Construction Of The Multi-gene Prokaryotic Expression Vector For Tea Caffeine Biosynthesis And Expression Regulation In Vitro

Caffeine is the main component of purine bases in tea, coffee and cocoa, which are the three major soft beverage plants. On one hand caffeine (1,3, 7-trimethylxanthine) is widely used in medical, food, chemical and other fields due to its varieties of of physiological functions; on the other hand long-term excessive caffeine intaking has some side effects on human body. Thus tea products with low-caffeine are becoming more and more popular.At present, commercial caffeine and low-caffeine products are mainly produced from chemical synthesis. These production methods have many drawbacks, such as harmful side-products and impurities, environmental contaminations, etc. And the method of extracting from tea and coffee or removing the natural caffeine, improves the raw material and manufacturing cost, and easily leads to the residue of hazardous substances.Therefore, using genetic engineering, technology biosynthesis of caffeine in vitro by constructing engineering bacteria has broad application prospects. Meanwhile, studying biological functions of key caffeine synthetic enzymes can realize the biosynthesis artificial regulations of caffeine, which provides important theoretical basis for the molecular breeding of low-caffeine tea plants.The final caffeine biosynthetic pathway mainly includes four steps, The details are as following:xanthosine(XR)→ 7-methylxanthosine(7mXR)→ 7-methylxanthine (7-MX)→theobromine(Tb)→affeine(Cf).Through the in vitro co-expressing of the key enzymes involve in caffeine biosynthesis in Escherichia coli and detecting of enzyme activity, we tried to establish a research platform for the in vitro biosynthesis of caffeine. Site-directed mutagenesis was performed to regulate the synthesis of theobromine and caffeine to find out the molecular regulating mechanism, then provided the theoretical basis for artificial regulation of caffeine biosynthesis.The results are as following: 1. The construction of the expression vectors pMAL-CaXMT and pMAL-TCS1 and study on the respective expression in vitro.Based on the ORF of the CaXMT and TCS1 genes, we designed two pairs of specific primers, and obtained the gene segments of CaXMT and TCS1 RT-PCR. The recombinant expression vectors pMAL-CaXMT and pMAL-TCSl 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. 2. The construction of the multi-gene fusion expression vector pMAL-CaXMT-TCS1 and study on its expression.The multi-gene fusion expression vector pMAL-CaXMT-TCSl was successfully constructed by ligating the digested products of pMAL-CaXMT and pMAL-TCSl,which were produced by the SalI Sbfl restriction enzyme digestion. The activity of the fusion proteins were assayed in vivo and in vitro respectively, and the reaction products were detected by HPLC and LC-MS. The results suggested that the combinant can catalyze the conversion from XR to caffeine via 7-MX and theobromine. The optimal reaction conditions were found as follow:Reactions with fusion pritein extracts containing 170 μmol/L of XR and 1140 μmol/L SAM at 25 ℃ for 4 h, and the maximum yield of caffeine was up to 43.87 μmol/L. 3. Site-directed mutagenesis for caffeine synthase (TCS1) of Camellia sinensis and study on expression in vitroThrough analysis of the protein tertiary structure and spatial structure models of TCSl and ICSI, we found four key amino acids in TCS1,which may increase its catalytic activity. Results were list as following:TM:225 Arg→His, TM2:317 Val→Met, TM3:271 Phe→Trp, and TM4:272 Ala→Pro. Then we constructed the recombinant vectors, and analyzed the enzyme activities of the expressed fusion proteins. The mutated enzyme showed significantly changes in theobromine and Caffeine production compared with the feral TCSl,especially the TMl can catalyze the second methylation step simply, converting 7-methylxanthine to 3,7-dimethylxanthine, while other mutants showed visible activity differences of 3-and 1-N-methyltransferase.Taken together, the results indicated that caffeine could be successfully produced by E.coli through co-expression of CaXMT and TCSl. Meanwhile the site-directed mutagenesis of tea caffeine synthase was performed to regulate the synthesis of theobromine and caffeine. The results were all performed the E.coli expression system, lying a foundation for the industrialized production of caffeine in the yeast system.