Study On Caffeine Biosynthetic Pathway And Molecular Regulation In Tea Plant (Camellia Sinensis (L.) O. Kuntze)

Caffeine (1,3,7-trimethylxanthine) as a natural alkaloid exsits in many plants, it isone of the most important purine alkaloid in tea plant. It has effects on multiplehuman physiologies, including excitation of central nervous system, aiding digestion,and diuresis.Caffeine is an important food additive and medicinal material. At present,commercial caffeine is mainly produced from chemical synthesis and natural productextraction. These production methods have many drawbacks, such as harmfulside-products and impurities, high cost, environmental contaminations, etc. Due tocaffeine’s role as a specific functional component of soft drinks, current negativeaspects of caffeine production may cause concerns in certain consumer groups, suchas young kids, pregnant women, and some disease patients. That determines both teaand coffee beverages with low-caffeine are also in great demand.Therefore, biosynthesis of caffeine by microbial fermentation through biologicalengineering technology has great potential application values. Scientifically, it isimportant to study site-directed mutagenesis of key caffeine synthetic enzymes, notonly for understanding molecular mechanisms and regulations of the catalytic activity,but also for providing important theoretical basis for molecular breeding oflow-caffeine tea plants.The final caffeine biosynthetic pathway can be divided into four steps. It includesthree methylation reactions catalyzed by N-methyltransferase and one nucleosidecleavage reaction catalyzed by nucleosidase. The details are as following: xanthosine(XR)→7-methylxanthosine(7mXR)→7-methylxanthine(7-MX)→theobromine(Tb)→Caffeine.This objective of the research was to establish a research platform for producingcaffeine by fermentation, and to find out the molecular regulating mechanism oncaffeine biosynthetic enzymes in tea plant. The key enzymes involves in caffeinebiosynthesis were expressed in Escherichia coli or Saccharomyces cerevisiae and theenzymatic activity were analyzed and confirmed. Site-directed mutagenesis of teacaffeine synthase was performed. The results are as following:1. Coffee xanthosine methyltransferase (CaXMT1) was cloned from the leaf ofCoffee arabica. It was shown to carry out the first and second step of caffeinesynthesis, simultaneously catalyze the methyl transfer of xanthosine and ribose removal of7-methylxanthoine. The two reactions could be coupled and catalyzed bythis single enzyme. These activities were detected by HPLC and confirmed byLC-MS, suggesting CaXMT1can directly catalyze the conversion from XR to7-MX.2. Tea caffeine synthase (TCS1) was cloned from the leaf of Camellia sinensis, andproved to catalyze the last two steps of caffeine synthesis, i.e. the convertion from7-MX to caffeine via theobromine.3. The two genes, CaXMT1and TCS1were both introduced into the yeast cells andinduced to express the functional proteins. After fed with substrate and prolongedfermentation, caffeine could be detected in the yeast medium, and the yield was0.38mg/L.4. Adenosyl-nucleosidase, inosine-/guanosine-nucleosidase and inosine-/guanosine-phospholipase were selected from established tea genomic library. Eventually, threecandidate tea nucleosidases were screened on the basis of semi-quantative PCRresults separately amplified in leaf and root of tea plant. However, enzymes activitieswere not detected under current experimental condition.5. Four amino acid residues of TCS1were selected for site-directed mutagenesis:225Arg→His,317Val→Met,271Phe→Trp, and272Ala→Pro. These amino acidswere selected based protein structure analysis and substrate binding modeling. Themutated enzyme showed significantly changes in theobromine production.6. The two genes, CaXMT1and TCS1were both introduced into E.coli cells andinduced to expression. Unlike in yeast systems, only7-MX but no caffeine could bedetected in enzymatic assays.Taken together, the results indicated that caffeine could be successfully producedby yeast fermentation through co-expression of CaXMT1and TCS1, even though theyield was low. To improve the accumulation of7-MX and regulate the caffeinebiosynthetic pathway, we also attempted to identify and characterize tea7-methylxanthine nucleosidase without success. We further co-expressed twobiosynthetic genes in the E.coli to engineer caffeine production too.The TCS1mutantsindeed increased the theobromine yields of the enzymatic assays. The research laid afoundation for industrially production of caffeine by fermentation, and informationfor regulating the caffeine biosynthesis at the molecular level.