Microbial enzymes during solid-state fermentation (SSF), which play important roles in the food, chemical, pharmaceutical and environmental fields, remain relatively unknown. In this work, the microbial communities and enzymes in SSF of Pu-erh tea, a well-known traditional Chinese tea, were investigated by integrated metagenomics/metaproteomics approach. The dominant bacteria and fungi were identified as Proteobacteria (48.42%) and Aspergillus (94.98%), through pyrosequencing-based analyses of the bacterial 16S and fungal 18S rRNA genes, respectively. In total, 335 proteins with at least two unique peptides were identified and classified into 28 Biological Processes and 35 Molecular Function categories using a metaproteomics analysis. The integration of metagenomics and metaproteomics data demonstrated that Aspergillus was dominant fungus and major host of identified proteins (50.45%). Enzymes involved in the degradation of the plant cell wall were identified and associated with the soft-rotting of tea leaves. Peroxiredoxins, catalase and peroxidases were associated with the oxidation of catechins. In conclusion, this work greatly advances our understanding of the SSF of Pu-erh tea and provides a powerful tool for studying SSF mechanisms, especially in relation to the microbial communities present.

An in-depth knowledge of the microbiota and metabolites in the solid-state fermentation (SSF) of Post-fermented Pu-erh tea (Pu-erh Shucha, PFPT), a Chinese traditional tea with various health benefits, is essential to develop modern fermentation technology. In this work, the microbial diversity and succession in two laboratory-developed SSF protocols for PFPT were investigated using pyrosequencing analyses of the bacterial 16S rRNA and fungal 18S rRNA genes. The active bacteria in the initial stages of SSF (seven days) were from the raw materials and environment, with a dominance of Proteobacteria in both the raw materials and SSF after seven days. The environmental bacteria were inoculated into the tea mass throughout the fermentation process and multiplied, with a dominance of Firmicutes at day 14 and 21, and then Firmicutes and Actinobacteria at the last stages of fermentation (day 28 and 35). The dominant fungi came from the raw material and were identified at the genus level as Aspergillus throughout the SSF process. The contents of tea polyphenols, free amino acids, gallic acid, theaflavin, thearubigin, and catechins decreased significantly (p < 0.05), while the level of theabrownin increased significantly (p < 0.05). The caffeine content showed no significant change (p > 0.05). In total, 30 bacterial and three fungal genera showed significant correlations to 1-8 and 3-4 identified tea compounds, respectively (p < 0.05). The dynamics of the microbiota and chemical compounds, and correlations between their changes in the SSF of PFPT were revealed, and present a foundation for further studies on the microbial effects on chemical compounds.

Although the pathway and transcription factor regulation of anthocyanin biosynthesis in tea plants [Camellia sinensis (L.) O. Ktze] are known, post-transcriptional regulation mechanisms involved in anthocyanin accumulation have not been comprehensively studied. We obtained the full-length transcriptome of a purple cultivar ('Zijuan') and a normal green cultivar ('Yunkang 10#') of C. sinensis var. asssamica (Masters) showing different accumulation of anthocyanins and catechins through PacBio isoform sequencing (Iso-Seq). In total, 577,557 mapped full-length cDNAs were obtained, and 2,600 average-length gene isoforms were identified in both cultivars. After gene annotations and pathway predictions, we found that 98 key genes in anthocyanin biosynthesis pathways could have undergone alternative splicing (AS) events, and identified a total of 238 isoforms involved in anthocyanin biosynthesis. We verified expression of the C4H, CHS, FLS, CCOM, F3'5'H, LAR, PAL, CCR, CYP73A13, UDP75L12, UDP78A15/UFGT, UDP94P1, GL3, MYB113, ANR, ANS, F3H, 4CL1, CYP98A3/C3H, CHI, DFR genes and their AS transcripts using qRT-PCR. Correlation analysis of anthocyanin biosynthesis and gene expression results revealed that C4H1, FLS1, PAL2, CCR2, UDP75L122 and MYB113-1 are crucial AS transcripts for regulating anthocyanin biosynthesis in C. sinensis var. assamica. Our results reveal post-transcriptional regulation of anthocyanin biosynthesis in tea plants, and provide more new insights into the regulation of secondary metabolism.

Post-fermented Pu-erh tea (PFPT) has several health benefits, however, little is known about the bioactive compounds. In this study, a PFPT compound was isolated by column chromatography and identified as Teadenol A by spectroscopic data analyses, including mass spectrometry and 1D and 2D NMR spectroscopy. Teadenol A in tea leaves was biotransformed by Aspergillus niger and A. tamari at 28˝C for 14 d at concentrations ranging from 9.85˘1.17 to 12.93˘0.38 mg/g. Additionally, the compound was detected in 22 commercial PFPTs at concentrations ranging from 0.17˘0.1 to 8.15˘0.1 mg/g. Teadenol A promoted the secretion of adiponectin and inhibited the expression of protein tyrosine phosphatase-1B. Antioxidant assays (e.g., 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity, total antioxidant capacity (T-AOC), hydrogen donating ability, and superoxide anion radical scavenging capacity) revealed that Teadenol A has antioxidant properties. Therefore, Teadenol A is an important bio-active component of PFPT.

Flavonoids are rich in tea plants (Camellia sinensis), and responsible for the flavor and healthful benefits of tea beverage. The anthocyanin levels in the purple tender shoots are higher than in the general green leaves of tea plant, which provide special materials to search metabolic mechanisms of flavonoid enrichment in plant. In this work, flavonoid differences between purple and green shoots from tea cultivars ‘Zijuan’ (ZJ) and ‘Yunkang10’ (YK-10) were investigated through metabolomic analysis, and mechanisms for their difference were surveyed by comparative transcriptomic and proteomic analysis. Levels of 34 flavonoids were different between ZJ and YK-10 shoots. Among them, eight and six were marker metabolites in ZJ and YK-10, respectively. The differentially expressed genes (DEGs), proteins (DEPs) and different-level metabolites (DLMs) between ZJ and YK-10 were researched, respectively; and interactions including DEG-DLM, DEP-DLM, DEG-DEP and DEG-DEP-DLM were analyzed; the contents of 18 characteristic flavonoids in tea leaves and expressions of 34 flavonoid metabolic genes were measured to verify the omics results. Integrated above analyses, a proposed model of flavonoids biosynthesis in tea shoots were established. The differential expression of the leucoanthocyanidin reductase (LAR), anthocyanidin synthase (ANS), anthocyanidin reductase (ANR), UDPG-flavonoid glucosyltransferase (UGT) 75L12 and 94P1 at gene level, and the ANS, ANR and UGT78A15 at protein level, were closely associated with differences in flavonoids between ZJ and YK-10 shoot. Together, this study provides new information on the flavonoid accumulation mechanism in tea plant.