Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 µM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 µM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 µM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

From Tea Leaves to Factories: A Review of Research Progress in l-Theanine Biosynthesis and Production.

l-Theanine is the most popular nonprotein amino acid contained in tea leaves. It is one of the umami components of green tea, contributing to the unique flavor of tea. Because of its various health functions, l-theanine has been commercially developed as a valuable ingredient easily used for various applications in food and pharmaceutical industries. Nowadays, l-theanine is mass-produced by plant extraction, chemical synthesis, or enzymatic transformation in factories. This review embodies the available up to date information on the l-theanine synthesis metabolism in the tea plant as well as approaches to produce it, placing emphasis on the biotransformation of l-theanine. It also gives insight into the challenges of l-theanine production on a large scale, as well as directions for future research. This review comprehensively summarizes information on l-theanine to provide an approach for an in-depth study of l-theanine production.

Time-series transcriptomic analysis reveals novel gene modules that control theanine biosynthesis in tea plant (Camellia sinensis).

Theanine (thea) is a unique non-protein amino acid in tea plant (Camellia sinensis) and one of the most important small molecular compounds for tea quality and health effects. The molecular mechanism that maintains thea biosynthesis is not clear but may be reflected in complicated biological networks as other secondary metabolites in plants. We performed an integrative transcriptomic analysis of tea seedlings bud and leave over the time-course of ethylamine (EA) treatment that activated thea pathway. We identified 54 consistent differentially expressed genes (cDEGs, 25 upregulated and 29 downregulated) during thea activation. Gene Ontology (GO) functional enrichment analysis of upregulated genes and downregulated genes showed that they may function as a cascade of biological events during their cooperative contribution to thea biosynthesis. Among the total cDEGs, a diversity of functional genes (e.g., enzymes, transcription factors, transport and binding proteins) were identified, indicating a hierarchy of gene control network underlying thea biosynthesis. A gene network associated with thea biosynthesis was modeled and three interconnected gene functional modules were identified. Among the gene modules, several topologically important genes (e.g., CsBCS-1, CsRP, CsABC2) were experimentally validated using a combined thea content and gene expression analysis. Collectively, we presented here for the first time a comprehensive landscape of the biosynthetic mechanism of thea controlled by a underling gene network, which might provide a theoretical basis for the identification of key genes that contribute to thea biosynthesis.

Exogenous melatonin improves tea quality under moderate high temperatures by increasing epigallocatechin-3-gallate and theanine biosynthesis in Camellia sinensis L.

Global warming has multifarious effects on crop growth and productivity. Nonetheless, the effects of moderate-high temperatures and melatonin on tea yield and quality remain unclear. In this study, we found that melatonin, a universal growth stimulatory molecule, not only promotes photosynthesis and biomass accumulation in tea plants (Camellia sinensis L.) but also improves tea quality under sub high temperature (SHT). SHT increased the dry biomass and photosynthesis by 40.8% and 28.1%, respectively, and exogenous melatonin caused a further improvement. Moreover, SHT increased the total polyphenol concentrations and decreased the free amino acid concentrations, leading to a significant increase (68.2%) in polyphenol to free amino acid ratio. However, melatonin decreased the polyphenol to free amino acid ratio by delicately improving the concentrations of polyphenols and amino acids. Consistent with the total polyphenol, melatonin increased the concentrations of (-)-catechin, (-)-gallocatechin (GC), and (-)-epigallocatechin-3-gallate (EGCG) in tea leaves. The qRT-PCR analysis revealed that melatonin increased the transcript levels of catechins biosynthesis genes, such as CsCHS, CsCH1, CsF3H, CsDFR, CsANS, CsLAR, and CsANR under SHT. Meanwhile, the theanine concentration was decreased by SHT, which was attributed to the attenuated expression of CsGS, CsGOGAT, CsGDH, and CsTS1. Nonetheless, melatonin significantly increased those transcripts and the content of theanine under SHT. Melatonin also increased the caffeine content by inducing the expression of CsTIDH, CssAMS, and CsTCS1. These results suggest that melatonin could positively alter tea growth and quality by modulating the photosynthesis and biosynthesis of polyphenols, amino acids, and caffeine in tea leaves under SHT.

The R2R3-MYB transcription factor CsMYB73 negatively regulates l-Theanine biosynthesis in tea plants (Camellia sinensis L.).

l-Theanine, a non-proteinaceous amino acid abundantly present in tea (Camellia sinensis), contributes to the umami flavor of tea and has beneficial effects on human health. While key l-theanine biosynthetic genes have been well documented, their transcriptional regulation remains poorly understood. In this study, we determined the l-theanine contents in tea leaves of two cultivars at three developmental stages and investigated the expression patterns of the l-theanine biosynthetic genes CsGS1 and CsGS2. Additionally, we identified an R2R3-MYB transcription factor, CsMYB73, belonging to subgroup 22 of the R2R3-MYB family. CsMYB73 expression negatively correlated with l-theanine accumulation during leaf maturation. We found that CsMYB73, as a nuclear protein, binds to the promoter regions of CsGS1 and CsGS2 via MYB recognition sequences and represses the transcription of CsGS1 and CsGS2 in tobacco leaves. Collectively, our results demonstrate that CsMYB73 is a transcriptional repressor involved in l-theanine biosynthesis in tea plants. Our findings might contribute to future tea plant breeding strategies.

Identification and expression profiling of MYB transcription factors related to l-theanine biosynthesis in Camellia sinensis.

The MYB proteins belong to a large family of transcription factors in plant genomes and play significant roles in primary and secondary metabolism. Although several CsMYB genes have been identified in Camellia sinensis, few CsMYBs involved in l-theanine biosynthesis have been analyzed. In this study, we screened and identified 20 CsMYBs related to l-theanine biosynthesis. Transcriptomic analysis revealed that the expression profiles of the CsMYBs were positively or negatively related to dynamic changes in the l-theanine content. Validation of selected l-theanine biosynthetic and CsMYB genes was conducted by qRT-PCR. The results illustrated that most of the structural and CsMYB genes were downregulated with a decrease in the l-theanine levels. Protein-protein interaction networks of CsMYB5, CsMYB12 and CsMYB94 proteins demonstrated that they might form complexes with bHLH and WD 40 proteins. Multiple DNA-binding sites of the R2R3-MYB protein were observed in promoter regions of structural genes, indicating CsMYB family proteins might be involved in l-theanine metabolism via the attachment of AC elements. Moreover, CsMYB73 demonstrated binding specificity to the promoter region of CsGDH2 (CsGDH2-pro). These findings provide fundamental understanding of specific members of the CsMYBs related to the l-theanine biosynthesis pathway.

Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons.

BACKGROUND: Catechins, caffeine, and theanine as three important metabolites in the tea leaves play essential roles in the formation of specific taste and shows potential health benefits to humans. However, the knowledge on the dynamic changes of these metabolites content over seasons, as well as the candidate regulatory factors, remains largely undetermined. RESULTS: An integrated transcriptomic and metabolomic approach was used to analyze the dynamic changes of three mainly metabolites including catechins, caffeine, and theanine, and to explore the potential influencing factors associated with these dynamic changes over the course of seasons. We found that the catechins abundance was higher in Summer than that in Spring and Autumn, and the theanine abundance was significantly higher in Spring than that in Summer and Autumn, whereas caffeine exhibited no significant changes over three seasons. Transcriptomics analysis suggested that genes in photosynthesis pathway were significantly down-regulated which might in linkage to the formation of different phenotypes and metabolites content in the tea leaves of varied seasons. Fifty-six copies of nine genes in catechins biosynthesis, 30 copies of 10 genes in caffeine biosynthesis, and 12 copies of six genes in theanine biosynthesis were detected. The correlative analysis further presented that eight genes can be regulated by transcription factors, and highly correlated with the changes of metabolites abundance in tea-leaves. CONCLUSION: Sunshine intensity as a key factor can affect photosynthesis of tea plants, further affect the expression of major Transcription factors (TFs) and structural genes in, and finally resulted in the various amounts of catechins, caffeine and theaine in tea-leaves over three seasons. These findings provide new insights into abundance and influencing factors of metabolites of tea in different seasons, and further our understanding in the formation of flavor, nutrition and medicinal function.

TeaCoN: a database of gene co-expression network for tea plant (Camellia sinensis).

BACKGROUND: Tea plant (Camellia sinensis) is one of the world's most important beverage crops due to its numerous secondary metabolites conferring tea quality and health effects. However, only a small fraction of tea genes (especially for those metabolite-related genes) have been functionally characterized to date. A cohesive bioinformatics platform is thus urgently needed to aid in the functional determination of the remaining genes. DESCRIPTION: TeaCoN, a database of gene co-expression network for tea plant, was established to provide genome-wide associations in gene co-expression to survey gene modules (i.e., co-expressed gene sets) for a function of interest. TeaCoN featured a comprehensive collection of 261 high-quality RNA-Seq experiments that covered a wide range of tea tissues as well as various treatments for tea plant. In the current version of TeaCoN, 31,968 (94% coverage of the genome) tea gene models were documented. Users can retrieve detailed co-expression information for gene(s) of interest in four aspects: 1) co-expressed genes with the corresponding Pearson correlation coefficients (PCC-values) and statistical P-values, 2) gene information (gene ID, description, symbol, alias, chromosomal location, GO and KEGG annotation), 3) expression profile heatmap of co-expressed genes across seven main tea tissues (e.g., leaf, bud, stem, root), and 4) network visualization of co-expressed genes. We also implemented a gene co-expression analysis, BLAST search function, GO and KEGG enrichment analysis, and genome browser to facilitate use of the database. CONCLUSION: The TeaCoN project can serve as a beneficial platform for candidate gene screening and functional exploration of important agronomical traits in tea plant. TeaCoN is freely available at http://teacon.wchoda.com .

Exploring plant metabolic genomics: chemical diversity, metabolic complexity in the biosynthesis and transport of specialized metabolites with the tea plant as a model.

The diversity and complexity of secondary metabolites in tea plants contribute substantially to the popularity of tea, by determining tea flavors and their numerous health benefits. The most significant characteristics of tea plants are that they concentrate the complex plant secondary metabolites into one leaf: flavonoids, alkaloids, theanine, volatiles, and saponins. Many fundamental questions regarding tea plant secondary metabolism remain unanswered. This includes how tea plants accumulate high levels of monomeric galloylated catechins, unlike the polymerized flavan-3-ols in most other plants, as well as how they are evolved to selectively synthesize theanine and caffeine, and how tea plants properly transport and store these cytotoxic products and then reuse them in defense. Tea plants coordinate many metabolic pathways that simultaneously take place in young tea leaves in response to both developmental and environmental cues. With the available genome sequences of tea plants and high-throughput metabolomic tools as great platforms, it is of particular interest to launch metabolic genomics studies using tea plants as a model system. Plant metabolic genomics are to investigate all aspects of plant secondary metabolism at the genetic, genome, and molecular levels. This includes plant domestication and adaptation, divergence and convergence of secondary metaboloic pathways. The biosynthesis, transport, storage, and transcriptional regulation mechanisms of all metabolites are of core interest in the plant as a whole. This review highlights relevant contexts of metabolic genomics, outstanding questions, and strategies for answering them, with aim to guide future research for genetic improvement of nutrition quality for healthier plant foods.

Identification of MYB Transcription Factors Regulating Theanine Biosynthesis in Tea Plant Using Omics-Based Gene Coexpression Analysis.

Theanine (thea) is the most abundant free amino acid in tea plant (Camellia sinensis) and one of the most important secondary metabolites conferring tea quality and health benefits. Great effort has recently been made to functionally dissect enzyme genes (e.g., GS, GDH, GOGAT) responsible for in vivo thea accumulation. However, the transcriptional regulation of its biosynthesis remains to be explored. Starting from publicly available (condition-independent) tea transcriptome data, we performed an exhaustive coexpression analysis between transcription factor (TF) genes and thea enzyme genes in tea plant. Our results showed that two typical plant-specialized (secondary) metabolites related TF families, such as MYB, bHLH, together with WD40 domain proteins, were prominently involved, suggesting a potential MYB-bHLH-WD40 (MBW) complex-mediated regulatory pattern in thea pathway. Aiming at the most involved MYB family, we screened seven MYB genes as thea candidate regulators through a stringent multistep selection (e.g., filtering with condition-specific nitrogen-treated transcriptome data). The control of MYB regulators in thea biosynthesis was further demonstrated using an integrated analysis of thea accumulation and MYB expression in several major tea tissues, including leave, bud, root, and stem. Our investigation aided tea researchers in having a comprehensive view of transcriptional regulatory landscape in thea biosynthesis, serving as the first platform for studying molecular regulation in thea pathway and a paradigm for understanding the characteristic components biosynthesis in nonmodel plants.

Efficient fermentative production of L-theanine by Corynebacterium glutamicum.

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.

Comparative Transcriptomic Analysis Reveals Regulatory Mechanisms of Theanine Synthesis in Tea (Camellia sinensis) and Oil Tea (Camellia oleifera) Plants.

Tea provides a rich taste and has healthy properties due to its variety of bioactive compounds, such as theanine, catechins, and caffeine. Theanine is the most abundant free amino acid (40%-70%) in tea leaves. Key genes related to theanine biosynthesis have been studied, but relatively little is known about the regulatory mechanisms of theanine accumulation in tea leaves. Herein, we analyzed theanine content in tea (Camellia sinensis) and oil tea (Camellia oleifera) and found it to be higher in the roots than in other tissues in both species. The theanine content was significantly higher in tea than oil tea. To explore the regulatory mechanisms of theanine accumulation, we identified genes involved in theanine biosynthesis by RNA-Seq analysis and compared theanine-related modules. Moreover, we cloned theanine synthase (TS) promoters from tea and oil tea plants and found that a difference in TS expression and cis-acting elements may explain the difference in theanine accumulation between the two species. These data provide an important resource for regulatory mechanisms of theanine accumulation in tea plants.

CsGOGAT Is Important in Dynamic Changes of Theanine Content in Postharvest Tea Plant Leaves under Different Temperature and Shading Spreadings.

We analyzed the changes of theanine content in postharvest tea leaves under high temperature (38 °C), low temperature (4 °C), and shading spreadings by using ultrahigh-performance liquid chromatography. The differentially expressed proteins (DEPs), CsFd-GOGAT and CsNADH-GOGAT, which are involved in theanine biosynthesis pathway, were identified from the corresponding proteome data. The protein-protein interactions of CsFd-GOGAT and CsNADH-GOGAT, CsTS1, or CsNiR were verified by yeast two-hybrid technology. The expression profiles of 17 genes in theanine metabolism, including CsFd-GOGAT and CsNADH-GOGAT, were analyzed by quantitative real-time polymerase chain reaction. The correlations between the dynamic changes of theanine content and expression profiles of related genes and DEPs were analyzed. This study preliminarily proved the importance of CsGOGAT in dynamic changes of theanine content in postharvest tea leaves during spreading.

Omega-3 fatty acids correlate with gut microbiome diversity and production of N-carbamylglutamate in middle aged and elderly women.

Omega-3 fatty acids may influence human physiological parameters in part by affecting the gut microbiome. The aim of this study was to investigate the links between omega-3 fatty acids, gut microbiome diversity and composition and faecal metabolomic profiles in middle aged and elderly women. We analysed data from 876 twins with 16S microbiome data and DHA, total omega-3, and other circulating fatty acids. Estimated food intake of omega-3 fatty acids were obtained from food frequency questionnaires. Both total omega-3and DHA serum levels were significantly correlated with microbiome alpha diversity (Shannon index) after adjusting for confounders (DHA Beta(SE) = 0.13(0.04), P = 0.0006 total omega-3: 0.13(0.04), P = 0.001). These associations remained significant after adjusting for dietary fibre intake. We found even stronger associations between DHA and 38 operational taxonomic units (OTUs), the strongest ones being with OTUs from the Lachnospiraceae family (Beta(SE) = 0.13(0.03), P = 8 × 10-7). Some of the associations with gut bacterial OTUs appear to be mediated by the abundance of the faecal metabolite N-carbamylglutamate. Our data indicate a link between omega-3 circulating levels/intake and microbiome composition independent of dietary fibre intake, particularly with bacteria of the Lachnospiraceae family. These data suggest the potential use of omega-3 supplementation to improve the microbiome composition.

Studies on the Biochemical Formation Pathway of the Amino Acid l-Theanine in Tea (Camellia sinensis) and Other Plants.

Tea (Camellia sinensis) is the most widely consumed beverage aside from water. The flavor of tea is conferred by certain metabolites, especially l-theanine, in C. sinensis. To determine why more l-theanine accumulates in C. sinensis than in other plants, we compare l-theanine contents between C. sinensis and other plant species (Camellia nitidissima, Camellia japonica, Zea mays, Arabidopsis thaliana, and Solanum lycopersicum) and use a stable isotope labeling approach to elucidate its biosynthetic route. We quantify relevant intermediates and metabolites by mass spectrometry. l-Glutamic acid, a precursor of l-theanine, is present in most plants, while ethylamine, another precursor of l-theanine, specifically accumulates in Camellia species, especially C. sinensis. Most plants contain the enzyme/gene catalyzing the conversion of ethylamine and l-glutamic acid to l-theanine. After supplementation with [2H5]ethylamine, all the plants produce [2H5]l-theanine, which suggests that ethylamine availability is the reason for the difference in l-theanine accumulation between C. sinensis and other plants.

Metabolic Flux Redirection and Transcriptomic Reprogramming in the Albino Tea Cultivar 'Yu-Jin-Xiang' with an Emphasis on Catechin Production.

In this study, shade-induced conversion from a young pale/yellow leaf phenotype to a green leaf phenotype was studied using metabolic and transcriptomic profiling and the albino cultivar 'Yu-Jin-Xiang' ('YJX') of Camellia sinensis for a better understanding of mechanisms underlying the phenotype shift and the altered catechin and theanine production. Shaded leaf greening resulted from an increase in leaf chlorophyll and carotenoid abundance and chloroplast development. A total of 1,196 differentially expressed genes (DEGs) were identified between the 'YJX' pale and shaded green leaves, and these DEGs affected 'chloroplast organization' and 'response to high light' besides many other biological processes and pathways. Metabolic flux redirection and transcriptomic reprogramming were found in flavonoid and carotenoid pathways of the 'YJX' pale leaves and shaded green leaves to different extents compared to the green cultivar 'Shu-Cha-Zao'. Enhanced production of the antioxidant quercetin rather than catechin biosynthesis was correlated positively with the enhanced transcription of FLAVONOL SYNTHASE and FLAVANONE/FLAVONOL HYDROXYLASES leading to quercetin accumulation and negatively correlated to suppressed LEUCOANTHOCYANIDIN REDUCTASE, ANTHOCYANIDIN REDUCTASE and SYNTHASE leading to catechin biosynthesis. The altered levels of quercetin and catechins in 'YJX' will impact on its tea flavor and health benefits.

Global transcriptome and gene regulation network for secondary metabolite biosynthesis of tea plant (Camellia sinensis).

BACKGROUND: Major secondary metabolites, including flavonoids, caffeine, and theanine, are important components of tea products and are closely related to the taste, flavor, and health benefits of tea. Secondary metabolite biosynthesis in Camellia sinensis is differentially regulated in different tissues during growth and development. Until now, little was known about the expression patterns of genes involved in secondary metabolic pathways or their regulatory mechanisms. This study aimed to generate expression profiles for C. sinensis tissues and to build a gene regulation model of the secondary metabolic pathways. RESULTS: RNA sequencing was performed on 13 different tissue samples from various organs and developmental stages of tea plants, including buds and leaves of different ages, stems, flowers, seeds, and roots. A total of 43.7 Gbp of raw sequencing data were generated, from which 347,827 unigenes were assembled and annotated. There were 46,693, 8446, 3814, 10,206, and 4948 unigenes specifically expressed in the buds and leaves, stems, flowers, seeds, and roots, respectively. In total, 1719 unigenes were identified as being involved in the secondary metabolic pathways in C. sinensis, and the expression patterns of the genes involved in flavonoid, caffeine, and theanine biosynthesis were characterized, revealing the dynamic nature of their regulation during plant growth and development. The possible transcription factor regulation network for the biosynthesis of flavonoid, caffeine, and theanine was built, encompassing 339 transcription factors from 35 families, namely bHLH, MYB, and NAC, among others. Remarkably, not only did the data reveal the possible critical check points in the flavonoid, caffeine, and theanine biosynthesis pathways, but also implicated the key transcription factors and related mechanisms in the regulation of secondary metabolite biosynthesis. CONCLUSIONS: Our study generated gene expression profiles for different tissues at different developmental stages in tea plants. The gene network responsible for the regulation of the secondary metabolic pathways was analyzed. Our work elucidated the possible cross talk in gene regulation between the secondary metabolite biosynthetic pathways in C. sinensis. The results increase our understanding of how secondary metabolic pathways are regulated during plant development and growth cycles, and help pave the way for genetic selection and engineering for germplasm improvement.

Occurrence and de novo biosynthesis of caffeine and theanine in seedlings of tea (Camellia sinensis).

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

Occurrence, biosynthesis and metabolism of theanine (γ-glutamyl-L-ethylamide) in plants: a comprehensive review.

Theanine (γ-glutamyl-L-ethylamide) is the most abundant non-protein amino acid in tea leaves. In addition to Camellia sinensis, theanine occurs in several plants belonging to the Ericales. Biosynthesis of theanine from glutamic acid and ethylamine by theanine synthetase is present in all organs of tea seedlings, but roots are the major site of theanine biosynthesis in adult tea trees. Theanine is transported from roots to young leaves via the xylem sap. Theanine is hydrolysed to glutamic acid and ethylamine in leaves. Ethylamine produced from theanine is predominantly used for catechin biosynthesis. Concentration of ammonia and light intensity influence the biosynthesis and degradation of theanine, respectively. Biosynthesis, translocation and degradation of theanine and related enzymes and genes are reviewed.

An overview of biological production of L-theanine.

L-Theanine (γ-glutamylethylamide) is a unique non-protein amino acid that is naturally found in tea plants. It contributes to the umami taste and unique flavor to green tea infusion, and thus its content in tea leaves highly impacts the tea quality and price. In addition to the graceful taste, it has been proved to have many beneficial physiological effects, especially promoting relaxation and improving concentration and learning ability. Based on these promising advantages, L-theanine has been commercially developed as a valuable ingredient for use in food and beverages to improve and/or maintain human health. L-Theanine can be obtained by chemical synthesis or isolation from tea, while chemical synthesis of L-theanine is hard to be accepted by consumers and is not allowed to use in food industry, and isolation of L-theanine in high purity generally involves time-consuming, cost-ineffective, and complicated operational processes. Accordingly, the biological production of L-theanine has recently attracted much attention. Four kinds of bacterial enzymes, including L-glutamine synthetase, γ-glutamylmethylamide synthetase, γ-glutamyltranspeptidase, and L-glutaminase, have been characterized to have L-theanine-producing ability. Herein, an overview of recent studies on the biological production of L-theanine was presented.