Cloning And Functional Verification Of Guanine Deaminase In Caffeine Biosynthesis Salvage Pathway

Caffeine(1,3,7-trimethyl xanthine)which is widely found in natural plants is a kind of xanthine alkaloid,and the most common plants with high content of caffeine include coffee,cocoa and tea.A large number of microbes flourish during the pile-fermenting process of dark tea.Variety of connotations in tea is changed by the extracellular enzymes and respiration heat of microorganisms and metabolic pathways in microbes.This is the cause of the unique flavor of dark tea.Different from the contents of amino acids and tea polyphenols decreased,the content of caffeine presents an upward trend because microbes synthesize caffeine through its own metabolism.There may be a salvage pathway of caffeine synthesis in microorganism witch different from the core pathway of caffeine synthesis in plants.Guanine is a basic metabolite of microorganism which can be recycled and the purine salvage pathway is asymmetric because adenine derivatives can be converted into guanine nucleotides,whereas the reverse is not possible.As a result,guanine deamination is the key step in this compensation path.This objective of the paper was to verify the salvage pathway of caffeine synthesis in microorganism from theoretical and practical aspects.The key enzymes were expressed and the enzymatic activity were analyzed and confirmed.The effects of different microbes,promoters and different expression systems on the activity of guanine deaminase were compared.And the synthetic path of caffeine in actual pile-fermenting process was traced by isotope labeling.The results are as following:(1)In the pile-fermenting process of dark tea,the endogenous enzyme of tea has been inactivated and the exogenous enzyme derived from microorganism becomes the most important biocatalyst in fermentation.Caffeine content has a rising trend and there is a new pathway as guanine → xanthine → caffeine during the fermentation process in the microorganism.(2)The two guanine deaminase genes EGUD and GUD1 were cloned from Escherichia coli and Saccharomyces cerevisiae respectively and the recombinant vector pMAL-GUD1 and pMAL-EGUD were successfully transformed into E.coli to express.Under the condition of adding guanine as substrate,the concentration of xanthine was 312 μg/mL and 267 μg/mL respectively after BL21/pMAL-GUD1 and BL21/pMAL-EGUD were cultured for 120 h,and the concentration of xanthine was 225 μg/mL and 191 μg/mL respectively,without adding substrate.(3)The recombinant vector pRSF-GUD1 was transferred into the Escherichia coli and the strain containing the recombinant vector pRSF-GUD1 has a higher conversion rate of guanine than that of the strain containing pMAL-GUD1.The xanthine synthesis rate of strain containing the recombinant vector pRSF-GUD1 was higher than 37.5% and 23.9% that of the strain containing pMAL-GUD1 respectively in the two cases were fed guanine as substrate and without substrate.It suggests that T7 promoter is more suitable for expression of guanine deaminase gene than Lac promoter.(4)The recombinant vector pZ8-GUD1 was transferred into Corynebacterium glutamatum,and the effects of different expression strains on the activity of GUD1 were compared.The xanthine synthesis rate of strain containing the recombinant vector pRSF-GUD1 was lower 44.8% and 6.7% respectively than BL21/pRSF-GUD1 and BL21/pMAL-GUD1 with guanine as substrate.And it was lower 24.7% and 6.7% without substrate.The results showed that Escherichia coli is more suitable than Corynebacterium glutamiens for GUD1 expression and GUD1 is more efficient in converting guanine into xanthine in E.coli.This paper will provide new ideas for the scientific research and biosynthesis of caffeine.It clarifies the cause of caffeine rise during the pile-fermentation of dark tea and reveals the salvage pathway of caffeine synthesis.