Production Of Glycosides Research Articles

Engineering of Cyclodextrin Glucosyltransferase from Paenibacillus macerans for Improved Regioselectivity and Product Specificity Toward Hydroxyflavone Glycosylation

The regioselective glycosylation and product specificity of hydroxyflavonoid compounds profoundly influences their biological activity and stability, offering significant therapeutic potential. However, most cyclodextrin glucosyltransferases (CGTases) inherently lack regioselectivity and product specificity for flavone glycosylation. Herein, a CGTase from Paenibacillus macerans was engineered for enhanced glycosylation regioselectivity and product specificity by combining molecular docking analysis and saturation mutagenesis strategies. K232L (favoring 4′-and 6-hydroxyflavones) and K232V (favoring 7-hydroxyflavone) were identified with distinct preferences. In addition, H233Y (preferring for 4′-hydroxyflavones), H233T (preferring for 6′-hydroxyflavones), and H233K (preferring for 7′-hydroxyflavones) also demonstrated distinct regioselectivity. These variants further exhibited enhanced hydrolytic activity, enabling the efficient production of short sugar-chain glycosides. Molecular dynamics (MDs) simulations revealed that the variants adopted optimized catalytic conformations with increased loop region flexibility near the binding pocket, enhancing substrate accessibility. These findings underscore the pivotal roles of K232 and H233 in broadening the substrate scope of CGTase and offer valuable guidance for enzyme engineering targeting regioselective glycosylation.

Discovery and mechanistic exploration of promiscuous xylosyltransferase based on protein engineering.

Glycosylation is an effective means to alter the structure and properties of plant compounds, influencing the pharmacological activity of natural products (NPs) to obtain highly active NPs. In nature, glucosides are the most widely distributed, while other glycosides such as xylosides are less common and present in lower quantities. This is due to the scarcity of xylosyltransferases with substrate promiscuity in nature, and the modification of their catalytic function is also quite challenging. In this study, we first performed a phylogenetic analysis of reported UDP-glycosyltransferases (UGTs) of plant and microbiological origin and identified a unique motif region from the UGTs of the Bacillus genus, which may be responsible for the broad sugar donor catalytic activity of the UGTs in the Bacillus genus. Then, utilizing protein engineering techniques, we have evolved a xylosyltransferase M3-2, which exhibited high substrate promiscuity, sugar donor promiscuity, and site selectivity, enabling the synthesis of a variety of O-glycosides. In addition, another mutant M3-1 has been engineered to alter the sugar donor specificity of the UGT, enabling the switch from UDP-Glc donor to UDP-Xyl. The improved enzymatic activity is likely attributed to stable hydrophobic interactions and hydrogen bonding interactions between the enzyme and the substrate. In order to synthesize xylosylated products more economically and efficiently, an in vitro synthetic pathway that utilizes NPs and inexpensive glucuronic acid as starting materials was designed. Through this pathway, we successfully synthesized a variety of unnatural xylosylated products belonging to O-glycosides, one of which 10a possesses excellent anti-inflammatory activity. We anticipate that this work will contribute to the future discovery and industrial production of unnatural glycosides.

Total biosynthesis of cotylenin diterpene glycosides as 14-3-3 protein-protein interaction stabilizers.

Cotylenins (CNs) are bioactive fungal diterpene glycosides that exhibit stabilizing activity on 14-3-3 protein-protein interactions (PPIs), which has significant therapeutic potential. Although CNs were isolated as early as 1970, their biosynthetic pathway has remained unclear, and their limited supply has hindered further research. Here, we report the identification of the biosynthetic gene cluster cty and elucidation of the biosynthetic pathway of CNs. Our investigation reveals the roles of glycosyltransferase, methyltransferase, and prenyltransferase enzymes in the assembly and modification of the saccharide moiety, as well as the multifunctional oxidation activity of the P450 enzyme CtyA. We leveraged this knowledge to achieve the total biosynthesis of not only key intermediates such as CN-C, E, F, and I, but also a novel, unnatural CN derivative using heterologous expression. This showcases the potential of pathway enzymes as catalytic tools to expand the structural diversity of diterpene glycosides. Furthermore, the stabilization effects of pathway intermediates on 14-3-3 PPIs underscore the importance of saccharide modifications in bioactivity. These findings provide a foundation for future rational synthesis of cotylenin A and other structurally diverse derivatives, broadening the scope of diterpene glycoside production.

Green synthesis of polymeric surfactants from recycling of plastic waste for applications on steel protection in the petroleum industry

ABSTRACT Green synthesis of four novel nonionic polymeric surfactants: from the recycled product of poly (ethylene terephthalate) plastic waste for application in corrosion protection of carbon steel. In this respect, PET waste was subjected to aminolysis and glycolysis via a reaction with Trimethylenediamine (TEDA) and Tri ethylene glycol (TEG) in the presence of suitable catalysts. The prepared materials were characterized by FT-IR, 1HNMR, and elemental analysis. It was evaluated as a corrosion inhibitor for steel (type-X60) used in the petroleum industry in the marine environment. Chemical, analytical, and electrochemical techniques were used for the evaluation of the corrosion inhibition efficiency of the prepared polymeric surfactants. The effect of concentration and temperature was studied. The maximum inhibition efficiency (IE) of 96% at an inhibitor concentration of 200 ppm and a temperature of 303 K. The glycoside and amide products were compared, the efficiency of amide products (96%) was higher than the efficiency of glycoside products (91%). According to potentiodynamic polarization data, the prepared surfactant boosts polarization resistance and inhibition performance by adsorbing on the metal/electrolyte interface. The surface morphology of steel was examined using SEM. A protective coating of inhibitor molecules forms on the steel surface.

Cooperative Catalysis in Stereoselective O- and N-Glycosylations with Glycosyl Trichloroacetimidates Mediated by Singly Protonated Phenanthrolinium Salt and Trichloroacetamide.

The development of small-molecule catalysts that can effectively activate both reacting partners simultaneously represents a pivotal pursuit in advancing the field of stereoselective glycosylation reactions. We report herein the development of the singly protonated form of readily available phenanthroline as an effective cooperative catalyst that facilitates the coupling of a wide variety of aliphatic alcohols, phenols, and aromatic amines with α-glycosyl trichloroacetimidate donors. The glycosylation reaction likely proceeds via an SN2-like mechanism, generating β-selective glycoside products. The developed protocol provides access to O- and N-glycosides in good yields with excellent levels of β-selectivity and enables late-stage functionalization of O- and N-glycosides via cross-coupling reactions. Importantly, this method exhibits excellent β-selectivity that is unattainable through a C2-O-acyl neighboring group participation strategy, especially in the case of glycosyl donors already containing a C2 heteroatom or sugar unit. Kinetic studies demonstrate that the byproduct trichloroacetamide group plays a previously undiscovered pivotal role in influencing the reactivity and selectivity of the reaction. A proposed mechanism involving simultaneous activation of the glycosyl donor and acceptor by the singly protonated phenanthrolinium salt catalyst with the assistance of the trichloroacetamide group is supported by kinetic analysis and preliminary computational studies. This cooperative catalysis process involves four consecutive hydrogen bond interactions. The first interaction occurs between the carbonyl oxygen of the trichloroacetamide group and the hydroxyl group of alcohol nucleophile (C═O···HO). The second involves the trichloroacetamide-NH2 forming a hydrogen bond with the nitrogen atom of the phenanthroline (NH···N). The third involves the donor trichloroacetimidate (═NH) engaging in a hydrogen bond interaction with the phenanthrolinium-NH (NH···N═H). Lastly, the protonated trichloroacetimidate-NH2 forms a hydrogen bond with the fluorine atom of the tetrafluoroborate ion.

High-level sustainable production of complex phenylethanoid glycosides from glucose through engineered yeast cell factories

Complex phenylethanoid glycosides (PhGs), such as verbascoside and echinacoside, comprise a vital family of natural products with renowned nutraceutical and pharmaceutical significance. Despite the high demand for these compounds across various industries, traditional plant extraction methods yield insufficient quantities, highlighting the need for alternative production methods. Therefore, this paper reports the successful engineering of Saccharomyces cerevisiae cell factories for the efficient production of complex PhGs from glucose. First, key pathway enzymes with enhanced catalytic activities in yeast were primarily screened from various verbascoside-producing plants. Second, intermediate osmanthuside B was produced with a titer of 21.5 ± 1.5 mg/L from glucose by overexpressing several enzymes, including glucosyltransferase RrUGT33 from Rhdiola rosea, acyltransferase SiAT, and 1,3-rhamnosyltransferase SiRT from Sesamum indicum, UDP-L-rhamnose synthase AtRHM2, and 4-coumarate: coenzyme A ligase At4CL1 from Arabidopsis thaliana in a p-coumaric acid-overproducing S. cerevisiae strain. Third, the production of osmanthuside B was further enhanced by increasing the copy number of SiAT and AtRHM2 in genome and diverting L-tyrosine into tyrosol biosynthesis by introducing an aromatic aldehyde synthase PcAAS from Petroselinum crispum with a titer of 320.6 ± 59.3 mg/L. Fourth, the biosynthesis of verbascoside was accomplished by integrating genes CYP98A20 and AtCPR1 into the chromosomes of the osmanthuside B-producing strain, the titer reached 184.7 ± 5.7 mg/L. Furthermore, the overexpression of the glucose-6-phosphate dehydrogenase (ZWF1) led to significantly enhanced verbascoside production to 230.6 ± 11.8 mg/L. The strains were further engineered to produce echinacoside with a titer of 184.2 ± 11.2 mg/L. Finally, the fed-batch fermentation in a 5-L bioreactor yielded 4497.9 ± 285.2 mg/L of verbascoside or 3617.4 ± 117.4 mg/L of echinacoside. This work provides a crucial foundation for the green, industrial, and sustainable production of verbascoside and echinacoside and sets an initial point for the microbial production of other complex PhG derivatives.

Effective and eco-friendly botanical insecticidal agents against Spodoptera frugiperda (Noctuidae: Lepidoptera) using the essential oil of Stevia rebaudiana

Using synthetic pesticides is the main strategy for controlling pests. However, these compounds have caused worry because of their harmful effects on health and their diminishing efficacy against pests that have developed resistance. Consequently, there is a growing interest in adopting more sustainable control methods. Stevia rebaudiana is a valuable medicinal plant used in the food industry for the production of steviol glycosides, a type of natural sweetener. An EO that could be useful for creating innovative insecticides may come from the industrially used plant biomass. In this study, gas chromatography-mass spectrometry (GC-MS) was used to analyse the chemical composition of S. rebaudiana leaves. Sesquiterpenes, or caryophyllene oxide (20.7 %), spathulenol (14.9 %), and (E)-nerolidol (8.0 %), and diterpenes, or phytol (9.2 %), made up the majority of the EO composition. The efficacy of the EO major constituents, namely Phytol, (E)-nerolidol, Spathulenol, and Caryophyllene oxide, was also tested against S. frugiperda. Phytol was the most effective LC50 = 14.38 mg/L, followed by (E)-nerolidol LC50 = 15.88 mg/L, Spathulenol LC50 = 18.42 mg/L, and Caryophyllene oxide LC50 = 23.41 mg/L. Furthermore, some of the biological and histological features of the extracts were also studied in a lab setting. Overall, Stevia rebaudiana (Bertoni) should be given more consideration in the development of environmentally safe and efficient pesticides.

Immobilization β-glucosidase from Dictyoglomus thermophilum on UiO-66-NH2: An efficient catalyst for enzymatic synthesis of kinsenoside via reverse hydrolysis reaction

Kinsenoside is a rare and valuable glycoside with extensive bioactivities. However, the enzymatic synthesis of kinsenoside has been a challenging task due to the limited enzyme toolbox and unsatisfactory yield. Herein, the β-glucosidase from Dictyoglomus thermophilum (DtBGL) was heterologously expressed, purified and enzymatically characterized. The purified DtBGL was successfully immobilized on the metal-organic frameworks of UiO-66-NH2. The DtBGL@UiO-66-NH2 was fully characterized using SEM, XRD, TGA and FTIR. The studies on enzymatic properties demonstrated that DtBGL@UiO-66-NH2 exhibited increased catalytic activity and stability compared to the free DtBGL. Particularly, DtBGL@UiO-66-NH2 could catalyze the synthesis of kinsenoside via the reverse hydrolysis reaction and the kinsenoside yield was 34.12 % under the optimized catalytic system, which was 1.9-fold higher compared with the free DtBGL. Moreover, DtBGL@UiO-66-NH2 displayed good reusability with a kinsenoside yield of 27.02 % after reuse for 3 times. The present work not only identifies and characterizes a highly active β-glucosidase with reverse hydrolysis activity, but also proposes the immobilized enzyme as an effective catalyst for the industrial production of glycosides.

Enzymatic Glycosylation of 4'-Hydroxychalcones: Expanding the Scope of Nature's Catalytic Potential.

Chalcones, including 4'-hydroxychalcones, have garnered significant attention in the area of drug discovery due to their diverse pharmacological properties, such as anti-inflammatory, antioxidative, and anticancer effects. However, their low water solubility and bioavailability limit their efficacy in vivo. Glycosylation presents a promising approach to enhance the water solubility, stability, and metabolic properties of chalcones. This study investigates the enzymatic glycosylation of eight chemically synthesized 4'-hydroxychalcones using a diverse set of sugar glucosyltransferases from bacterial, plant, and fungal sources, alongside Glycine max sucrose synthase (GmSuSy) in a cascade reaction. Among the tested enzymes, five exhibited a remarkable versatility for glycoside production, and for large-scale biotransformation, flavonoid 7-O-glucosyltransferase Sbaic7OGT from Scutellaria baicalensis was selected as the most effective. As a result of the experiments conducted, eight trans-chalcone glycosides were obtained. During the purification of the reaction products, we also observed the isomerization of the products by simple sunlight exposure, which resulted in eight additional cis-chalcone glycosides. This study highlights the novel use of a cascade reaction involving Glycine max sucrose synthase (GmSuSy) for the efficient glycosylation of trans-4'-hydroxychalcones, alongside the unexpected discovery of cis-chalcone glycosides during the purification process.

Enhanced catalytic activity and reusability of sucrose phosphorylase@magnetic nanoparticles by surface-coating amorphous ZIF-67

Sucrose phosphorylase (SPase) is a highly efficient glycosyltransferase which has a wide range of substrate specificity and excellent application prospects in the cosmetics, food, and medicine fields. The application of free SPase is limited due to its high cost, poor stability and poor reusability. Immobilization of enzymes can solve these problems. In this study, SPase was firstly immobilized by magnetic nanoparticles (MNPs) and surface-coating amorphous ZIF-67 for enhancing catalytical activity and reusability. ZIF-67@SPase@MNPs with MNPs core and ZIF-67 shell was characterized by TEM, SEM, XRD, FT-IR and VSM. Compared with free SPase, the catalytic activity of ZIF-67@SPase@MNPs increased by 30 %. Coated with amorphous ZIF-67, the immobilized enzyme retained 70 % relative activity after 12 cycles and 80 % relative activity after 15 days of storage. In addition, ZIF-67@SPase@MNPs had strong magnetic properties and the saturation magnetization was 52.07 emu/g. Surface-coating amorphous ZIF-67 on SPase@MNPs is a promising method for immobilizing enzymes, which can improve catalytic activity and reusability, therefore showing great application potential in biocatalysts and product separation. It has a good application prospect in the production of high viscosity glycosides.

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.

Naringenin chalcone carbon double-bond reductases mediate dihydrochalcone biosynthesis in apple leaves.

Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85% to 95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g. in biofermentation and biopharming.

Production of neohesperidin from hesperetin by an engineered strain of Escherichia coli

Neohesperidin is a flavonoid glycoside widely used in the food and pharmaceutical industries. The current production of neohesperidin mainly relies on extraction from plants. Microbial fermentation demonstrates a promising prospect as an environmentally friendly, efficient, and economical method. In this study, we designed and constructed the biosynthetic pathway of neohesperidin in an Escherichia coli strain by introducing the glycosyltransferase UGT73B2 from Arabidopsis thaliana, rhamnose synthase VvRHM-NRS from Vitis vinifera, and rhamnose transferase Cm1,2RhaT from Citrus maxima. After optimization of the module and the uridine diphosphate (UDP)-glucose synthetic pathway, the engineered strain produced 4.64 g/L neohesperidin in a 5 L bioreactor, and the molar conversion rate of hesperetin was 45.8%. This has been the highest titer reported to date for the biosynthesis of neohesperidin in microorganisms. This study lays a foundation for the construction and application of strains with high yields of neohesperidin and provides a potential choice for the microbial production of other flavonoid glycosides.

Genome-wide identification of the phenylalanine ammonia-lyase gene from Epimedium Pubescens Maxim. (Berberidaceae): novel insight into the evolution of the PAL gene family

BackgroundPhenylalanine ammonia-lyase (PAL) serves as a key gateway enzyme, bridging primary metabolism and the phenylpropanoid pathway, and thus playing an indispensable role in flavonoid, anthocyanin and lignin biosynthesis. PAL gene families have been extensively studied across species using public genomes. However, a comprehensive exploration of PAL genes in Epimedium species, especially those involved in prenylated flavonol glycoside, anthocyanin, or lignin biosynthesis, is still lacking. Moreover, an in-depth investigation into PAL gene family evolution is warranted.ResultsSeven PAL genes (EpPAL1-EpPAL7) were identified. EpPAL2 and EpPAL3 exhibit low sequence identity to other EpPALs (ranging from 61.09 to 64.38%) and contain two unique introns, indicating distinct evolutionary origins. They evolve at a rate ~ 10 to ~ 54 times slower compared to EpPAL1 and EpPAL4-7, suggesting strong purifying selection. EpPAL1 evolved independently and is another ancestral gene. EpPAL1 formed EpPAL4 through segmental duplication, which lead to EpPAL5 and EpPAL6 through tandem duplications, and EpPAL7 through transposed duplication, shaping modern EpPALs. Correlation analysis suggests EpPAL1, EpPAL2 and EpPAL3 play important roles in prenylated flavonol glycosides biosynthesis, with EpPAL2 and EpPAL3 strongly correlated with both Epimedin C and total prenylated flavonol glycosides. EpPAL1, EpPAL2 and EpPAL3 may play a role in anthocyanin biosynthesis in leaves. EpPAL2, EpPAL3, EpPAL6, and EpPAL7 might be engaged in anthocyanin production in petals, and EpPAL2 and EpPAL3 might also contribute to anthocyanin synthesis in sepals. Further experiments are needed to confirm these hypotheses. Novel insights into the evolution of PAL gene family suggest that it might have evolved from a monophyletic group in bryophytes to large-scale sequence differentiation in gymnosperms, basal angiosperms, and Magnoliidae. Ancestral gene duplications and vertical inheritance from gymnosperms to angiosperms likely occurred during PAL evolution. Most early-diverging eudicotyledons and monocotyledons have distinct histories, while modern angiosperm PAL gene families share similar patterns and lack distant gene types.ConclusionsEpPAL2 and EpPAL3 may play crucial roles in biosynthesis of prenylated flavonol glycosides and anthocyanins in leaves and flowers. This study provides novel insights into PAL gene family evolution. The findings on PAL genes in E. pubescens will aid in synthetic biology research on prenylated flavonol glycosides production.

Ecological factors impacting genetic characteristics and metabolite accumulations of Gastrodia elata

ObjectiveThe investigation of the correlation between ecological factors and the genetic characteristics or metabolites of plants offers valuable insights into the regional causes of genetic and metabolic diversity. Here, Gastrodia elata, a medicinal plant, is employed as a model to explore the environmental factors that influence its genetic characteristics and metabolic accumulations. MethodsA total of 23 G. elata populations from six cultispecies and 11 cultivated regions were selected based on the predictions of the global geographic information system. The genetic characteristics of these populations were evaluated using highly polymorphic simple sequence repeat markers. Additionally, the metabolic accumulations and antioxidant capacity of mature tubers were measured employing colorimetry and high performance liquid chromatography (HPLC). Ecological data of each region were obtained from the WorldClim-global climate database and harmonized world soil database. To assess the influence of ecological factors on the genetic characteristics and metabolic profiles of G. elata, Pearson’s correlation analysis was conducted. ResultsGenetic variation among G. elata populations exceeded that within populations. Genetic diverisity, distance and structure manifested regional and species-specific patterns. Metabolic profiling and antioxidant capacity exhibited regional variations. Notably, the Lueyang region demonstrated that a content range of total polysaccharide, total protein, and phenolic glycosides was 9.34 %−189.67 % higher than the average. Similarly, in the Hubei region, total phenolic content, p-hydroxybenzyl alcohol content, and antioxidant indicators were observed to be higher than the average levels, by 106.57 %, 136.47 % and 12.50 %−91.14 %, respectively. Furthermore, ecological factors had a significant comprehensive impact on G. elata genetic characteristics (r > 0.256 and P<0.05). Multivariate metabolite accumulations in G. elata were influenced by dominant ecological factors. Temperature notably impacted the accumulation of total protein (|r| > 0.528 and P<0.05). Moisture, encompassing precipitation and soil content, significantly affected the production of phenolic glycosides (|r| > 0.503 and P<0.05). ConclusionThe genetic characteristics of G. elata manifested regional and species-specific patterns, with the metabolic accumulations and antioxidant capacity of mature tubers exhibited regional variations. Specifically, multivariate ecological factors comprehensively influenced genetic characteristics. Temperature and moisture played pivotal roles in regulating the accumulations of proteins and phenolic glycosides, respectively. These findings underscore the significant impact of ecological factors on the shaping of G. elata, highlighting their crucial role in enhancing the quality of Chinese medicinal materials.

Advances in glycosyltransferase-mediated glycodiversification of small molecules.

Currently, numerous glycosides have been synthesized and used in clinical applications, neutraceuticals, cosmetics, and food processing. Structurally, a glycoside is composed of aglycone attaching to one or several sugar moieties so-called glycone. It is found that biochemical or biopharmaceutical properties of glycoside are mainly determined by its sugar part and thereby alternation of this glycone resulting in novel structure and characteristics as well. The use of traditional production methods of glycosides such as direct extraction and purification from plants, animals, or microorganisms is very challenging (laborious, time-consuming, technique, high price, low yield, etc.). Alternatively, the use of enzymatic methods for the biosynthesis of glycosides has become a highly promising tool. Particularly, the diverse structure of glycosides can be obtained using the promiscuous catalytic activity of glycosyltransferases (GT) mined from bioresources (plants, fungi, microorganisms, etc.). In addition, the exploration of GT catalytic promiscuity toward diverse aglycones, and glycones has indeed been interesting and played a key role in the production of novel glycosides. This review described the recent advances in glycosyltransferase-mediated glycodiversification of small molecules (flavonoids, steroids, terpenoids, etc.). Mostly, references were collected from 2014 to 2023.

Single-Molecule Identification and Quantification of Steviol Glycosides with a Deep Learning-Powered Nanopore Sensor.

Steviol glycosides (SGs) are a class of high-potency noncalorie natural sweeteners made up of a common diterpenoid core and varying glycans. Thus, the diversity of glycans in composition, linkage, and isomerism results in the tremendous structural complexity of the SG family, which poses challenges for the precise identification and leads to the fact that SGs are frequently used in mixtures and their variances in biological activity remain largely unexplored. Here we show that a wild-type aerolysin nanopore can detect and discriminate diverse SG species through the modulable electro-osmotic flow effect at varied applied voltages. At low voltages, the neutral SG molecule was drawn and stuck in the pore entrance due to an energy barrier around R220 sites. The ensuing binding events enable the identification of the majority of SG species. Increasing the voltage can break the barrier and cause translocation events, allowing for the unambiguous identification of several pairs of SGs differing by only one hydroxyl group through recognition accumulation from multiple sensing regions and sites. Based on nanopore data of 15 SGs, a deep learning-based artificial intelligence (AI) model was created to process the individual blockage events, achieving the rapid, automated, and precise single-molecule identification and quantification of SGs in real samples. This work highlights the value of nanopore sensing for precise structural analysis of complex glycans-containing glycosides, as well as the potential for sensitive and rapid quality assurance analysis of glycoside products with the use of AI.

Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS.

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

Contents of paeoniflorin and albiflorin in two Korean landraces of Paeonia lactiflora and characterization of paeoniflorin biosynthesis genes in peony.

Paeoniflorin and albiflorin are monoterpene glycosides that exhibit various medicinal properties in Paeonia species. This study explored the terpene biosynthesis pathway and analyzed the distribution of these compounds in different tissues of two Korean landraces of Paeonia lactiflora to gain insights into the biosynthesis of monoterpene glycosides in P. lactiflora and their potential applications. Two Korean landraces, Hongcheon var. and Hwacheon var, of P. lactiflora were used for the analyses. Contents of the paeoniflorin and albiflorin were analyzed using HPLC. RNA was extracted, sequenced, and subjected to transcriptome analysis. Differential gene expression, KEGG, and GO analyses were performed. Paeoniflorin biosynthesis genes were isolated from the transcriptomes using the genes in Euphorbia maculata with the NBLAST program. Phylogenetic analysis of of 1-Deoxy-D-xylulose 5-phosphate synthase (DOXPS), geranyl pyrophosphate synthase (GPPS), and pinene synthase (PS) was carried out with ClustalW and MEGA v5.0. Analysis of paeoniflorin and albiflorin content in different tissues of the two P. lactiflora landraces revealed significant variation. Transcriptome analysis yielded 36,602 unigenes, most of which were involved in metabolic processes. The DEG analysis revealed tissue-specific expression patterns with correlations between landraces. The isolation of biosynthetic genes identified 173 candidates. Phylogenetic analysis of the key enzymes in these pathways provides insights into their evolutionary relationships. The sequencing and analysis of DOXPS, GPPS, PS revealed distinct clades and subclades, highlighting their evolutionary divergence and functional conservation. Our findings highlight the roots as the primary sites of paeoniflorin and albiflorin accumulation in P. lactiflora, underscoring the importance of tissue-specific gene expression in their biosynthesis. this study advances our understanding of monoterpene glycoside production and distribution in Paeonia, thereby guiding further plant biochemistry investigations.

Palladium-Catalyzed Regio- and Stereoselective Glycosylation of Azole Heterocycles Enables Access to Diverse Heterocyclic N-Glycosides.

An efficient and practical glycosylation platform for synthesizing N-glycosides by leveraging palladium catalysis is disclosed. This approach enables facile access to diverse heterocyclic N-glycosides with excellent regio- and stereoselectivities and high site selectivity of multiple N atoms. The reaction exhibits a broad substrate scope (65 examples), high functional group tolerance, and easy scalability. Its synthetic utility is demonstrated through late-stage functionalization of pharmaceutically relevant molecules and various diastereoselective transformations of the glycoside products. Overall, our method provides a handy tool for efficient and stereocontrolled synthesis of valuable N-glycosylated heterocycles.