Abstract
The widespread use of caffeine in food and drug industries has caused great environmental pollution. Herein, an efficient caffeine-degrading strain Paraburkholderia caffeinilytica CF1 isolated from a tea garden in China can utilize caffeine as its sole carbon and nitrogen source. Combination of chromatographic and spectrophotometric techniques confirmed that strain CF1 adopts N-demethylation pathway for caffeine degradation. Whole genome sequencing of strain CF1 reveals that it has two chromosomes with sizes 3.62 Mb and 4.53 Mb, and a 174-kb mega-plasmid. The plasmid P1 specifically harbors the genes essential for caffeine metabolism. By analyzing the sequence alignment and quantitative real-time PCR data, the redundant gene cluster of caffeine degradation was elucidated. Genes related to catalyzing the N1-demethylation of caffeine to theobromine, the first step of caffeine degradation were heterologously expressed, and methylxanthine N1-demethylase was purified and characterized. Above all, this study systematically unravels the molecular mechanism of caffeine degradation by Paraburkholderia.
Key Points
• Caffeine degradation cluster in Paraburkholderia caffeinilytica CF1 was located in mega-plasmid P1.
• The whole genome and the caffeine degrading pathway of P. caffeinilytica CF1 were sequenced and elucidated, respectively.
• This study succeeded in heterologous expression of methylxanthine N1-demethylase (CdnA) and Rieske oxygenase reductase (CdnD) and illuminated the roles of CdnA and CdnD in caffeine degradation of P. caffeinilytica CF1.
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References
Ashihara H, Crozier A (1999) Biosynthesis and metabolism of caffeine and related purine alkaloids. Adv Bot Res 30:117–205. https://doi.org/10.1016/S0065-2296(08)60228-1
Burnet M, Lafontaine PJ, Hanson AD (1995) Assay, purification, and partial characterization of choline monooxygenase from Spinach. Plant Physiol 108:581–588. https://doi.org/10.1104/pp.108.2.581
Burnet MW, Goldmann A, Message B, Drong R, El Amrani A, Loreau O, Slightom J, Tepfer D (2000) The stachydrine catabolism region in Sinorhizobium meliloti encodes a multi-enzyme complex similar to the xenobiotic degrading systems in other bacteria. Gene 244:151–161. https://doi.org/10.1016/S0378-1119(99)00554-5
Cafaro V, Scognamiglio R, Viggiani A, Izzo V, Passaro I, Notomista E, Piaz FD, Amoresano A, Casbarra A, Pucci P, Di-Donato A (2002) Expression and purification of the recombinant subunits of toluene o-xylene monooxygenase and reconstitution of the active complex. Eur J Biochem 269:5689–5699. https://doi.org/10.1046/j.1432-1033.2002.03281.x
Cai S, Chen L, Ai Y, Qiu J, Wang C, Shi C, He J, Cai T (2017) Degradation of diphenyl ether in Sphingobium phenoxybenzoativorans SC_3 is initiated by a novel ring cleavage dioxygenase. Appl Environ Microbiol 83:e00104–e00117. https://doi.org/10.1128/AEM.00104-17
Chao H, Chen Y, Fang T, Xu Y, Huang W, Zhou N (2015) HipH catalyzes the hydroxylation of 4-hydroxyisophthalate to protocatechuate in 2,4-xylenol catabolism by Pseudomonas putida NCIMB 9866. Appl Microbiol Biotechnol 82:724–731. https://doi.org/10.1128/AEM.03105-15
Chen Y, Chao H, Zhou N (2014) The catabolism of 2,4-xylenol and p-cresol share the enzymes for the oxidation of para-methyl group in Pseudomonas putida NCIMB 9866. Appl Microbiol Biotechnol 98:1349–1356. https://doi.org/10.1007/s00253-13-5001-z
Chin CS, Alexander DH, Marks P, Klammer AA, Drake J, Heiner C, Clum A, Copeland A, Huddleston J, Eichler EE, Turner SW, Korlach J (2013) Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat Methods 10:563–569. https://doi.org/10.1038/nmeth.2474
Dash SS, Gummadi SN (2006) Catabolic pathways and biotechnological applications of microbial caffeine degradation. Biotechnol Lett 28:1993–2002. https://doi.org/10.1007/s10529-006-9196-2
Dehio C, Meyer M (1997) Maintenance of broad-host-range incompatibility group P and group Q plasmids and transposition of Tn5 in Bartonella henselae following conjugal plasmid transfer from Escherichia coli. J Bacteriol 179:538–540
Delcher AL, Bratke KA, Powers EC, Salzberg SL (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23:673–679. https://doi.org/10.1093/bioinformatics/btm009
Fan H, Chen L, Sun H, Wang H, Liu Q, Ren Y, Wei D (2017) Characterization of a novel nitrilase, BGC4, from Paraburkholderia graminis showing wide-spectrum substrate specificity, a potential versatile biocatalyst for the degradation of nitriles. Biotechnol Lett 39:1725–1731. https://doi.org/10.1007/s10529-017-2410-6
Gao Z, Yuan Y, Xu L, Liu R, Chen M, Zhang C (2016) Paraburkholderia caffeinilytica sp. nov., isolated from the soil of a tea plantation. Int J Syst Evol Microbiol 66:4185–4190. https://doi.org/10.1099/ijsem.0.001333
Gu T, Zhou C, Sørensen SR, Zhang J, He J, Yu P, Yan X, Lia S (2013) The novel bacterial N-demethylase PdmAB is responsible for the initial step of N, N-dimethyl-substituted phenylurea herbicide degradation. Appl Environ Microbiol 79:7846–7856. https://doi.org/10.1128/AEM.02478-13
Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) Abroad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86. https://doi.org/10.1016/S0378-1119(98)00130-9
Lee Y, Jeon CO (2018) Paraburkholderia aromaticivorans sp. nov., an aromatic hydrocarbon-degrading bacterium, isolated from gasoline-contaminated soil. Int J Syst Evol Microbiol 68:1251–1257. https://doi.org/10.1099/ijsem.0.002661
Li J, Zhang D, Song M, Jiang L, Wang Y, Luo C, Zhang G (2017) Novel bacteria capable of degrading phenanthrene in activated sludge revealed by stable-isotope probing coupled with high-throughput sequencing. Biodegradation 8:423–436. https://doi.org/10.1007/s10532-017-9806-9
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2△△CT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Ma YX, Wu XH, Wu HS, Dong ZB, Ye JH, Zheng XQ, Liang YR, Lu J (2018) Different catabolism pathways triggered by various methylxanthines in caffeine-tolerant bacterium Pseudomonas putida CT25 isolated from tea garden soil. J Microbiol Biotechnol 28:1147–1155. https://doi.org/10.4014/jmb.1801.01043
Madyastha KM, Sridhar GR (1998) A novel pathway for the metabolism of caffeine by a mixed culture consortium. Biochem Biophys Res Commun 249:178–181. https://doi.org/10.1006/bbrc.1998.9102
Madyastha KM, Sridhar GR, Vadiraja BB, Madhavi YS (1999) Purification and partial characterization of caffeine oxidase—a novel enzyme from a mixed culture consortium. Biochem Biophys Res Commun 263:460–464. https://doi.org/10.1006/bbrc.1999.1401
Mazzafera P (2004) Catabolism of caffeine in plants and microorganisms. Front Biosci 9:1348–1359. https://doi.org/10.2741/1339
Mazzafera P, Olsson O, Sandberg G (1996) Degradation of caffeine and related methylxanthines by Serratia marcescens isolated from soil under coffee cultivation. Microb Ecol 31:199–207. https://doi.org/10.1007/BF00167865
Min J, Zhang J, Zhou N (2014) The gene cluster for para-nitrophenol catabolism is responsible for 2-chloro-4-nitrophenol degradation in Burkholderia sp. strain SJ98. Appl Environ Microbiol 80:6212–6222. https://doi.org/10.1128/AEM.02093-14
Mohanty SK, Yu CL, Das S, Louie TM, Gakhar L, Subramanian M (2012) Delineation of the caffeine C-8 oxidation pathway in Pseudomonas sp. strain CBB1 via characterization of a new trimethyluric acid monooxygenase and genes involved in trimethyluric acid metabolism. J Bacteriol 194:3872–3882. https://doi.org/10.1128/JB.00597-12
Mohapatra BR, Harris N, Nordin R, Mazumder A (2006) Purification and characterization of a novel caffeine oxidase from Alcaligenes species. J Biotechnol 125:319–327. https://doi.org/10.1016/j.jbiotec.2006.03.018
Nathanson JA (1984) Caffeine and related methylxanthines: possible naturally occurring pesticides. Science 226:184–187. https://doi.org/10.1126/science.6207592
Obranić S, Babić F, Maravić-Vlahoviček G (2013) Improvement of pBBR1MCS plasmids, a very useful series of broad-host-range cloning vectors. Plasmid 70:263–267. https://doi.org/10.1016/j.plasmid.2013.04.001
Retnadhas S, Gummadi SN (2018) Identification and characterization of oxidoreductase component (NdmD) of methylxanthine oxygenase system in Pseudomonas sp. NCIM5235. Appl Microbiol Biotechnol 102:7913–7926. https://doi.org/10.1007/s00253-018-9224-x
Shao YW, Guo LZ, Zhang YQ, Yu H, Zhao BS, Pang HQ, Lu WD (2018) Glycine betaine monooxygenase, an unusual Rieske-type oxygenase system, catalyzes the oxidative N-demethylation of glycine betaine in Chromohalobacter salexigens DSM 3043. Appl Environ Microbiol 18:84. https://doi.org/10.1128/AEM.00377-18
Sideso OFP, Marvier AC, Katerelos NA, Goodenough PW (2001) The characteristics and stabilization of a caffeine demethylase enzyme complex. Int J Food Sci Technol 36:693–698. https://doi.org/10.1046/j.1365-2621.2001.00496.x
Summers RM, Louie TM, Yu CL, Gakhar L, Louie KC, Subramanian MV (2012) Novel, highly specific N-demethylases enable bacteria to live on caffeine and related purine alkaloids. J Bacteriol 194:2041–2049. https://doi.org/10.1128/JB.06637-11
Summers RM, Seffernick JL, Quandt EM, Yu CL, Barrick JE, Subramaniana MV (2013) Caffeine junkie: an unprecedented glutathione s-transferase-dependent oxygenase required for caffeine degradation by Pseudomonas putida CBB5. J Bacteriol 195:39331–33939. https://doi.org/10.1128/JB.00585-13
Summers RM, Mohanty SK, Gopishetty S, Subramanian MV (2015) Genetic characterization of caffeine degradation by bacteria and its potential applications. Microb Biotechnol 8:369–378. https://doi.org/10.1111/1751-7915.12262
Waller GR (1989) Biochemical frontiers of allelopathy. Biol Plant 31:418–447. https://doi.org/10.1007/BF02876217
Wang C, Chen Q, Wang R, Shi C, Yan X, He J, Hong Q, Li S (2014) A novel angular dioxygenase gene cluster encoding 3-phenoxybenzoate 1′, 2′-dioxygenase in Sphingobium wenxiniae JZ-1. Appl Environ Microbiol 80:3811–3818. https://doi.org/10.1128/AEM.00208-14
Yu CL, Kale Y, Gopishetty S, Louie TM, Subramanian M (2008) A novel caffeine dehydrogenasein Pseudomonas sp. strain CBB1 oxidizes caffeine to trimethyluric acid. J Bacteriol 190:772–776. https://doi.org/10.1128/JB.01390-07
Yu CL, Louie TM, Summers R, Kale Y, Gopishettey S, Subramanian M (2009) Two distinct pathways for metabolism of theophylline and caffeine are coexpressed in Pseudomonas putida CBB5. J Bacteriol 191:4624–4632. https://doi.org/10.1128/JB.00409-09
Funding
This study was supported by the ‘Thirteenth Five-Year Plan’ for the National Key Research and Development Program (Grant No. 2016YFD0400903) and State Ethnic Affairs Commission & Ministry of Education, China and the Science and technology research project of Liaoning Provincial Department of Education (2017 J005).
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LX and GZ conceived and designed research. SD, YX, and LB conducted experiments. ZC contributed new reagents. ZC and HW analyzed data. GZ wrote the manuscript. All authors read and approved the manuscript.
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Figure S1 The two caffeine catabolic pathways of N-demethylation and C8-oxidation in caffeine-degrading bacterial isolates. Figure S2 UPLC analysis of metabolites generated during caffeine degradation by strain CF1. Figure S3 SDS-PAGE (a) and MALDI-TOF/TOF (b) analysis of CdnA-His6. Figure S4 Neighbour-joining tree showing the phylogenetic relationship of CdnA and α-subunits of 38 characterized ROs. Table S1 List of primers. Table S2 Strain CF1 and its mutants grew with methylxanthines as the sole carbon and nitrogen source. Table S3 Putative genes related to caffeine metabolism in P. caffeinilytica CF1. Table S4 Effects of metal ions on methylxanthine N1-demethylase activity of CdnA-His6. (PDF 600 kb).
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Sun, D., Yang, X., Zeng, C. et al. Novel caffeine degradation gene cluster is mega-plasmid encoded in Paraburkholderia caffeinilytica CF1. Appl Microbiol Biotechnol 104, 3025–3036 (2020). https://doi.org/10.1007/s00253-020-10384-7
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DOI: https://doi.org/10.1007/s00253-020-10384-7