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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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