Transcriptome Sequencing And Functional Analysis Of Differently Expressed Genes For Caffeine Stress Tolerance In Paraburkholderia Caffeinilytica CF1

The recycling of the tea residue that produced during the tea processing was hindered due to the caffeine existing.Microbial degradation of caffeine is considered to be the most effective way for future tea residue utilization.Caffeine is toxic to most microorganisms.However,some bacteria such as Paraburkholderia caffeinilytica CF1 are able to metabolize this alkaloid.Understanding the genes and enzymes involved in caffeine metabolism can lead to applications such as production of methylxanthines and environmental waste remediation.In order to identify genes involved in caffeine degradation,a global transcription analysis of strain CF1 growing under caffeine free and caffeine addition condition was carried out.The next generation sequencing technology,RNA-seq,was used to obtain the transcriptome profiles.To our knowledge,this is the first report about transcriptome analysis using RNA-seq to screen functional gene of caffeine degradation.The research presented here may help us understand caffeine stress tolerance mechanisms and formulate new strategies to enhance industrial application of microbial caffeine degradation.Two transcripts(including the experimental group and the control group)by differential transcriptome sequencing on the CF1 strain.123 Unigenes were significantly up-regulated by screening,and 2669 Unigenes were significantly down-regulated.Among them,Unigene 1974_All(log2 Ratio = 7.5)and Unigene2111_All(log2 Ratio = 9.0)were analyzed by GO and Pathway enrichment analysis.Functional explanations are methyl xanthine N3-demethylase and methyl xanthine N-demethylase electron transfer protein.A similar strain sequence was found in the NCBI database based on the Unigene2111_All and Unigene 1974_All sequences obtained by sequencing of the transcriptome,and the specific primers were designed.The gene amplification products were ligated to the cloning vector,and then was sequenced to obtain the exact sequences of the genes cdnB(methyl xanthine N3-demethylase)and cdnD(methyl xanthine N-demethylase electron transfer protein).The expression levels of cdnB and cdnD in experimental group and control group were detected by qRT-PCR.The results were consistent with the results of differential transcription group sequencing.The expression vectors Rosetta(DE3)-pET32a-cdnB and Rostta-gami-pLysS-pET3 2a-cdnD were obtained by transposing pET32a-cdnB and pET32a-cdnD expression vectors into Rosetta(DE3)and Rostta-gami-pLysS,respectively.The induction conditions and extraction conditions of CdnB and CdnD were determined by optimization.The activity of CdnB was detected by HPLC.When 3-methylxanthine and 3,7-dimethylxanthine were used as substrates,CdnB was detected to have N3-demethylation activity in the presence of CdnD.