Nontargeted and targeted metabolomics analysis for evaluating the effect of "golden flora" amount on the sensory quality, metabolites, and the alpha-amylase and lipase inhibitory activities of Fu brick tea.

To investigate the effects of "golden flora" amount on the sensory quality, metabolites and bioactivities of Fu brick tea (FBT), FBT samples with different "golden flora" amounts were prepared from the same materials by adjusting the water content before pressing. With the increase of "golden flora" in samples, the tea liquor color changed from yellow to orange red and the astringent taste gradually diminished. Targeted analysis demonstrated that (-)-epigallocatechin gallate, (-)-epicatechin gallate, and most amino acids gradually decreased as the increase of "golden flora". Seventy differential metabolites were identified by untargeted analysis. Among them, sixteen compounds including two Fuzhuanins and four EPSFs were positively correlated with "golden flora" amount (P < 0.05). The FBT samples with "golden flora" exhibited significantly higher inhibitory potency on α-amylase and lipase than the samples without "golden flora". Our results provide a theoretical basis of guiding FBT processing based on desired sensory quality and metabolites.

Widely targeted metabolomics using UPLC-QTRAP-MS/MS reveals chemical changes during the processing of black tea from the cultivar Camellia sinensis (L.) O. Kuntze cv. Huangjinya.

Huangjinya is a light-sensitive mutant tea cultivar that produces fresh leaves with a yellow phenotype, and the leaves also be used to produce black tea with special sensory characteristics. To thoroughly explore the chemical changes that occur during the processing of Huangjinya black tea, tea samples were collected from each processing step to perform quantitative and qualitative analyses by high-performance liquid chromatography and ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS). Compared to fresh tea leaves, only approximately 20% of the catechins remained at the end of processing, while theaflavins levels peaked at the rolling step and were slightly reduced in the fermentation and drying processes. The levels of amino acids derived from protein hydrolysis increased significantly in the withering and rolling processes. Altogether, 620 differential metabolites were identified from 11 subclasses using widely targeted metabolomics based on UPLC-HRMS for the four steps used to process Huangjinya black tea. Flavonoids, phenolic acids, and lipids were the three major classes of differential metabolites, accounting for 52.4% of the differential compounds. The greatest changes in the metabolite profile occurred during the rolling step, with 292 metabolites showing increases or decreases. Two glycoconjugates of the amino acid were first identified in tea, which was sharply increased in the drying stage. The present study provides comprehensive information on the chemical changes during the processing of Huangjinya black tea, and this information is valuable for optimizing manufacturing process and utilization of the Huangjinya tea plant.

Isolation and Functional Characterization of Multiple NADPH-Cytochrome P450 Reductase Genes from Camellia sinensis in View of Catechin Biosynthesis.

Catechins are critical constituents for the sensory quality and health-promoting benefits of tea. Cytochrome P450 monooxygenases are required for catechin biosynthesis and are dependent on NADPH-cytochrome P450 reductases (CPRs) to provide reducing equivalents for their activities. However, CPRs have not been identified in tea, and their relationship to catechin accumulation also remains unknown. Thus, three CsCPR genes were identified in this study, all of which had five CPR-related conserved domains and were targeted to the endoplasmic reticulum. These three recombinant CsCPR proteins could reduce cytochrome c using NADPH as an electron donor. Heterologous co-expression in yeast demonstrated that all the three CsCPRs could support the enzyme activities of CsC4H and CsF3'H. Correlation analysis indicated that the expression level of CsCPR1 (or CsCPR2 or CsCPR3) was positively correlated with 3',4',5'-catechin (or total catechins) content. Our results indicate that the CsCPRs are involved in the biosynthesis of catechins in tea leaves.

CsUGT78A15 catalyzes the anthocyanidin 3-O-galactoside biosynthesis in tea plants.

Anthocyanins are a group of natural water-soluble pigments in plants that contribute to the pink-purple color of a range of tissues. Because anthocyanins have various biological activities in human health, there is great research interest in the development of anthocyanin-rich foods and beverages, including purple shoot tea. Anthocyanidin 3-O-galactosides have been identified as one of the main anthocyanin components in purple shoot tea, but the enzyme responsible for their biosynthesis remains unclear. UDP-galactose anthocyanidin 3-O-galactosyltransferase (UA3GalT) is presumed to catalyze the galactosylation of anthocyanidin. Therefore, we assayed the UA3GalT activity in five tea samples with varying degrees of purple color and found that its activity was strongly positively correlated (r = 0.929, p < 0.05) with anthocyanin content. Phylogenetic analysis and sequence alignment suggested that CsUGT78A15 encoded a UA3GalT enzyme. Enzymatic assays indicated that rCsUGT78A15 could catalyze the synthesis of cyanidin 3-O-galactoside and delphinidin 3-O-galactoside using UDP-galactose as a sugar donor, and it showed higher catalytic efficiency towards delphinidin than cyanidin. These results indicate that CsUGT78A15 acts as a UA3GalT in vitro. Subcellular localization showed that CsUGT78A15 was located in the endoplasmic reticulum (ER) and nucleus, consistent with the location of anthocyanin synthesis. Transient overexpression of CsUGT78A15 in the fruit of mature 'Granny Smith' apples showed that the upregulation of CsUGT78A15 promoted cyanidin 3-O-galactoside accumulation in apple skins. These results suggested that CsUGT78A15 could catalyze galactosylation of anthocyanidins in planta. Our findings provide insight into the biosynthesis of anthocyanins in tea plants.

[Engineering of a flavonoid 3'-hydroxylase from tea plant (Camellia sinensis) for biosynthesis of B-3',4'-dihydroxylated flavones].

Objective: A flavonoid 3'-hydroxylase from tea plant was engineered to synthesize B-3',4'-dihydroxylated flavones such as eriodictyol, dihydroquercetin and quercetin. Methods: Four articifical P450 constructs harboring both flavonoid 3'-hydroxylase gene from Camellia sinensis (CsF3'H) and P450 reductase gene from Arabidopsis thaliana (ATR1 or ATR2) were introduced into Escherichia coli strains TOP10, DH5α and BL21, resultantly engineering strains S1 to S12. The plasmid pYES-Dest52-CsF3'H harboring CsF3'H gene was introduced into yeast Saccharomyces cerevisiae WAT11 designated as strain S13. The plasmid pES-HIS-CsF3H::AtFLS 9 AA was constructed through fusing flavanone 3-hydroxylase gene from Camellia sinensis (CsF3H) and flavonol synthase gene from Arabidopsis thaliana (AtFLS). Plasmid pES-URA-CsF3'H and pES-HIS-CsF3H::AtFLS 9 AA were then co-introduced into yeast S. cerevisiae WAT11 designated as strain S14. Results: Strain S6 generated highest bioconversion efficiency at 25℃ among all E. coli strains during 24 h fernentation. Supplemented with 1000 µmol/L naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by strain S13 were 734.32 µmol/L, 446.07 µmol/L and 594.64 µmol/L respectively. Supplemented with 5 mmol/L naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by strain S14 were 1412.16 µmol/L, 490.25 µmol/L, 445.75 µmol/L, 66.75 µmol/L and 73.50 µmol/L during 36-48 h fermentaion respectively. Conclusion: CsF3'H was engineered for biosynthesis of B-3',4'-dihydroxylated flavone.

Cloning and Characterization of a Flavonoid 3'-Hydroxylase Gene from Tea Plant (Camellia sinensis).

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3'-hydroxylase (F3'H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3'H, designated as CsF3'H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3'H was highly homologous with the characterized F3'Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3'H-specific conserved motifs were discovered in the protein sequence of CsF3'H. Enzymatic analysis of the heterologously expressed CsF3'H in yeast demonstrated that tea F3'H catalyzed the 3'-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent Km values for these substrates were 17.08, 143.64 and 68.06 µM, and their apparent Vmax values were 0.98, 0.19 and 0.44 pM·min(-1), respectively. Transcription level of CsF3'H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3',4'-flavan-3-ols, 3',4',5'-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3'H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3'H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3'H in the biosynthesis of 3',4'-catechins, 3',4',5'-catechins and flavan 3-ols in tea leaves.