LC Domain-Mediated Coalescence Is Essential for Otu Enzymatic Activity to Extend Drosophila Lifespan.

In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.

L-Theanine Prolongs the Lifespan by Activating Multiple Molecular Pathways in Ultraviolet C-Exposed Caenorhabditis elegans.

L-theanine, a unique non-protein amino acid, is an important bioactive component of green tea. Previous studies have shown that L-theanine has many potent health benefits, such as anti-anxiety effects, regulation of the immune response, relaxing neural tension, and reducing oxidative damage. However, little is known concerning whether L-theanine can improve the clearance of mitochondrial DNA (mtDNA) damage in organisms. Here, we reported that L-theanine treatment increased ATP production and improved mitochondrial morphology to extend the lifespan of UVC-exposed nematodes. Mechanistic investigations showed that L-theanine treatment enhanced the removal of mtDNA damage and extended lifespan by activating autophagy, mitophagy, mitochondrial dynamics, and mitochondrial unfolded protein response (UPRmt) in UVC-exposed nematodes. In addition, L-theanine treatment also upregulated the expression of genes related to mitochondrial energy metabolism in UVC-exposed nematodes. Our study provides a theoretical basis for the possibility that tea drinking may prevent mitochondrial-related diseases.

Microbial-Transferred Metabolites of Black Tea Theaflavins by Human Gut Microbiota and Their Impact on Antioxidant Capacity.

Theaflavins (TFs), the primary bioactive components in black tea, are poorly absorbed in the small intestine. However, the biological activity of TFs does not match their low bioavailability, which suggests that the gut microbiota plays a crucial role in their biotransformation and activities. In this study, we aimed to investigate the biotransferred metabolites of TFs produced by the human gut microbiota and these metabolites' function. We profiled the microbial metabolites of TFs by in vitro anaerobic human gut microbiota fermentation using liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. A total of 17 microbial metabolites were identified, and their corresponding metabolic pathways were proposed. Moreover, full-length 16S rRNA gene sequence analysis revealed that the TFs altered the gut microbiota diversity and increased the relative abundance of specific members of the microbiota involved in the catabolism of the TFs, including Flavonifractor_plautii, Bacteroides_uniformis, Eubacterium_ramulus, etc. Notably, the antioxidant capacity of the TF sample increased after fermentation compared to the initial sample. In conclusion, the results contribute to a more comprehensive understanding of the microbial metabolites and antioxidant capacity of TFs.

Over-expression of human PP5 gene in mice induces corneal hyperplasia and leads to ocular surface squamous neoplasia.

Protein phosphatase 5 (PP5) plays an important role in cell proliferation, differentiation, and development. Transgenic PP5 mice (Tg-hPP5 mice) overexpressing human PP5 gene were successfully generated by embryo injection. Tg-hPP5 mice spontaneously developed corneal hyperplasia and ocular surface squamous neoplasia (OSSN). To investigate the mechanism behind PP5-induced corneal hyperplasia, we performed immunohistochemistry, quantitative real-time PCR, and Western Blotting analyses on the corneas of Tg-hPP5 mice at 2 months and 9 months of age. We provide the first demonstration that Tg-hPP5 mice develop corneal hyperplasia at 9-months of age demonstrated via histological analysis and in vitro co-transfection investigation. We also present data that the expression of p53 is significantly reduced while the expression of FGF-7 is significantly increased in Tg-hPP5 mice with corneal hyperplasia. Co-transfection of PP5, p53, and FGF-7-promoter-driven luciferase revealed that PP5 promotes while p53 inhibits FGF-7 expression, which indicates PP5 overexpression inhibits p53 phosphorylation, thereby reducing its tumor suppressor function and increasing FGF-7 expression. In conclusion, PP5 plays a pivotal role in corneal hyperplasia development and its downregulation is a potential target for corneal hyperplasia and OSSN treatment.

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice.

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

The roles of kinetochore of micronucleus in mitosis of HeLa cells: a live cell imaging study.

BACKGROUND: Micronuclei (MNi) are extensively used to evaluate genotoxic effects and chromosome instability. However, the roles of kinetochore of MN in mitosis have not been completely addressed. METHODS: The HeLa CENP B-GFP H2B-mCherry cells are applied to address these questions via the long-term live-cell imaging. In the cells, the kinetochore-positive micronucleus (K+MN) contained CENP B-GFP, while the kinetochore-negative micronucleus (K-MN) did not. RESULTS: K-MN-bearing cells produced much more chromosome fragments than did MN-free cells. Most of the chromosome fragments eventually merged into K-MNi. K+MN-bearing cells yielded more kinetochore-positive lagging chromosomes (K+LCs) and K+MNi than MN-free cells did. The results suggested the differences in the fates of K+MNi and K-MNi in mitosis. The cycle of K-MN → Chromosome fragment → K-MN may occur in generations of K-MN-bearing cells, while part of K+MNi might reincorporate into the main nucleus. The K+MN-bearing cells prolonged significantly duration of mitosis compared with MN-free cells. The presence of micronuclei, regardless of K-MN and K+MN, enhanced apoptosis cell death. And K+MN-bearing cells were inclined to apoptosis more than K-MN-bearing cells. The results suggested differences in fates between K-MN-bearing and K+MN-bearing cells. CONCLUSIONS: Kinetochore determined the fates of micronuclei. Kinetochore in micronuclei indirectly prolonged the duration of mitosis. Kinetochore enhanced cytotoxicity of micronuclei. Our data are direct evidences showing the roles of kinetochore of micronucleus in mitosis of HeLa cells.

Epigallocatechin-3-gallate inhibits the growth and increases the apoptosis of human thyroid carcinoma cells through suppression of EGFR/RAS/RAF/MEK/ERK signaling pathway.

BACKGROUND: Thyroid cancer is the most common type of endocrine malignancy and the incidence rate is rapidly increasing worldwide. Epigallocatechin-3-gallate (EGCG) could suppress cancer growth and induce apoptosis in many types of cancer cells. However, the mechanism of action of EGCG on the growth of human thyroid carcinoma cells has not been fully illuminated. METHODS: Cell proliferation and viability were detected by EdU and MTS assays. Cell cycle distribution was measured by flow cytometry. Migration and invasion were evaluated by scratch and transwell assays. Apoptotic levels were detected by TUNEL staining and western blotting. The protein levels of EGFR/RAS/RAF/MEK/ERK signaling pathway were detected by western blotting. The in vivo results were determined by tumor xenografts in nude mice. The in vivo proliferation, tumor microvessel density, and apoptosis were detected by immunohistochemistry. RESULTS: EGCG inhibited the proliferation, viability, and cell cycle progression in human thyroid carcinoma cells. EGCG decreased the migration and invasion, but increased the apoptosis of human thyroid carcinoma cells. EGCG reduced the protein levels of phospho (p)-epidermal growth factor receptor (EGFR), H-RAS, p-RAF, p-MEK1/2, and p-extracellular signal-regulated protein kinase 1/2 (ERK1/2) in human thyroid carcinoma cells. EGCG inhibited the growth of human thyroid carcinoma xenografts by inducing apoptosis and down-regulating angiogenesis. CONCLUSIONS: EGCG could reduce the growth and increase the apoptosis of human thyroid carcinoma cells through suppressing the EGFR/RAS/RAF/MEK/ERK signaling pathway. EGCG can be developed as an effective therapeutic agent for the treatment of thyroid cancer.

Peptide P11 suppresses the growth of human thyroid carcinoma by inhibiting the PI3K/AKT/mTOR signaling pathway.

Thyroid carcinoma is the most common endocrine malignancy, and the incidence of thyroid carcinoma is increasing in recent decades. CYYGQSKYC (P6), a nonapeptide with anti-lymphangiogenic effect by its binding to VEGFR-3 and selectively inhibiting VEGF-C binding to VEGFR-3, could suppress the migration and invasion of cancer cells. LSPPRYP (P9) acts as an effective bFGF/FGFR antagonist and inhibits the growth of the murine melanoma B16-F10 cells. In order to increase the anti-tumor effects of P6 and P9, we connected P6 with P9 via a flexible linker Gly-Gly-Gly (GGG) to reconstruct a novel peptide P11, CYYGQSKYCGGGLSPPRYP. In the present study, the mechanism of action of peptide P11 on the growth of human thyroid carcinoma cells both in vitro and in vivo was determined. Our results showed that peptide P11 inhibited the proliferation, viability, migration, and invasion of human thyroid carcinoma cells. Peptide P11 increased the apoptosis and decreased the protein levels of p-PI3K, p-AKT, and p-mTOR in human thyroid carcinoma cells. In addition, P11 could effectively inhibit the growth of human thyroid carcinoma xenograft tumors in nude mice. In conclusion, peptide P11 could inhibit the growth of human thyroid carcinoma by inhibiting the PI3K/Akt/mTOR signaling pathway. Novel peptides can be designed and applied for the treatment of various types of cancer.

MicroRNAs in the pathogenesis of type 2 diabetes: new research progress and future direction.

miRNA is a short non-coding RNA that can influence mRNA processing at the post-transcriptional level. A large number of miRNAs have been found in virtually all species so far, and these small molecules play an important role in many different physiological processes and various pathologic conditions, such as cell metabolism, cancer, autoimmune disease, and diabetes mellitus. T2D arises from a dysregulated response to the elevated glucose level in the circulation. The prevalence of T2D has increased dramatically in all age groups, and T2D in older adults is associated with more T2D complications and higher mortality. Despite the existing findings describing the pathological mechanism, T2D pathology is more complex and the pathophysiology of the disease is still not fully elucidated. In this review, we summarize the current understanding of miRNA-mediated modulation of gene expression in T2D pathogenesis, as well as related signaling pathways, and insight into the important role of miRNA in various T2D complications. Furthermore, the potential therapeutic value of miRNA for T2D patients is also discussed in detail.

Correction to: Critical Roles of microRNAs in the Pathogenesis of Fatty Liver: New Advances, Challenges, and Potential Directions.

The original version of this article unfortunately contained an error in the Acknowledgement section. The corrected text has been given below.

Critical Roles of microRNAs in the Pathogenesis of Fatty Liver: New Advances, Challenges, and Potential Directions.

In this review, we summarize the current understanding of microRNA (miRNA)-mediated modulation of the gene expression in the fatty liver as well as related signaling pathways. Because of the breadth and diversity of miRNAs, miRNAs may have a very wide variety of biological functions, and much evidence has confirmed that miRNAs are involved in the pathogenesis of fatty liver. In the pathophysiological mechanism of fatty liver, miRNAs may be regulated by upstream regulators, and have their own regulatory targets. miRNAs display important roles in the pathological mechanisms of alcoholic liver disease and non-alcoholic fatty liver disease. At present, most of the miRNA studies are focused on cell and tissue levels, and in vivo studies will help us elucidate the regulation of miRNAs and help us evaluate the potential of miRNAs as diagnostic markers and therapeutic targets. Furthermore, there is evidence that miRNAs are involved in the mechanism of natural medicine treatment in fatty liver. Given the important roles of miRNAs in the pathogenesis of fatty liver, we predict that studies of miRNAs in the pathogenesis of fatty liver will contribute to the elucidation of fatty liver pathology and the treatment of fatty liver patients.

Mineralocorticoid receptor agonists induce mouse aortic aneurysm formation and rupture in the presence of high salt.

OBJECTIVE: Elevated plasma aldosterone concentrations in patients have been linked to a spectrum of cardiovascular diseases. Mineralocorticoid receptor antagonists provide additional benefits in patients with heart failure. However, whether aldosterone and the mineralocorticoid receptor are involved in aortic aneurysm is unknown. APPROACH AND RESULTS: We report that administration of deoxycorticosterone acetate (DOCA) and salt or aldosterone and salt, but not DOCA or salt alone, to C57BL/6 male mice induced abdominal and thoracic aortic aneurysm formation and rupture in an age-dependent manner. DOCA and salt- or aldosterone and salt-induced aortic aneurysm mimicked human aortic aneurysm with respect to elastin degradation, inflammatory cell infiltration, smooth muscle cell degeneration and apoptosis, and oxidative stress. Aortic aneurysm formation did not correlate with the increase in blood pressure induced by DOCA and salt. Systemic administration of the angiotensin-converting enzyme inhibitor, enalapril, or angiotensin type 1 receptor antagonist, losartan, did not affect DOCA and salt-induced aortic aneurysm. In contrast, the mineralocorticoid receptor antagonists, spironolactone or eplerenone, significantly attenuated DOCA and salt- or aldosterone and salt-induced aortic aneurysm. CONCLUSIONS: The current study describes a novel aortic aneurysm animal model induced by mineralocorticoid receptor agonist and high salt, and reveals a previously unrecognized but potentially significant role of aldosterone in the pathogenesis of aortic aneurysm. These findings imply that mineralocorticoid receptor antagonists may be effective in the treatment of some aortic aneurysms.

Inhibition of ferroptosis underlies EGCG mediated protection against Parkinson's disease in a Drosophila model.

Ferroptosis, a new type of cell death accompanied by iron accumulation and lipid peroxidation, is implicated in the pathology of Parkinson's disease (PD), which is a prevalent neurodegenerative disorder that primarily occurred in the elderly population. Epigallocatechin-3-gallate (EGCG) is the major polyphenol in green tea with known neuroprotective effects in PD patients. But whether EGCG-mediated neuroprotection against PD involves regulation of ferroptosis has not been elucidated. In this study, we established a PD model using PINK1 mutant Drosophila. Iron accumulation, lipid peroxidation and decreased activity of GPX, were detected in the brains of PD flies. Additionally, phenotypes of PD, including behavioral defects and dopaminergic neurons loss, were ameliorated by ferroptosis inhibitor ferrostatin-1 (Fer-1). Notably, the increased iron level, lipid peroxidation and decreased GPX activity in the brains of PD flies were relieved by EGCG. We found that EGCG exerted neuroprotection mainly by restoring iron homeostasis in the PD flies. EGCG inhibited iron influx by suppressing Malvolio (Mvl) expression and simultaneously promoted the upregulation of ferritin, the intracellular iron storage protein, leading to a reduction in free iron ions. Additionally, EGCG downregulated the expression of Duox and Nox, two NADPH oxidases that produce reactive oxygen species (ROS) and increased SOD enzyme activity. Finally, modulation of intracellular iron levels or regulation of oxidative stress by genetic means exerted great influence on PD phenotypes. As such, the results demonstrated that ferroptosis has a role in the established PD model. Altogether, EGCG has therapeutic potentials for treating PD by targeting the ferroptosis pathway, providing new strategies for the prevention and treatment of PD and other neurodegenerative diseases.

(-)-Gallocatechin Gallate Mitigates Metabolic Syndrome-Associated Diabetic Nephropathy in db/db Mice.

Metabolic syndrome (MetS) significantly predisposes individuals to diabetes and is a prognostic factor for the progression of diabetic nephropathy (DN). This study aimed to evaluate the efficacy of (-)-gallocatechin gallate (GCG) in alleviating signs of MetS-associated DN in db/db mice. We administered GCG and monitored its effects on several metabolic parameters, including food and water intake, urinary output, blood glucose levels, glucose and insulin homeostasis, lipid profiles, blood pressure, and renal function biomarkers. The main findings indicated that GCG intervention led to marked improvements in these metabolic indicators and renal function, signifying its potential in managing MetS and DN. Furthermore, transcriptome analysis revealed substantial modifications in gene expression, notably the downregulation of pro-inflammatory genes such as S100a8, S100a9, Cd44, Socs3, Mmp3, Mmp9, Nlrp3, IL-1ß, Osm, Ptgs2, and Lcn2 and the upregulation of the anti-oxidative gene Gstm3. These genetic alterations suggest significant effects on pathways related to inflammation and oxidative stress. In conclusion, GCG demonstrates therapeutic efficacy for MetS-associated DN, mitigating metabolic disturbances and enhancing renal health by modulating inflammatory and oxidative responses.

Microbial-Transferred Metabolites and Improvement of Biological Activities of Green Tea Catechins by Human Gut Microbiota.

Green tea catechins (GTCs) are dietary polyphenols with broad bioactivities that undergo extensive microbial metabolism in the human gut. However, microbial-transferred metabolites and their health benefits are not fully understood. Herein, the microbial metabolism of GTCs by human fecal microbiota and dynamic alteration of the microbiota were integrally investigated via in vitro anaerobic fermentation. The results showed that the human gut microbiota exhibited a strong metabolic effect on GTCs via UHPLC-MS/MS analysis. A total of 35 microbial-transferred metabolites were identified, far more than were identified in previous studies. Among them, five metabolites, namely EGCG quinone, EGC quinone, ECG quinone, EC quinone, and mono-oxygenated EGCG, were identified for the first time in fermented GTCs with the human gut microbiota. Consequently, corresponding metabolic pathways were proposed. Notably, the antioxidant, α-amylase, and α-glucosidase inhibitory activities of the GTCs sample increased after fermentation compared to those of the initial unfermented sample. The results of the 16S rRNA gene sequence analysis showed that the GTCs significantly altered gut microbial diversity and enriched the abundancy of Eubacterium, Flavonifractor, etc., which may be further involved in the metabolisms of GTCs. Thus, these findings contribute to a better understanding of the interactions between GTCs and gut microbiota, as well as the health benefits of green tea consumption.

Large-leaf yellow tea protein derived-peptides alleviated dextran sodium sulfate-induced acute colitis and restored intestinal microbiota balance in C57BL/6 J mice.

Large-leaf yellow tea (LYT)-derived peptides (TPP) are rich in amino acids required for damage repair, such as Glu, Arg, and Pro, and can be used to alleviate acute colitis. However, its effect and mechanisms against colitis remain unclear. This study utilized TPP to intervene in dextran sodium sulfate-induced acute colitis in C57BL/6 J mice. Results confirmed that TPP ameliorated acute colitis symptoms by inhibiting pro-inflammatory cytokines, restoring gut microbiota dysbiosis, particularly by increasing the abundance of beneficial bacteria Akkermansia and Lactobacillus while declining harmful microbiota Escherichia-Shigella. Besides, TPP intervention reshaped the gut microbiota phenotype by increasing the aerobic phenotype and reducing the potentially pathogenic phenotype. Levels of short-chain fatty acids, including acetic acid, propanoic acid, isobutyric acid, and butyric acid, were also enhanced in a dose-dependent manner to help restore gut microbiota equilibrium. This study supports using TPP as a viable plant protein-derived dietary resource for alleviating inflammatory bowel disease.

Uncovering the Dynamic Alterations of Volatile Components in Sweet and Floral Aroma Black Tea during Processing.

Aroma is an indispensable factor that substantially impacts the quality assessment of black tea. This study aims to uncover the dynamic alterations in the sweet and floral aroma black tea (SFABT) throughout various manufacturing stages using a comprehensive analytical approach integrating gas chromatography electronic nose, gas chromatography-ion mobility spectrometry (GC-IMS), and gas chromatography-mass spectrometry (GC-MS). Notable alterations in volatile components were discerned during processing, predominantly during the rolling stage. A total of 59 typical volatile compounds were identified through GC-IMS, whereas 106 volatile components were recognized via GC-MS throughout the entire manufacturing process. Among them, 14 volatile compounds, such as linalool, ß-ionone, dimethyl sulfide, and 1-octen-3-ol, stood out as characteristic components responsible for SFABT with relative odor activity values exceeding one. This study serves as an invaluable theoretical platform for strategic controllable processing of superior-quality black tea.

Long-term drinking of green tea combined with exercise improves hepatic steatosis and obesity in male mice induced by high-fat diet.

Dietary habits and exercise play an important role in the well-being of human health. Currently, how long of drinking tea combined with exercise could efficiently ameliorate hepatic steatosis and obesity still needs to be investigated. Here, short-term and long-term green tea drinking combined with exercise were studied to improve hepatic steatosis and obesity in high-fat diet-induced (HF) mice. Our results showed that Yunkang 10 green tea (GT) combined with exercise (Ex) exhibited synergistic prevention effects on ameliorating hepatic steatosis and obesity. Especially, 22-week intervention with GT or Ex improved all symptoms of obesity, which indicated that long-term intervention exhibited profound preventive effects than the short term. Moreover, the combined intervention of 22 weeks inhibited the activation of NF-κB pathway and the expression of proinflammatory cytokines, which suggests that tea combined exercise may improve liver steatosis mainly by inhibiting inflammation. The key molecules for regulating lipid and glucose metabolism SCD1 were obviously downregulated, and GLU2 and PPARγ were significantly upregulated by GT and exercise in the liver of high-fat diet-induced mice. This study demonstrated that long-term intervention with GT and exercise effectively relieved hepatic steatosis and obesity complications by ameliorating hepatic inflammation, reducing lipid synthesis, and accelerating glucose transport.

Large Yellow Tea Polysaccharide Alleviates HFD-Induced Intestinal Homeostasis Dysbiosis via Modulating Gut Barrier Integrity, Immune Responses, and the Gut Microbiome.

Long-term high-fat diet (HFD) will induce dysbiosis and a disturbance of intestinal homeostasis. Large yellow tea polysaccharide (LYP) has been shown to improve obesity-associated metabolic disease via modulation of the M2 polarization. However, the contribution of LYP to intestinal barrier impairment and improvement mechanisms in obesity caused by an HFD are still not clear. In this study, we evaluated the impacts of LYP on the mucosal barrier function and microbiota composition in HFD-feeding mice. Results exhibited that dietary LYP supplement could ameliorate the physical barrier function via maintaining intestinal mucosal integrity and elevating tight-junction protein production, strengthen the chemical barrier function via up-regulating the levels of glucagon-like peptide-1 and increasing mucin-producing goblet cell numbers, and enhance the intestinal immune barrier function though suppressing immune cell subsets and cytokines toward pro-inflammatory phenotypes. Moreover, LYP reshaped the constitution and metabolism of intestinal flora by enriching probiotics that produce short-chain fatty acids. Overall, LYP might be used as a critical regulator of intestinal homeostasis to improve host health by promoting gut barrier integrity, modulating intestinal immune response, and inhibiting bowel inflammation.

Rolling forms the diversities of small molecular nonvolatile metabolite profile and consequently shapes the bacterial community structure for Keemun black tea.

The detailed dynamics of small molecular nonvolatile chemical and bacterial diversities, as well as their relationship are still unclear in the manufacturing process of Keemun black tea (KMBT). Herein, mass spectrometry-based untargeted metabolomics, Feature-based Molecular Networking (FBMN) and bacterial DNA amplicon sequencing were used to investigate the dense temporal samples of the manufacturing process. For the first time, we reveal that the pyrogallol-type catechins are oxidized asynchronously before catechol-type catechins during the black tea processing. Rolling is the key procedure for forming the small molecular nonvolatile metabolite profile (SMNMetProf), increasing the metabolite richness, and then shaping the bacterial community structure in the KMBT manufacturing process, which decreases both molecular weight and molecular polarity of the small molecular nonvolatile metabolites. The SMNMetProf of black tea is formed by the endogenous enzymatic oxidation of tea leaves, rather than bacterial fermentation.