An efficient C-glycoside production platform enabled by rationally tuning the chemoselectivity of glycosyltransferases.

Despite the broad potential applications of C-glycosides, facile synthetic methods remain scarce. Transforming glycosyltransferases with promiscuous or natural O-specific chemoselectivity to C-glycosyltransferases is challenging. Here, we employ rational directed evolution of the glycosyltransferase MiCGT to generate MiCGT-QDP and MiCGT-ATD mutants which either enhance C-glycosylation or switch to O-glycosylation, respectively. Structural analysis and computational simulations reveal that substrate binding mode govern C-/O-glycosylation selectivity. Notably, directed evolution and mechanism analysis pinpoint the crucial residues dictating the binding mode, enabling the rational design of four enzymes with superior non-inherent chemoselectivity, despite limited sequence homology. Moreover, our best mutants undergo testing with 34 substrates, demonstrating superb chemoselectivities, regioselectivities, and activities. Remarkably, three C-glycosides and an O-glycoside are produced on a gram scale, demonstrating practical utility. This work establishes a highly selective platform for diverse glycosides, and offers a practical strategy for creating various types of glycosylation platforms to access pharmaceutically and medicinally interesting products.

Comparison between bacterial bio-formulations and gibberellic acid effects on Stevia rebaudiana growth and production of steviol glycosides through regulating their encoding genes.

Stevia rebaudiana is associated with the production of calorie-free steviol glycosides (SGs) sweetener, receiving worldwide interest as a sugar substitute for people with metabolic disorders. The aim of this investigation is to show the promising role of endophytic bacterial strains isolated from Stevia rebaudiana Egy1 leaves as a biofertilizer integrated with Azospirillum brasilense ATCC 29,145 and gibberellic acid (GA3) to improve another variety of stevia (S. rebaudiana Shou-2) growth, bioactive compound production, expression of SGs involved genes, and stevioside content. Endophytic bacteria isolated from S. rebaudiana Egy1 leaves were molecularly identified and assessed in vitro for plant growth promoting (PGP) traits. Isolated strains Bacillus licheniformis SrAM2, Bacillus paralicheniformis SrAM3 and Bacillus paramycoides SrAM4 with accession numbers MT066091, MW042693 and MT066092, respectively, induced notable variations in the majority of PGP traits production. B. licheniformis SrAM2 revealed the most phytohormones and hydrogen cyanide (HCN) production, while B. paralicheniformis SrAM3 was the most in exopolysaccharides (EPS) and ammonia production 290.96 ± 10.08 mg/l and 88.92 ± 2.96 mg/ml, respectively. Treated plants significantly increased in performance, and the dual treatment T7 (B. paramycoides SrAM4 + A. brasilense) exhibited the highest improvement in shoot and root length by 200% and 146.7%, respectively. On the other hand, T11 (Bacillus cereus SrAM1 + B. licheniformis SrAM2 + B. paralicheniformis SrAM3 + B. paramycoides SrAM4 + A. brasilense + GA3) showed the most elevation in number of leaves, total soluble sugars (TSS), and up-regulation in the expression of the four genes ent-KO, UGT85C2, UGT74G1 and UGT76G1 at 2.7, 3.3, 3.4 and 3.7, respectively. In High-Performance Liquid Chromatography (HPLC) analysis, stevioside content showed a progressive increase in all tested samples but the maximum was exhibited by dual and co-inoculations at 264.37% and 289.05%, respectively. It has been concluded that the PGP endophytes associated with S. rebaudiana leaves improved growth and SGs production, implying the usability of these strains as prospective tools to improve important crop production individually or in consortium.

Biotransformation of Xanthohumol by Entomopathogenic Filamentous Fungi.

Xanthohumol (1) is a major prenylated flavonoid in hops (Humulus lupulus L.) which exhibits a broad spectrum of health-promoting and therapeutic activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer effects. However, due to its lipophilic nature, it is poorly soluble in water and barely absorbed from the gastrointestinal tract, which greatly limits its therapeutic potential. One method of increasing the solubility of active compounds is their conjugation to polar molecules, such as sugars. Sugar moiety introduced into the flavonoid molecule significantly increases polarity, which results in better water solubility and often leads to greater bioavailability. Entomopathogenic fungi are well known for their ability to catalyze O-glycosylation reactions. Therefore, we investigated the ability of selected entomopathogenic filamentous fungi to biotransform xanthohumol (1). As a result of the experiments, one aglycone (2) and five glycosides (3-7) were obtained. The obtained (2″E)-4″-hydroxyxanthohumol 4'-O-ß-D-(4‴-O-methyl)-glucopyranoside (5) has never been described in the literature so far. Interestingly, in addition to the expected glycosylation reactions, the tested fungi also catalyzed chalcone-flavanone cyclization reactions, which demonstrates chalcone isomerase-like activity, an enzyme typically found in plants. All these findings undoubtedly indicate that entomopathogenic filamentous fungi are still an underexploited pool of novel enzymes.

Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H+-ATPase.

Fusicoccin-A (FC-A) is a diterpene glucoside produced by a pathogenic fungus. Since its discovery, FC-A has been widely recognized as a phytotoxin that induces stomatal opening and leaf wilting, eventually leading to plant death. In this study, we present the first evidence that FC-A enhances plant growth by stabilizing the protein-protein interaction between plasma membrane (PM) H+-ATPase and 14-3-3 in guard cells. Long-term treatment of Arabidopsis plants with FC-A resulted in ~ 30% growth enhancement. Structurally similar fusicoccin-J (FC-J) showed a similar degree of growth-promotion activity as FC-A, whereas the more hydrophilic fusicoccin-H (FC-H) exhibited no effect on plant growth, indicating that the enhancement of plant growth observed with FC-A and FC-J involves upregulation of the protein-protein interaction between PM H+-ATPase and 14-3-3 in guard cells, which promotes stomatal opening and photosynthesis.

Improving organoleptic and antioxidant properties by inhibition of novel miRstv_7 to target key genes of steviol glycosides biosynthetic pathway in Stevia rebaudiana Bertoni.

Stevioside (5-10%) and rebaudioside-A (2-4%) are well-characterized diterpene glycosides found in leaves of Stevia rebaudiana known to have natural sweetening properties with zero glycaemic index. Stevioside has after-taste bitterness, whereas rebaudioside-A is sweet in taste. The ratio of rebaudioside-A to stevioside needs to be changed in order to increase the effectiveness and palatability of this natural sweetener. Plant-specific miRNAs play a significant role in the regulation of metabolic pathways for the biosynthesis of economically important secondary metabolites. In this study inhibition of miRNA through antisense technology was employed to antagonize the repressive action of miRstv_7 on its target mRNAs involved in the steviol glycosides (SGs) biosynthesis pathway. In transgenic plants expressing anti-miRstv_7, reduced expression level of endogenous miRstv_7 was observed than the non-transformed plants. As a result, enhanced expression of target genes, viz. KO (Kaurene oxidase), KAH (Kaurenoic acid-13-hydroxylase), and UGT76G1 (UDP-glycosyltransferase 76G1) led to a significant increase in the rebaudioside-A to stevioside ratio. Furthermore, metabolome analysis revealed a significant increase in total steviol glycosides content as well as total flavonoids content. Thus, our study can be utilized to generate more palatable varieties of Stevia with improved nutraceutical values including better organoleptic and antioxidant properties.

Gene isolation and enzymatic characterization of UDP-glycosyltransferases of terpene glucoside biosynthesis involved in linalyl-glycoside accumulation in Coffea arabica.

Terpenes, one of the secondary metabolites produced by plants, have diverse physiological functions. They are volatile compounds with physiological bioactivities (e.g., insect repellent, attracting enemies, and interacting with other plants). Terpenoids are also essential for flavor and aroma in plant-derived foods. In coffee, its aroma decides the value of coffee beans. Linalool, one of the volatile terpene compounds, is dominant in the coffee aroma. Coffee, with its good flavor and aroma, has high demand worldwide. Because terpenoids generally accumulate as glycosides in plant cells, glycosylation is catalyzed by UDP-glycosyltransferases (UGTs). Two linalyl-diglycosides have been identified: terpenoids reflected as necessary for coffee flavor. However, these UGTs and their action mechanisms are unknown in the Coffea genus. To obtain knowledge of terpene UGTs and elucidate the mechanism of terpene glycosylation in coffee, this study isolated terpene UGT genes and analyzed their functions. In silico screening based on the sequence of UGT85K11, which catalyzes terpene glycosylation from Camellia sinensis, was performed to obtain sequence information on five candidate UGT genes (CaUGT4, CaUGT5, CaUGT10, CaUGT15, and CaUGT20). These genes were isolated by reverse transcription-polymerase chain reaction, and the recombinant enzymes were produced with the Escherichia coli expression system. In functional analysis using radioisotopes, CaUGT4 showed critical activity against linalool, which had a higher affinity for its substrate than that of UGT85A84 from Osmanthus fragrans. Liquid chromatography-tandem mass spectrometry also revealed that CaUGT4 mainly produces linalyl glucoside. In this study, the first linalyl UGT was isolated from coffee. These findings can be used to elucidate the fundamental mechanism of the chemical defense in plants and apply aroma precursors for the plant-derived food industry in the future.

Comparative Proteomic Analysis Reveals the Effect Mechanisms of Glucose on the Biomass and Phenolic Glycoside Esters Synthesis Activity of Candida Parapsilosis ACCC 20221 Whole-Cell Catalyst.

A new Candida parapsilosis ACCC 20221 (C. parapsilosis ACCC 20221) whole-cell catalyst with a high phenolic glycoside esters synthesis activity and large biomass was obtained after culture with glucose. The possible mechanisms were revealed by using comparative proteomics. It found the expression of proteins involved in post-translational modification, protein turnover, and chaperone, and RNA processing and modification was upregulated, which ensured the metabolic balance and accurate translation, correct folding, and post-translational modification of proteins, thus enhancing the production of lipases in C. parapsilosis ACCC 20221 cultured with glucose. Moreover, the glycolysis pathway, tricarboxylic acid cycle, and fatty acids synthesis were enhanced, while the ß-oxidation of fatty acids was weakened in C. parapsilosis ACCC 20221 cells cultured with glucose, which led to an increase in energy generation and cell membrane synthesis; thus, large biomass was obtained. In addition, CCE40476.1 and CAC86400.1, which were likely to exert arbutin esters synthesis activity in C. parapsilosis ACCC 20221, were screened, and it was found that vinyl propionate could easily enter the catalytic pocket of CCE40476.1 and form hydrogen bonding interactions with Leu191 and Ser266.

The genetic architecture of the pepper metabolome and the biosynthesis of its signature capsianoside metabolites.

Capsicum (pepper) is among the most economically important species worldwide, and its fruits accumulate specialized metabolites with essential roles in plant environmental interaction and human health benefits as well as in conferring their unique taste. However, the genetics underlying differences in metabolite presence/absence and/or accumulation remain largely unknown. In this study, we carried out a genome-wide association study as well as generating and characterizing a novel backcross inbred line mapping population to determine the genetic architecture of the pepper metabolome. This genetic analysis provided over 1,000 metabolic quantitative trait loci (mQTL) for over 250 annotated metabolites. We identified 92 candidate genes involved in various mQTLs. Among the identified loci, we described and validated a gene cluster of eleven UDP-glycosyltransferases (UGTs) involved in monomeric capsianoside biosynthesis. We additionally constructed the gene-by-gene-based biosynthetic pathway of pepper capsianoside biosynthesis, including both core and decorative reactions. Given that one of these decorative pathways, namely the glycosylation of acyclic diterpenoid glycosides, contributes to plant resistance, these data provide new insights and breeding resources for pepper. They additionally provide a blueprint for the better understanding of the biosynthesis of species-specific natural compounds in general.

The case for sporadic cyanogenic glycoside evolution in plants.

Cyanogenic glycosides are α-hydroxynitrile glucosides present in approximately 3000 different plant species. Upon tissue disruption, cyanogenic glycosides are hydrolyzed to release toxic hydrogen cyanide as a means of chemical defense. Over 100 different cyanogenic glycosides have been reported, with structural diversity dependent on the precursor amino acid, and subsequent modifications. Cyanogenic glycosides represent a prime example of sporadic metabolite evolution, with the metabolic trait arising multiple times throughout the plant lineage as evidenced by recruitment of different enzyme families for biosynthesis. Here, we review the latest developments within cyanogenic glycoside biosynthesis, and argue possible factors driving sporadic evolution including shared intermediates and crossovers with other metabolic pathways crossovers, and metabolite multifunctionality beyond chemical defense.

Efficient and green production of flavone-5-O-glycosides by glycosyltransferases in Escherichia coli.

O-Glycosylflavonoids exhibit diverse biological activities but their low content in plants is difficult to extract and isolate, and chemical synthesis steps are cumbersome, which are harmful to the environment. Therefore, the biosynthesis of O-glycosylflavonoids represents a green and sustainable alternative strategy, with glycosyltransferases playing a crucial role in this process. However, there are few studies on flavone 5-O-glycosyltransferases, which limits the synthesis of rare flavone 5-O glycosides by microorganisms. In this study, we characterized a highly regioselectivity flavone 5-O glycosyltransferase from Panicum hallii. Site-directed mutagenesis at residue P141 switches glucosylation to xylosylation. Using a combinatorial strategy of metabolic engineering, we generated a series of Escherichia coli recombinant strains to biocatalyze glycosylation of the typical flavone apigenin. Ultimately, further optimization of transformation conditions, apigenin-5-O-glucoside and apigenin-5-O-xyloside were biosynthesized for the first time so far, and the yields were 1490 mg/L and 1210 mg/L, respectively. This study provides a biotechnological component for the biosynthesis of flavone-5-O-glycosides, and established a green and sustainable approach for the industrial production of high-value O-glycosylflavones by engineering, which lays a foundation for their further development and application in food and pharmaceutical fields.

BvCGT1-mediated differential distribution of flavonoid C-glycosides contributes to plant's response to UV-B stress.

C-glycosides are a predominant class of flavonoids that demonstrate diverse medical properties and plant physiological functions. The chemical stability, structural diversity, and differential aboveground distribution of these compounds in plants make them ideal protectants. However, little is known about the transcriptional regulatory mechanisms that play these diverse roles in plant physiology. In this study, chard was selected from 69 families for its significantly different flavonoid C-glycosides distributions between the aboveground and underground parts to investigate the role and regulatory mechanism of flavonoid C-glycosides in plants. Our results indicate that flavonoid C-glycosides are affected by various stressors, especially UV-B. Through cloning and validation of key biosynthetic genes of flavonoid C-glycosides in chard (BvCGT1), we observed significant effects induced by UV-B radiation. This finding was further confirmed by resistance testing in BvCGT1 silenced chard lines and in Arabidopsis plants with BvCGT1 overexpression. Yeast one-hybrid and dual-luciferase assays were employed to determine the underlying regulatory mechanisms of BvCGT1 in withstanding UV-B stress. These results indicate a potential regulatory role of BvDof8 and BvDof13 in modulating flavonoid C-glycosides content, through their influence on BvCGT1. In conclusion, we have effectively demonstrated the regulation of BvCGT1 by BvDof8 and BvDof13, highlighting their crucial role in plant adaptation to UV-B radiation. Additionally, we have outlined a comprehensive transcriptional regulatory network involving BvDof8 and BvDof13 in response to UV-B radiation.

Non-target and target quantitative metabolomics with quantitative aroma evaluation reveal the influence mechanism of withering light quality on tea aroma and volatile metabolites evolution.

Withering is the first and key process that influences tea quality, with light quality being a key regulatory factor. However, effects of withering light quality (WLQ) on transformation and formation pathways of tea aroma and volatile metabolites (VMs) remain unclear. In the present study, four WLQs were set up to investigate their effects on tea aroma and VMs. The results showed that blue and red light reduced the grassy aroma and improved the floral and fruity aroma of tea. Based on GC-MS/MS, 83 VMs were detected. Through VIP, significant differences, and OAV analysis, 13 key differential VMs were screened to characterize the differential impacts of WLQ on tea aroma. Further analysis of the evolution and metabolic pathways revealed that glycoside metabolism was the key pathway regulating tea aroma through WLQ. Blue light withering significantly enhanced glycosides hydrolysis and amino acids deamination, which was beneficial for the enrichment of floral and fruity VMs, such as geraniol, citral, methyl salicylate, 2-methyl-butanal, and benzeneacetaldehyde, as well as the transformation of grassy VMs, such as octanal, naphthalene, and cis-3-hexenyl isovalerate, resulting in the formation of tea floral and fruity aroma. The results provide theoretical basis and technical support for the targeted processing of high-quality tea.

Genes to specialized metabolites: accumulation of scopoletin, umbelliferone and their glycosides in natural populations of Arabidopsis thaliana.

BACKGROUND: Scopoletin and umbelliferone belong to coumarins, which are plant specialized metabolites with potent and wide biological activities, the accumulation of which is induced by various environmental stresses. Coumarins have been detected in various plant species, including medicinal plants and the model organism Arabidopsis thaliana. In recent years, key role of coumarins in maintaining iron (Fe) homeostasis in plants has been demonstrated, as well as their significant impact on the rhizosphere microbiome through exudates secreted into the soil environment. Several mechanisms underlying these processes require clarification. Previously, we demonstrated that Arabidopsis is an excellent model for studying genetic variation and molecular basis of coumarin accumulation in plants. RESULTS: Here, through targeted metabolic profiling and gene expression analysis, the gene-metabolite network of scopoletin and umbelliferone accumulation was examined in more detail in selected Arabidopsis accessions (Col-0, Est-1, Tsu-1) undergoing different culture conditions and characterized by variation in coumarin content. The highest accumulation of coumarins was detected in roots grown in vitro liquid culture. The expression of 10 phenylpropanoid genes (4CL1, 4CL2, 4CL3, CCoAOMT1, C3'H, HCT, F6'H1, F6'H2,CCR1 and CCR2) was assessed by qPCR in three genetic backgrounds, cultured in vitro and in soil, and in two types of tissues (leaves and roots). We not only detected the expected variability in gene expression and coumarin accumulation among Arabidopsis accessions, but also found interesting polymorphisms in the coding sequences of the selected genes through in silico analysis and resequencing. CONCLUSIONS: To the best of our knowledge, this is the first study comparing accumulation of simple coumarins and expression of phenylpropanoid-related genes in Arabidopsis accessions grown in soil and in liquid cultures. The large variations we detected in the content of coumarins and gene expression are genetically determined, but also tissue and culture dependent. It is particularly important considering that growing plants in liquid media is a widely used technology that provides a large amount of root tissue suitable for metabolomics. Research on differential accumulation of coumarins and related gene expression will be useful in future studies aimed at better understanding the physiological role of coumarins in roots and the surrounding environments.

The fusicoccin story revisited.

Fusicoccin (FC) is one of the most studied fungal metabolites to date. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as a high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase-14-3-3 receptor complex has taken center stage in explaining all FC-induced physiological effects. However, a more critical review shows that this is not evident for a number of FC-induced effects. This review challenges the notion that all FC-affected processes start with the binding to and activation of the plasma membrane ATPase, and raises the question of whether other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule. In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile making a renewed attempt with modern improved technology to answer this question; the answer might upgrade FC or its structural analogue(s) to the classification of plant hormone.

An efficient flavonoid glycosyltransferase NjUGT73B1 from Nardostachys jatamansi of alpine Himalayas discovered by structure-based protein clustering.

Tilianin and linarin, two rare glycosylated flavonoids in the aromatic endangered medicinal plant Nardostachys jatamansi (D.on)DC., play an important role in the fields of medicine, cosmetics, food and dye industries. However, there remains a lack of comprehensive understanding regarding their biosynthetic pathway. In this study, the phytochemical investigation of N. jatamansi resulted in the isolation of linarin. With help of AlphaFold2 to cluster the entire glycosyltransferase family based on predicted structure similarities, we successfully identified a flavonoid glycosyltransferase NjUGT73B1, which could efficiently catalyze the glucosylation of acacetin at 7-OH to produce tilianin, also the key precursor in the biosynthesis of linarin. Additionally, NjUGT73B1 displayed a high degree of substrate promiscuity, enabling glucosylation at 7-OH of many flavonoids. Molecular modeling and site-directed mutagenesis revealed that H19, H21, H370, F126, and F127 play the crucial roles in the glycosylation ability of NjUGT73B1. Notably, comparation with the wild NjUGT73B1, mutant H19K led to a 50% increase in the activity of producing tilianin from acacetin.

Construction of lignan glycosides biosynthetic network in Escherichia coli using mutltienzyme modules.

BACKGROUND: Due to the complexity of the metabolic pathway network of active ingredients, precise targeted synthesis of any active ingredient on a synthetic network is a huge challenge. Based on a complete analysis of the active ingredient pathway in a species, this goal can be achieved by elucidating the functional differences of each enzyme in the pathway and achieving this goal through different combinations. Lignans are a class of phytoestrogens that are present abundantly in plants and play a role in various physiological activities of plants due to their structural diversity. In addition, lignans offer various medicinal benefits to humans. Despite their value, the low concentration of lignans in plants limits their extraction and utilization. Recently, synthetic biology approaches have been explored for lignan production, but achieving the synthesis of most lignans, especially the more valuable lignan glycosides, across the entire synthetic network remains incomplete. RESULTS: By evaluating various gene construction methods and sequences, we determined that the pCDF-Duet-Prx02-PsVAO gene construction was the most effective for the production of (+)-pinoresinol, yielding up to 698.9 mg/L after shake-flask fermentation. Based on the stable production of (+)-pinoresinol, we synthesized downstream metabolites in vivo. By comparing different fermentation methods, including "one-cell, one-pot" and "multicellular one-pot", we determined that the "multicellular one-pot" method was more effective for producing (+)-lariciresinol, (-)-secoisolariciresinol, (-)-matairesinol, and their glycoside products. The "multicellular one-pot" fermentation yielded 434.08 mg/L of (+)-lariciresinol, 96.81 mg/L of (-)-secoisolariciresinol, and 45.14 mg/L of (-)-matairesinol. Subsequently, ultilizing the strict substrate recognition pecificities of UDP-glycosyltransferase (UGT) incorporating the native uridine diphosphate glucose (UDPG) Module for in vivo synthesis of glycoside products resulted in the following yields: (+)-pinoresinol glucoside: 1.71 mg/L, (+)-lariciresinol-4-O-D-glucopyranoside: 1.3 mg/L, (+)-lariciresinol-4'-O-D-glucopyranoside: 836 µg/L, (-)-secoisolariciresinol monoglucoside: 103.77 µg/L, (-)-matairesinol-4-O-D-glucopyranoside: 86.79 µg/L, and (-)-matairesinol-4'-O-D-glucopyranoside: 74.5 µg/L. CONCLUSIONS: By using various construction and fermentation methods, we successfully synthesized 10 products of the lignan pathway in Isatis indigotica Fort in Escherichia coli, with eugenol as substrate. Additionally, we obtained a diverse range of lignan products by combining different modules, setting a foundation for future high-yield lignan production.

Metabolomics study of dietary Pleurotus eryngii ß-type glycosidic polysaccharide on colitis induced by dextran sodium sulfate in mice - Exploration for the potential metabolic indicators in urine and serum.

Pleurotus eryngii, an edible mushroom recognized for its potent polysaccharides, demonstrates significant regulatory effects on metabolic processes. ß-glucan (WPEP) derived from P. eryngii has been noted for its therapeutic potential, exhibiting notable benefits in alleviating colonic inflammation and restructuring gut microbiota in mice treated with dextran sodium sulfate (DSS). This study focuses on utilizing DSS-induced colitis mice to explore the efficacy and underlying mechanisms of WPEP in ameliorating colitis, employing a metabolomics approach analyzing urine and serum. The findings reveal that WPEP administration effectively regulates metabolic imbalances in DSS mice, impacting purine metabolism, pentose and glucuronic acid interconversion, amino acid metabolism, primary bile acid biosynthesis, citric acid cycle, and lipid metabolism. Furthermore, WPEP demonstrates a capacity to modulate colitis by regulating diverse metabolic pathways, consequently influencing intestinal barrier integrity, motility, inflammation, oxidative stress, and immunity. These insights suggest that WPEP is a promising food component for managing inflammatory bowel diseases.

Untargeted metabolomic profiling of Prinsepia utilis Royle leaves by Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight-Mass Spectrometry.

Prinsepia utilis Royle, native to the Himalayas, is esteemed in Chinese and Indian folk medicine for its diverse medicinal benefits, targeting arthritis, pain relief, bone disorders, and joint discomfort. This study examined the 25% aqueous methanol extract of P. utilis leaves using UPLC-Q-TOF-MS/MS, identifying 78 metabolites, 76 of which were reported for the first time in P. utilis. These included 64 phenolics represented by 56 flavonoids, 5 phenolic acids, 3 phenolic glycosides, 4 terpenoids, 2 lignan glycosides, and 8 other compounds, expanding the knowledge of its chemical composition. These findings lay a foundation for further research, providing insights into potential bioactive compounds and opening avenues for applications in natural product drug discovery, traditional medicine, and nutraceutical development, leveraging the plant's established traditional uses.

Enzymatic Synthesis of Novel Terpenoid Glycoside Derivatives Decorated with N-Acetylglucosamine Catalyzed by UGT74AC1.

The addition of the O-linked N-acetylglucosamine (O-GlcNAc) is a significant modification for active molecules, such as proteins, carbohydrates, and natural products. However, the synthesis of terpenoid glycoside derivatives decorated with GlcNAc remains a challenging task due to the absence of glycosyltransferases, key enzymes for catalyzing the transfer of GlcNAc to terpenoids. In this study, we demonstrated that the enzyme mutant UGT74AC1T79Y/L48M/R28H/L109I/S15A/M76L/H47R efficiently transferred GlcNAc from uridine diphosphate (UDP)-GlcNAc to a variety of terpenoids. This powerful enzyme was employed to synthesize GlcNAc-decorated derivatives of terpenoids, including mogrol, steviol, andrographolide, protopanaxadiol, glycyrrhetinic acid, ursolic acid, and betulinic acid for the first time. To unravel the mechanism of UDP-GlcNAc recognition, we determined the X-ray crystal structure of the inactivated mutant UGT74AC1His18A/Asp111A in complex with UDP-GlcNAc at a resolution of 1.66 Å. Through molecular dynamic simulation and activity analysis, we revealed the molecular mechanism and catalytically important amino acids directly involved in the recognition of UDP-GlcNAc. Overall, this study not only provided a potent biocatalyst capable of glycodiversifying natural products but also elucidated the structural basis for UDP-GlcNAc recognition by glycosyltransferases.

The bioavailability, absorption, metabolism, and regulation of glucolipid metabolism disorders by quercetin and its important glycosides: A review.

Quercetin and its glycosides (QG), vitally natural flavonoid, have been popular for health benefits. However, the absorption and metabolism affect their bioavailability, and the metabolic transformation alters their biological activities. This review systematically summarizes the bioavailability and pathways for the absorption and metabolism of quercetin/QG in vivo and in vitro, the biological activities and mechanism of quercetin/QG and their metabolites in treating glucolipid metabolism are discussed. After oral administration, quercetin/QG are mainly absorbed by the intestine, undergo phase II metabolism in the small intestine and liver to form conjugates and are metabolized into small phenolic acids by intestinal microbiota. Quercetin/QG and their metabolites exert beneficial effects on regulating glucolipid metabolism disorders, including improving insulin resistance, inhibiting lipogenesis, enhancing thermogenesis, modulating intestinal microbiota, relieving oxidative stress, and attenuating inflammation. This review enhances understanding of the mechanism of quercetin/QG regulate glucolipid metabolism and provides scientific support for the development of functional foods.