Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that releases floral scent and plays an important role in the sweet flowery aroma of tea. During the withering process for white tea producing, MeSA was generated by salicylic acid carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral scent was formed. In this study, we first cloned a CsSAMT from tea leaves (GenBank accession no. MG459470) and used Escherichia coli and Saccharomyces cerevisiae to express the recombinant CsSAMT. The enzyme activity in prokaryotic and eukaryotic expression systems was identified, and the protein purification, substrate specificity, pH, and temperature optima were investigated. It was shown that CsSAMT located in the chloroplast, and the gene expression profiles were quite different in tea organs. The obtained results might give a new understanding for tea aroma formation, optimization, and regulation and have great significance for improving the specific quality of white tea.Keywords: characterization; CsSAMT; MeSA; white tea; withering;

We designed a novel method for rapid detection of glufosinate, based on quantum dots (QDs) as a fluorescence probe. To date, no studies have been published on the detection of glufosinate using QDs. The innovation of this fluorescence system, which was constructed using CuInS2 QDs and Cu(IDA)·2H2O, is based on the use of intermolecular hydrogen bonds between Cu(IDA)·2H2O and carboxyl group (–COOH) to detect glufosinate. In the fluorescence “turn-on” step, the presence of glufosinate induces the release of Cu(IDA)·2H2O from the surface of the QDs, resulting in fluorescence intensity recovery. This method was performed in relatively clean aqueous solutions. Under optimal conditions, the calibration curve of the method showed good linearity for glufosinate, with a correlation coefficient (R2) of 0.99597. The method was employed to detect glufosinate on fresh tea-leaves, with satisfactory results.

•Developed a portable and low-cost VIS-NIR spectroscopy system•Evaluation of color sensory quality of black tea samples using this spectra system•Comparing the performance of models from spectra and color informationInstrumental test of black tea samples instead of human panel test is attracting massive attention recently. This study focused on an investigation of the feasibility for estimation of the color sensory quality of black tea samples using the VIS-NIR spectroscopy technique, comparing the performances of models based on the spectra and color information. In model calibration, the variables were first selected by genetic algorithm (GA); then the nonlinear back propagation-artificial neural network (BPANN) models were established based on the optimal variables. In comparison with the other models, GA-BPANN models from spectra data information showed the best performance, with the correlation coefficient of 0.8935, and the root mean square error of 0.392 in the prediction set. In addition, models based on the spectra information provided better performance than that based on the color parameters. Therefore, the VIS-NIR spectroscopy technique is a promising tool for rapid and accurate evaluation of the sensory quality of black tea samples.Download high-res image (206KB)Download full-size image

Caffeine (Cf, 1,3,7-trimethylxanthine), a major secondary metabolite of many higher plants, is widely used in popular non-alcoholic beverages, and in the pharmaceutical and health industries. Currently, this valuable chemical is mainly manufactured by chemical synthesis. In this study, we developed a novel approach for de novo caffeine production in metabolically engineered Escherichia coli. Xanthine-to-caffeine conversion was first achieved by the expression of a plant-derived gene encoding tea caffeine synthase (TCS1). Caffeine accumulation was then increased using two metabolic strategies: higher-level expression of the target enzymes, and enhancement of xanthine and S-adenosyl-L-methionine biosynthesis. The final strain (BL21/pRSF-eCS1-SAM2-vgb-eGUD1) produced up to 21.46 ± 1.03 mg L−1 caffeine from 20 g L−1 of glucose in shake flask culture, yielding caffeine up to 2.96 mg g−1 glucose, which represents the highest titer of caffeine produced by fermentation reported to date. This novel microbial conversion also represents an innovative approach to produce value-added methylxanthine chemicals from cheap carbon sources.

In addressing caffeine toxicity to the producing cells, engineering a transporter that can move caffeine from cytoplasm across the cell membrane to the extracellular space, thus enhancing caffeine resistance and potentially increasing the yield in yeast, is important. An ABC-transporter bfr1 from Schizosaccharomyces pombe was cloned and transformed into S. cerevisiae, resulting in enhancing caffeine resistance. Afterward, a library of randomly mutagenized bfr1 mutants through error-prone PCR was generated. One mutant was identified with drastically increased caffeine resistance (15 mg/mL). Sequencing and structural analysis illustrated that many of the mutations occurred at the cytosolic domain. Site-directed mutagenesis of these mutations confirmed at least one amino acid that conferred enhancing caffeine resistance in the mutated bfr1. These data demonstrated engineering ABC-transporters can be an efficient way to reduce product toxicity in heterologous systems.Keywords: ABC-transporter; caffeine; error-prone PCR; metabolic engineering;

Theanine synthetase (TS) is a key enzyme involved in theanine biosynthesis. In our recent study, it has been revealed that theanine biosynthesis derived from nitrogen metabolism in tea (Camellia sinensis) plants can be influenced by shading treatment. The expression patterns of CsTS protein in the roots and shoots of tea seedlings were examined by western blot using a self-prepared polyclonal antibody with high specificity and sensitivity. The effect of long-term shade treatment on the levels of theanine synthesis was also investigated using roots and shoots of tea seedlings. Levels of theanine and total free amino acids gradually increased in shoots, reaching their maximum after 22 days of treatment (DOT). The immunoblotting analysis suggested that CsTS protein levels increased gradually up to 22 DOT and expression remained at a high level, except after 1 DOT where levels were low in both roots and shoots. The increased theanine concentration we observed in the shading treatment may be due to increased nitrogen assimilation and reduced theanine catabolism under shade conditions.

Theanine, an amino acid contained in green tea, is known to possess many pharmacological functions. In this paper, we investigated the absorption of theanine in the human intestinal epithelium, using a Caco-2 monolayer model. Different concentrations of either pure theanine or green tea extracts were administered to Caco-2 cells. The theanine content in the samples was analysed by high-performance liquid chromatography, coupled with fluorescence detection. Cell permeation was also measured. The data revealed that the transport of pure theanine occurred in a manner consistent with passive diffusion. Surprisingly, pure theanine showed good absorption, whereas theanine in the green tea extract was poorly absorbed in the Caco-2 cell model. Furthermore, the transport of theanine in green tea extract in the basolateral (BL) to apical (AP) direction was much greater than that in the AP–BL direction, suggesting that green tea components profoundly affect the trans-epithelial transport of theanine in this Caco-2 cell model.