Cloning And Expression Of Key Genes For Caffeine Degradation In Paraburkholderia Caffeinilytica CF1

With the rapid development of tea industry,the processing and reuse of its by-product tea residue has become a hotspot,and the composition of tea residue is caffeine.Traditional physical and chemical methods to remove caffeine have great drawbacks,such as chemical residues,time and effort waste,capital investment,environmental pollution.However,the microbial degradation of caffeine excludes the above disadvantages and increases the protein in the tea residue,with the purpose of improving the greater significance of re-use.The strain Paraburkholderia caffeinilytica CF1(CF1)screened by our laboratory has the ability to degrade high concentrations of caffeine and grow in a medium with caffeine as the sole carbon and nitrogen source.To obtain a functional gene directly related to caffeine degradation in caffeine stress,the strain was sequenced by high throughput sequencing and the functional analysis of these gene expression products was carried out.The aim of this study is to explore the enzyme and functional gene resources of caffeine-degrading bacteria,and to lay the foundation for the study of expression factors and the mechanism of stress proteins involved in cell resistance to degradation of caffeine.It has a practical significance for the development of caffeine degradation engineering bacteria preparation and improving the utilization efficiency of tea residue.The strain CF1 was sequenced by differential transcriptome,including two transcripts(including the experimental and control groups).The significant up-regulation sequence was 123,and significant down-regulation sequence was 2669(FDR≤0.001 AND |log2Ratiol≥1),in which Unigenel4_All(log2 Ratio=8.4)and Unigene 1922_All(log2 Ratio=9.7)were the contents of this study.A significant enrichment analysis of GO and Pathway was performed for these two differential sequences.Moreover,the functional explanations were methyl xanthine N1-demethylase and methyl xanthine N7-demethylase,respectively.The expression levels of cdnA and cdnC in the experimental group and the control group were detected by qRT-PCR and the results were consistent with the results of the differential transcriptome.Using the sequences of Unigene 14_All and Unigene 1922_All sequences as reference sequences,the specific primers were designed with the sequence of similar strains in NCBI database to obtain the full length of cdnA and N7 demethylase cdnC gene of methyl xanthine N1 demethylase.Furthermore,the CdnA and CdnC target proteins were successfully expressed by Rosetta(DE3)-pET32a-cdnA and Rosetta-gami-pLysS-pET32a-cdnC system,respectively.When caffeine,1,3-dimethylxanthine and 1,7-dimethylxanthine and 1-methylxanthine were used as substrates,CdnA was detected with N1-type demethylase active in the presence of CdnD.