Cloning And Functional Analysis On Caffeine-Degradation-Related Genes In Pseudomonas Strains From Tea Garden Soil

Caffeine biodegradation mediated by microbes and exogenous enzyme fermentation is one of the most important research areas of decaffeinated tea products processing. Base on optimizing the parameters for construction of genome DNA library, genomic library with5-8kb inserted sequence from a high caffeine tolelant Pseudomonas sp. CT25was successfully constructed. The caffeine-degradation-related gene clusters were obtained after caffeine tolerant selection of the library and inserted sequence analysis. The effect of caffeine nitrogen source on the gene expression of CT25was also investigated through transcriptome comparison. The main findings can be concluded as following:Optimized procedure for constructing genomic library through partial enzyme digestion could be described as, mixing2ug genomic DNA with0.2-0.4U Sau3A I and incubating at37℃for5min, recovering the DNA fragments with different molecular size from agarose gel after electrophoresis, ligating the revovered DNA fragments into the linearized and dephosphorylated vectors respectively, and then transforming into the competent Escherichia coli through thermal shock method. Efficient construction of genomic library also could be achieved according to the protocol, in which the DNA fragments with prospective sizes were obtained by treatment of genomic DNA with shear machine, and ligated with the vectors and then electro-transformed into competent E. coli.Around1000transformants were randomly chosen from genomic library (15,000transformants with5-8kb insertion) for caffeine degradation tests. Seven transformants with high caffeine degradation capability (CTG-004, CTG-005, Al, A13, F17,03and P16) were achieved. After sequencing and bioinformatic comparison of these transfromants, several genes, including frataxin-like Fe-S cluster protein and Fe-S oxidoreductase on CTG-004, HAMP-domain contained protein and signal-transduction-related protein on CTG-005, nitrogen metabolism transcription and regulation protein on Al, BAND7family proteins on A13, Fic/DOC protein reltated to folate metabolism on F17, radical SAM protein/molybdenum cofactor synthesis protein on03and, bifunnctional selenocysteine lyase/cysteine desulfurase and Fe-S metabolism associated protein on P16, might be responsible for the enhanced caffeine degradation capability. This indicated that higher caffeine tolerance of CT25might be synergistically modulated by many gene loci associated with signal transduction, transcription regulation and oxidative degradation of caffeine.Transcriptome difference was investigated when CT25strain was incubated in the mediums with caffeine or peptone as sole N-and C-sources. Results showed that when CT25strain was incubated in caffeine-contained medium it could sense the N-and C-source variation and down-regulate expression of many transcription factors and their regulators, consequently decrease the transcription activity of many genes related to DNA replication and reparation, matter transportation, amino acids metabolism, protein synthesis, tRNA processing and lipogenesis, meanwhile, it could also recognize the caffeine signal mediated by chemiotaxis factor, and up-regulate expression of HTH-type transcription factor, then enhance the transcription of the genes related to stress response, redox reaction, nitrogen metabolism, fat acids degradation as well as ferric ion translocation. This finding suggested that the CT25can systematically respond to high-caffeine and low-nutrition environment.