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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).