxanthines prompted us to investigate the degradation of these compounds in detail. We isolated a unique caffeine-degrading bacterium, P. putida CBB5, from soil via enrichment with caffeine as the sole source of carbon and nitrogen. Here we describe a detailed study of the metabolism of theophylline, caffeine, and related di- and monomethylxanthines by CBB5. Our results indicate that CBB5 initially N demethylated caffeine to produce theobromine (major product) and paraxanthine (minor product) before the pathways converged to 7-methylxanthine and xanthine. Surprisingly, CBB5 was also capable of utilizing theophylline as a sole carbon and nitrogen source. CBB5 N demethylated theophylline to 1-methylxanthine and 3-methylxanthine, which were further N demethylated to xanthine. Theophylline N-demethylase activity was detected in cell extracts prepared from theophylline-grown CBB5 cells. 1-Methylxanthine and 3-methylxanthine were detected as products of this NAD(P)H-dependent reaction. To our knowledge, this is the first report of a theophylline degradation pathway in bacteria and coexpression of distinct caffeine and theophylline degradation pathways.     

The mechanism of action of hexahydro-1,3,5-triethyl-s-triazine

Summary The mechanism of antimicrobial action of hexahydro-1,3,5-triethyl-s-triazine (HHTT) was studied using the HHTT-resistant isolate,Pseudomonas putida 3-T-15², its HHTT-sensitive, novobiocin-cured derivative,P. putida 3-T-15² 11:21,P. putida ATCC 12633,Pseudomonas aeruginosa PA01 andEscherichia coli J53 (RP4). HHTT was oxidized byP. putida 3-T-15², while respiration ofP. putida 3-T-15² 11:21 was inhibited by HHTT. Chemical assays showed that HHTT released formaldehyde.P. putida 3-T-15² was highly resistant to formaldehyde, whileP. putida 3-T-15² 11:21 was highly sensitive to formaldehyde. Both HHTT and formaldehyde acted similarly to inhibit proline uptake in bacterial cells and to inhibit the synthesis of the inducible enzymes, -galactosidase and glucose-6-phosphate dehydrogenase. HHTT did not have uncoupler-like activity.P. putida 3-T-15² used either HHTT or ethylamine, a component of HHTT, as a nitrogen source for growth, but neither HHTT, ethylamine or formaldehyde served as a carbon and energy source for growth. We concluded that a major mechanism of antimicrobial action of HHTT was through its degradation product, formaldehyde.     

Characteristics of a newly created bioluminescent Pseudomonas putida harboring TOL plasmid for use in analysis of a bioaugmentation system

Insertion of a bacterial lux operon into the chromosome of Pseudomonas putida mt-2 holding TOL plasmid, yielded a new bioluminescent strain of P. putida BLU. Both in the cultures containing toluene and m-toluic acid as the sole carbon sources, P. putida BLU showed the same specific growth rate and cell yield as those of the wild strain. The bioluminescence output in the cell growth phases correlated with the cell concentration, indicating that the bioluminescent P. putida BLU can be monitored and quantified in a mixed culture in real time by the luminescence detection.     

In silico genome-scale metabolic analysis of Pseudomonas putida KT2440 for polyhydroxyalkanoate synthesis,degradation of aromatics and anaerobic survival

Genome-scale metabolic models have been appearing with increasing frequency and have been employed in a wide range of biotechnological applications as well as in biological studies. With the metabolic model as a platform, engineering strategies have become more systematic and focused, unlike the random shotgun approach used in the past. Here we present the genome-scale metabolic model of the versatile Gram-negative bacterium Pseudomonas putida, which has gained widespread interest for various biotechnological applications. With the construction of the genome-scale metabolic model of P. putida KT2440, PpuMBEL1071, we investigated various characteristics of P. putida, such as its capacity for synthesizing polyhydroxyalkanoates (PHA) and degrading aromatics. Although P. putida has been characterized as a strict aerobic bacterium, the physiological characteristics required to achieve anaerobic survival were investigated. Through analysis of PpuMBEL1071, extended survival of P. putida under anaerobic stress was achieved by introducing the ackA gene from Pseudomonas aeruginosa and Escherichia coli.