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Poster 制作说明: 1、所有提交的poster,请按照此模版制作; 2、科研成果最好为2015年新成果; 3、如果提交的内容为2015年9月份参加“农业部 重点实验室和观察实验站”展出并已获奖的poster 内容,在该次展示不参与评奖。 请同学们踊跃制作,展示自己的成果! 茶树生物学与资源利用国家重点实验室2015年学术年会 Combinatorial Biosynthesis of Caffeine and the Cloning and Prokaryotic Expression of Xanthosine Nucleosidase Gene in Camellia sinensis Meng-Meng Li , Xin Wu , Cheng Deng, Wei-Wei Deng, Zheng-Zhu Zhang * National Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, P.R.China Abstract Among many metabolites in tea plant, caffeine is used widely in medical, food, chemical and other fields due to its physiological functions. Despite its importance, the low yield of caffeine limits large-scale development of the plant natural product industry. In caffeine biosynthetic pathway, xanthosine is considered as the most effective precursor substance, whose and the well-known metabolic pathway is as follows: xanthosine (XR) → 7-methylxanthosine (7mXR) → 7-methylxanthine (7-MX) → theobromine (Tb) → Caffeine (Cf). In this research, we provide a new approach of the combinatorial biosynthesis of caffeine. We demonstrated the biosynthetic pathway from xanthosine to caffeine by the co-expression of Coffea Arabica xanthosine methyltransferase gene (CaXMT) and Camellia sinensis caffeine synthase gene (TCS1) in the prokaryotic cell system. The recombinant strain of E.coli that simultaneously overexpressed the key genes produced 0.85 mg caffeine/L. Furthermore, we selected adenosyl-nucleosidase, inosine-/guanosinenucleosidase and inosine-/guanosine-phospholipase from established tea genomic library, on the basis of semi-quantative PCR results separately amplified in leaf and root of tea plant, we isolated three candidate tea xanthosine nucleosidases clones. After expression in E.coli, one of them was found to encode a protein possessing methyl transfer and nucleoside cleavage activity and was designated as tea xanthosine nucleosidase (TXN). The open reading frame (ORF) of TXN is 723 bp in length, which encoded a 240 amino acids sequence with the molecular mass of approximately 26.24 kDa and an isoelectric point of 4.96. It shows similarity to Ricinus communis mta/sah nucleosidase (78%) and Coffea Arabica xanthosine methyltransferase (40%). The results were all performed in the E.coli expression system, laid a foundation for the industrialized production of caffeine in vivo in largescale. Co-expression of CaXMT and TCS1 in E. coli system Figure 2. In vitro functional analysis of pMAL-CaXMT-TCS1 by HPLC (CK). Standard compounds; (CT). Products detection of enzymatic assays with recombinant CaXMT-TCS1; Screening, cloning and functional analysis of tea xanthosine nucleosidase candidate genes On the basis of semi-quantative PCR results separately amplified in leaf and root of tea plant, we isolated three candidate tea xanthosine nucleosidases clones. After expression in E.coli, one of them was found to encode a protein possessing methyl transfer and nucleoside cleavage activity and was designated as tea xanthosine nucleosidase (TXN). It shows similarity to Ricinus communis mta/sah nucleosidase (78%) and Coffea Arabica xanthosine methyltransferase (40%). Results Construction of CaXMT and TCS1 vectors for over-expression in E. coli system We designed two pairs of specific primers, and obtained the gene fragments of CaXMT from coffee and TCS1 from tea. The recombinant expression vectors pMALCaXMT and pMAL-TCS1 were then constructed by inserting the CaXMT and TCS1 genes into pMAL-c5X vector. Finally the enzymatic reactions in vitro suggested CaXMT can directly catalyze the conversion from XR to 7-MX ,while TCS1 can catalyze the conversion from 7-mx to caffeine via theobromine (Fig.1). Figure 3. In vitro functional analysis of TXN by UPLC 1.Standard compounds; 2. Products detection of enzymatic assays with recombinant MBP; 3. Products detection of enzymatic assays with TXN Conclusion Figure 1. In vitro functional analysis of CaXMT and TCS1 by HPLC 1.Standard compounds; 2. Products detection of enzymatic assays with recombinant MBP; 3. Products detection of enzymatic assays with CaXMT and TCS1 separately. The results indicated that caffeine could be successfully produced by E.coli through co-expression of CaXMT and TCS1. Meanwhile the screening, cloning and functional analysis of tea xanthosine nucleosidase candidate genes was performed to regulate the synthesis of 7-methylxanthine in tea plant. The results were all performed the E.coli expression system, lying a foundation for the industrialized production of caffeine in the yeast system. Acknowledgments Alignment of CADs and phylogenetic relationship analysis This study was supported by national natural science foundation of China "In Vitro Combinatorial Biosynthesis and Gene Regulation of Caffeine Biosynthesis in Camellia sinensis"(3110649).