Functional Identification Of Relevant Genes Involved In Theanine Synthesis From Camellia Sinensis(L.)O. Kuntze

L-theanine(γ-N-ethyl-glutamine)is a unique non-protein amino acid present in Camellia sinensis(L.)O.Kuntze,which is also a major contributor to impart a sweet taste to the tea and to promote the secretion of body fluid.It has multiple effects on human physiologies,including promoting sleep improvement,medicating blood pressure and advancing the immunity.L-theanine classified as generally recognized as safe(GRAS)by FDA is widely used in food and pharmaceutical industry.Traditionally it can be obtained through natural extraction and purification from tea,while the method requires high cost and considerable time and its efficiency is relatively low.The chemical synthesis of L-theanine is hard to be accepted by consumers due to the deficiency of the fundamental data such as its L,D isomer ratio and related safety evaluation.The development of biological synthesis by microbial fermentation to produce theanine has been an increasing trend in recent years.Different from other plants,theanine anabolism is closely related to the nitrogen metabolism of tea plant.In the accepted synthetic pathway of theanine,the major enzymes include alanine decarboxylase,glutamate synthase(GOGAT),glutamine synthetase,theanine synthetase,etc.There have been limited numbers of reports on theanine synthetase so far.Yukitata et al.from Japan screened two sequences from a cDNA library of tea,and their translated protein are confirmed to be capable of synthesizing theanine after prokaryotic expression and purification.Their homology with glutamine synthetases at amino acid level is as high as 90%,suggesting they are likely to belong to the glutamine synthetase gene family.Besides the transgenic enzymes from other species can produce theanine through biological fermentation,which include glutamine synthetase,glutamyl-methylamine synthetase,gamma glutamyl transpeptidase(GGT).So it need further study to determine whether tea synthesize theanine using glutamine synthase or γ-glutamyl transferase under specific condition or theanine synthesis is catalyzed by specific enzymes.This study I conducted focused on three genes that are probable to be involved in theanine synthesis,among them two(named CsGS1-1,CsGS1-2)were screened from the young root cDNA library of tea,while the other(denominated as CsGGT)was acquired from the data of tea genome.The genes were either expressed in Escherichia coli or Saccharomyces cerevisiae and the enzymatic activities were determined.The target of thestudy is to explore the theanine synthesis mechanism.The results are as following:1.CsGS1-1 was cloned from the root of Camellia sinensis.After the prokaryotic vector was constructed by Infusion technology,it was induced to expression in Escherichia coli,fed with substrate,the UPLC-MS-MS results showed theanine was detected in the medium while the control group had none of theanine.It can be inferred that CsGS1-1 can be very likely to synthesize theanine.2.CsGS1-2 was cloned from the root of Camellia sinensis.After the yeast expression vector was constructed by Gateway technology,it was induced to expression,and enzyme activities analysis was performed.The results showed the glutamine concentration of the treatment group(carrying CsGS1-2)is appropriate two times as much as that in the control group(empty vector),suggesting CsGS1-2 is capable of synthesizing the glutamine.3.The prokaryotic vector of CsGS1-2 was constructed by Infusion technology.After the induction,the results of SDS-PAGE analysis showed soluble recombination protein was detected in the total protein,which will make a preparation for the enzyme activity determination in vitro.Moreover the expression vectors of CsGS1-1 in soybean hairy root system had been successfully constructed,which will make a preparation for the expression of CsGS1-1 in soybean hairy root and the determination of the gene.The results above will provide a foundation for further illumination of the theanine and glutamine synthesis mechanism,the differences and the correlation between them and up to the elucidation of the anabolic pathway of theanine.