Prokaryotic Expression And Functional Analysis Of Key Genes Involved In Caffeine Biosynthesis In Tea Plant (Camellia Sinensis)

Caffeine is a xanthine alkaloid found widesly in a variety of plant as a common purine base.Caffeine plants for human consumption include coffee,tea,cocoa.Caffeine is not only the important taste compound,but also the major component of tea alkaloids.Caffeine can cause some negative effects on paticular group,such as spontaneous abortion of pregnant women and premature birth children.However,appropriate caffeine have positive effects on human body,for example,refreshing,protecting liver function,diuresis,improving digestion.Most caffeine used in the food and pharmaceutical industry,while partly applied to industry.Chemically synthesized caffeine has toxic side effects as well as polluting environment.There are still some problems in natural extracts,such as expensive equipment and toxic residues.Caffeine biosynthesis has many advantages like non-pollution,non-toxic side effects.Domestic and foreign scholars have almost mastered caffeine biosynthetic pathway and major donor route,which laid the foundation for caffeine biosynthesis.The detailed biosynthesis process follows:adenine nucleotides(AMP)? Inosine-5'-monophosphate(IMP)?xanthosine-5-monophosphate(XMP)? xanthosine(XR)? 7-methylxanthosine(7-MXR)?7-methylxanthine(7-MX)?theobromine(Tb)? caffeine(Cf).Mutants mutated site-directedly by tea caffeine synthase(TCS1)through analyzing protein space model were TM1(673CGT ? CAT),TM2(949GTT ? ATG),TM3(811TTT ? TGG)and TM4(814GCA ? CCA)respectively compared with TCS1.Caffeine synthase mutants(TMX)expressed individually and the test results of in vitro activity showed that: TM1 can only catalyze the convertion from7-methylxanthine to theobromine,TM2/3/4 can catalyze the convertion from7-methylxanthine to caffeine,but the caffeine content was low(this part of work has been done by State Key Laboratory of Tea Plant Biology and Utilization).Based on individual expression of mutants(TMX),this study constructed co-expression vectors by mutants(TMX)and coffee xanthosine methyltransferase(CaXMT)as well as attempting to produce caffeine by Escherichia coli in vitro and finding a cheap synthetic substrate.The main results are as following:(1)This research concatenated CaXMT and TMX respectively in the same expression vector pMAL-c5 X,then induced coexpression of the fusion protein after transforming into E.coli cells.Then added xanthosine and S-adenosyl-L-methionine in the obtained crude enzyme solution,the in vitro enzymatic reaction products weredetected by HPLC.The result showed that there was only theobromine,but no caffeine generated in the products of pMAL-CaXMT-TM2/3/4.There was only 7-MX in the products of pMAL-CaXMT-TM1.(2)The expression vector(pRSF-Duet1)had two promoters,which were linked with CaXMT and TMX,respectively.Then transformed the co-expression vector into E.coli cells.In vitro enzyme reaction assay showed that there was theobromine in the products of pRSF-CaXMT-TM1,and there were caffeine in the products of pRSF-CaXMT-TM2/3/4.The products of pRSF-CaXMT-TM4 had the largest generation of caffeine(1.89 ?g/mL).(3)Inosine monophosphate dehydrogenase(TIDH)was cloned in young leaves of Camellia sinensis,which proved to catalyze the convertion from Inosine-5'-monophosphate to xanthosine-5-monophosphate via Nicotinamide adenine dinucleotide.In this study,CaXMT and TMX were demonstrated coexpression in the same vector(pRSF-Duet1)and caffeine could be synthesized in vitro on the basis of E.coli expression system.TIDH can catalyse the Inosine-5'-monophosphate(IMP)convert to xanthosine-5-monophosphate(XMP),a precursor of xanthosine,through coenzyme NAD+.The research laid a foudation for the construction of coexpression vector which was used for the biosynthesis of caffeine,and provided a theoretical basis for exploring cheap substrate in the caffeine biosynthesis pathway.