Studies On The Bfrl Mutant Gene (Mutbfr1)Increased Caffeine Tolerance In Engineering Strains

Caffeine(2,6-dioxo 1,3,7-trimethylxanthine),which is an important alkaloid in a variety of higher plants,is the most abundant purine in tea.Caffeine has been widely used in medicines and foods because of its functions of excitement,digestion and anti-tumor.Presently,plant extraction and chemical synthesis are the main sources of commercial caffeine,but these methods have drawbacks.Therefore,microbial fermentation has become a new way to produce natural and pollution-free caffeine.However,in the process of construction of engineering bacteria fermentation,the production of caffeine is limited because of the antibacterial activity of caffeine,which affects the fermentation efficiency of engineering bacteria.In addition,the piling fermentation of pu' erh tea is the key process for the formation of tea quality,in which a large number of microorganisms are involved,and it causes the change of some characteristic components during fermentation process.It has been reported that the caffeine content has a significant growth trend during the process of fermentation in pu'erh tea,so is this increase trend related to microorganisms?The previous studies have shown that there is a caffeine synthesis pathway in microorganisms,and the methylation sequence was N3?N1?N7.Based on the study of caffeine metabolism in microorganisms,this paper explored whether there are biosynthesis pathways of caffeine in microorganisms,which participated in the fermentation process of pu'erh tea,and the strategies to increase the production of caffeine.Through isotope tracer method,stable isotope xanthine was added to tea samples to imitated pile fermentation,which explore the pathway of microbial caffeine metabolism,and further clarify the biological mechanism of the increase of caffeine in the process of piling fermentation;the mutBfr1 was cloned and analyzed by bioinformatics,and transferred into E.coli expression to verify the ability of caffeine resistance;the TCS1 gene and mutBfr1 gene were tandem expressed by genetic engineering to explore the ability to increase the production of caffeine.The main obtain results of this paper are as follows:(1)The results of imitated piling fermentation of pu'erh tea with exogenous purine precursor supplementation showed:the tea samples with xanthine(0.1 mg/g)added,theobromine increased by 49.89%,while theobromine increased by 36.57%;xanthine(1 mg/g)was added to the tea samples,theobromine and caffeine increased by 53.56%and 37.86%,respectively.Meanwhile,theobromine and theobromine increased by 43.56%and 32.56%respectively in the tea samples without exogenous xanthine supplementation.Then,the tea leaves were sterilized,and the content of theobromine and caffeine remained basically constant during the pile fermentation process.The results showed that the microorganisms were the main factor for caffeine increased in pu'erh tea.In the 15N-xanthine tracer experiment,15N-theophylline and 15N-caffeine were detected in the pu'erh tea,while isotopically labeled substances were not detected in tea samples without supplement 15N-xanthine.The results showed that there maight was a microbial catalyzed caffeine metabolic pathway from xanthine to theobromine to caffeine during the fermentation process of pu'erh tea.(2)Bioinformatics analysis of the mutBfr1 gene obtained in the previous study group,the amino acid sequence analysis of the mutBfr1 gene was as follows:mutBfr1 gene has 13 bases mutation compared with the nucleotide sequence of the wild-type bfrl gene.The predicted value of mutBfr1 protein GRAVY was-0.161,indicating that the protein is a hydrophilic protein;there are 12 transmembrane features with higher predicted transmembrane helix region;the mutBfr1 gene was expressed in E.coli,based on plate spotting and the growth curve of the strain,mutBfr1 was capable of conferring 8 mg/mL caffeine tolerance in E.coli,which reduced the caffeine sensitivity of the strain by caffeine efflux.(3)Using the homologous recombination technique,the mutBfr1 and tea caffeine synthase genes(TCS1)were tandem expressed in E.coli.The results showed that the target strains BL21/pMAL-eCSl and BL21/pMAL-eCS1-mutBfr1 were fermented without supplement substrate,and caffeine was detected extracellularly,but no caffeine was detected in the cells.The addition of theophylline to the medium was detected by UPLC,and the caffeine was detected in the recombinant strains compared to BL21/pMAL-c5X.According to the standard curve of caffeine,the caffeine accumulated in recombinant strains BL21/pMAL-TCS1 BL21/pMAL-TCS1-mutBfr1,BL21/pMAL-eCS1 and BL21/pMAL-eCSl-mutBfr1 was(6.28±1.21)?g/mL,(6.15±1.15)?g/mL,(22.28±1.21)?g/mL and(20.84±1.74)?g/mL,respectively.The results showed the strains which were expressed in tandem with mutBfr1 and the caffeine synthase gene produced no higher caffeine content than the strains expressing the single caffeine synthase gene.Meanwhile,the caffeine was not detected in intracellular by UPLC analysis.