UDP-glucose Research Articles

Pharmacometabolomics Approach to Explore Pharmacokinetic Variation and Clinical Characteristics of a Single Dose of Desvenlafaxine in Healthy Volunteers

This study investigated the effects of a single dose of desvenlafaxine via oral administration on the pharmacokinetic parameters and clinical and laboratory characteristics in healthy volunteers using a pharmacometabolomics approach. In order to optimize desvenlafaxine’s therapeutic use and minimize potential adverse effects, this knowledge is essential. Methods: Thirty-five healthy volunteers were enrolled after a health trial and received a single dose of desvenlafaxine (Pristiq®, 100 mg). First, liquid chromatography coupled to tandem mass spectrometry was used to determine the main pharmacokinetic parameters. Next, ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry was used to identify plasma metabolites with different relative abundances in the metabolome at pre-dose and when the desvenlafaxine peak plasma concentration was reached (pre-dose vs. post-dose). Results: Correlations were observed between metabolomic profiles, such as tyrosine, sphingosine 1-phosphate, and pharmacokinetic parameters, as well as acetoacetic acid and uridine diphosphate glucose associated with clinical characteristics. Our findings suggest that desvenlafaxine may have a broader effect than previously thought by acting on the proteins responsible for the transport of various molecules at the cellular level, such as the solute carrier SLC and adenosine triphosphate synthase binding cassette ABC transporters. Both of these molecules have been associated with PK parameters and adverse events in our study. Conclusions: This altered transporter activity may be related to the reported side effects of desvenlafaxine, such as changes in blood pressure and liver function. This finding may be part of the explanation as to why people respond differently to the drug.

Synthesis of value-added uridine 5'-diphosphate-glucose from sucrose applying an engineered sucrose synthase counteracts the activity-stability trade-off

The one-step deconstruction of sucrose into uridine 5′-diphosphate-glucose (UDP-Glc), an important sugar donor for transglycosylation, employing sucrose synthase (Susy) is emerging as a valuable sucrose utilization process. The insufficient activity and stability of Susy limit the productivity of UDP-Glc from sucrose. Here, an engineered Susy (SusyM6) that counteracted the activity-stability trade-off was developed with the half-life time and activity being 43-fold and 1.4-fold of wild-type, respectively. Tighter hydrophobic patches and stabilization of the SSN2 domain contributed to greater activity and stability. The use of SusyM6 in UDP-Glc production resulted in a satisfactory space-time yield of 73 g/L/h within 1 h. The cascade of different biocatalysts with SusyM6, focusing on utilizing two products of sucrose decomposition, fructose and UDP-Glc, expanded sucrose utilization, efficiently promoting the UDP-Glc productivity and giving a cost-effective method for UDP-galactose (UDP-Gal) synthesis. This study demonstrated promising green pathways for producing multiple value-added products from sucrose using Susy.

Increased sugar content impairs pollen fertility and reduces seed-setting in high-photosynthetic-efficiency rice

Crop yield depends on biomass, which is primarily associated with photosynthesis. We previously demonstrated that two photorespiratory bypasses, i.e., GOC (glycolate oxidase + oxalate oxidase + catalase) and GCGT (glycolate oxidase + catalase + glyoxylate carboligase + tartronic semialdehyde reductase), significantly increased photosynthesis, biomass, and grain yield, but decreased seed-setting rates in rice. This study explored the underlying mechanism of how elevated photosynthetic efficiency impacted the seed-setting. First, pollen germination assessed in vivo and in vitro, revealed a reduced germination rate in GCGT rice. Subsequent analysis found that photosynthates highly accumulated in the leaves and stems; sucrose and soluble sugar levels were increased but the starch level was reduced in the anthers. Uridine diphosphate glucose (UDP-Glc) was increased but uridine diphosphate galactose (UDP-Gal) was unaltered, thus causing an imbalance in the UDP-Glc/UDP-Gal ratio in GCGT anthers. Most anthers in GCGT plants had two locules in contrast to four in the wild-type (WT). Pollen tapetum was developmentally abnormal, and genes related to sucrose synthesis, transport, and tapetal programmed cell death (PCD) were upregulated, whereas those involved in starch synthesis and conversion were downregulated in GCGT anthers. Taken together, our results demonstrated that an increase in sugar content was the primary factor causing reduced seed-setting rates in high photosynthetic efficiency rice, during which metabolic disorder of sugars and UDP sugar imbalance in anthers lead to impaired pollen fertility.

Glucosyltransferase TeGSS from Thermosynechococcus elongatus produces an α-1,2-glucan

Here, we enzymatically produced a novel α-1,2-glucan, glucosylsucrose, that has a chemical structure significantly different from that of other glucans. This structural difference suggests its potential to modulate new physiological activities compared to known glucans. The enzyme TeGSS catalyzes the synthesis of this α-1,2-glucan from sucrose and UDP-glucose (UDPG). Using NMR spectroscopy, we elucidated the chemical structures of TeGSS-synthesized glucosylsucrose tri-, tetra-, and penta-saccharides in which the monosaccharide units are linked by α-1,2-glycosidic bonds. We also report the crystal structures of TeGSS co-crystallized with UDP and glucosylsucrose tri- and tetra-saccharides. Site-directed mutagenesis of residues in and around the TeGSS catalytic center has allowed us to propose a concerted SNi mechanism of action. Finally, we developed an enzyme-coupled reaction involving TeGSS and SuSyAc that allows production of UDPG for the synthesis of α-1,2-glucan.

Efficient synthesis of salidroside from tyrosol based on UDPG recycling system

Salidroside is a functional ingredient with wide applications in food and pharmaceutical fields. It is conventionally produced by extraction from plants, the application of which is limited by the scarcity of raw materials and cumbersome process. This study achieved the efficient production of salidroside by biosynthesis with tyrosol as the substrate. While utilizing glycosyltransferases for tyrosol glycosylation, we introduced sucrose synthase to construct the uridine diphosphate glucose (UDPG) recycling system. The glycosyltransferase UGT33 and sucrose synthase AtSUS were screened out by comparison, and the recombinant strain Escherichia coli BL21/pETDuet-AtSUS-UGT33 was constructed. The copy number of the gene was optimized and the optimal copy number ratio of glycosyltransferase to sucrose synthase was determined to be 3:1. The whole-cell transformation conditions (temperature, pH, inoculum amount, substrate concentration, and concentrations of metal ions) of the recombinant strain were optimized, and the highest yield of salidroside reached 8.17 g/L after fermentation under the optimal conditions in a 5 L fermenter for 24 h. This study provides a reference for the efficient production of salidroside by microorganisms.

Efficient nata de coco production of Komagataeibacter nataicola driven by microbiota-fermented coconut water: Biological and structural characteristics

Nata de coco is a traditional fermented food, chemically known as bacterial cellulose (BC), widely used in the food industry, personal care, and biomaterial areas. In the nata de coco industry, microbiota-fermented coconut water (FCW) has been regarded as a highly efficient medium for improving BC synthesis by Komagataeibacter spp. However, its effects on the biological properties of the strains of Komagataeibacter and structural characteristics of BC remain unknown, resulting in unstable production and BC quality. This study aimed to explore the biological properties of K. nataicola, the characteristics of BC structure and reasons for the efficiency under the effect of FCW. The results showed that FCW could increase the BC yield of K. nataicola by 2.03–4.75 times, the uridine diphosphate glucose pyrophosphorylase activity by 10–45.3 times, and the hexokinase, phosphofructokinase, or pyruvate kinase activities by 2 times. The extracellular metabolite profile of K. nataicola showed that its glycolysis/gluconeogenesis pathway was significantly stimulated and the utilization of erythritol obviously increased. These phenomena might be attributed to the use of ethanol, lactate, xylitoland mannitol in FCW by K. nataicola. In addition, the structural characteristics of BC produced in FCW included higher number of hydrogen bonds, thinner fibrils, a looser three-dimensional network, improved porosity and greater mechanical strength. This study offered novel insights into the BC synthesis of K. nataicola and the BC structural characteristics cultivated in FCW, providing valuable references for the efficient production and potential applications of both nata de coco and BC.

Unlocking Industrial Potential: Phase-Transition Coimmobilization of Multienzyme Systems for High-Efficiency Uridine Diphosphate Galactose Production.

Transitioning from batch to continuous industrial production often improves the economic returns and production efficiency. Immobilization is a critical strategy that can facilitate this shift. This study refined the previously established method for synthesizing uridine diphosphate galactose (UDP-Gal) by employing thermophilic enzymes. Three thermophilic enzymes (galactokinase, uridine diphosphate glucose pyrophosphorylase, and inorganic pyrophosphatase) were coimmobilized on the pH-responsive carrier Eudragit S-100, promoting enzyme recovery and reuse while their industrial potential was assessed. The coimmobilization system efficiently catalyzed UDP-Gal production, yielding 13.69 mM in 1.5 h, attaining a UTP conversion rate of 91.2% and a space-time yield (STY) of 5.16 g/L/h. Moreover, the system exhibited exceptional reproducibility, retaining 58.9% of its initial activity after five cycles. This research highlighted promising prospects for coimmobilization in industrial synthesis and proposed a novel methodology for enhancing UDP-Gal production in the industry. In addition, the phase-transition property of Eudragit S-100 paves the way for further exploration with the one-pot synthesis of poorly soluble galactosides.

Fermentation broth of a novel endophytic fungus enhanced maize salt tolerance by regulating sugar metabolism and phytohormone biosynthesis or signaling

Soil salinization is a major environmental factor that severely affects global agriculture. Root endophytes can enter root cells, and offer various ecological benefits, such as promoting plant growth, improving soil conditions, and enhancing plant resistance. Su100 is a novel strain of endophytic fungus that was characterized from blueberry roots. In this study, we focused on evaluating the effects of Su100 secretion on maize growth. The results demonstrated that maize treated with Su100 fermentation broth (SFB) exhibited significantly stronger salt tolerance than the control. It is worth mentioning that the treated root system not only had an advantage in terms of biomass but also a change in root structure with a significant increase in lateral roots (LRs) compared to the control. Transcriptome analysis combined with hormone content measurements indicated that SFB upregulated the auxin signaling pathway, and also caused alterations in brassinosteroids (BR) and jasmonic acid (JA) biosynthesis and signaling pathways. Transcriptome analyses also indicated that SFB caused significant changes in the sugar metabolism of maize roots. The major changes included: enhancing the conversion and utilization of sucrose in roots; increasing carbon flow to uridine diphosphate glucose (UDPG), which acted as a precursor for producing more cell wall polysaccharides, mainly pectin and lignin; accelerating the tricarboxylic acid cycle, which were further supported by sugar content determinations. Taken together, our results indicated that the enhanced salt tolerance of maize treated with SFB was due to the modulation of sugar metabolism and phytohormone biosynthesis or signaling pathways. This study provided new insights into the mechanisms of action of endophytic fungi and highlighted the potential application of fungal preparations in agriculture.

Insights from Structure-Based Simulations into the Persulfidation of Uridine Diphosphate-Glycosyltransferase71c5 Facilitating the Reversible Inactivation of Abscisic Acid.

The action of abscisic acid (ABA) is closely related to its level in plant tissues. Uridine diphosphate-glycosyltransferase71c5 (UGT71C5) was characterized as a major UGT enzyme to catalyze the formation of the ABA-glucose ester (ABA-GE), a reversible inactive form of free ABA in Arabidopsis thaliana (thale cress). UGTs function in a mode where the catalytic base deprotonates an acceptor to allow a nucleophilic attack at the anomeric center of the donor, achieving the transfer of a glucose moiety. The proteomic data revealed that UGT71C5 can be persulfidated. Herein, an experimental method was employed to detect the persulfidation site of UGT71C5, and the computational methods were further used to identify the yet unknown molecular basis of ABA glycosylation as well as the regulatory role of persulfidation in this process. Our results suggest that the linker and the U-shaped loop are regulatory structural elements: the linker is associated with the binding of uridine diphosphate glucose (UPG) and the U-shaped loop is involved in binding both UPG and ABA.It was also found that it is through tuning the dynamics of the U-shaped loop that is accompanied by the movement of tyrosine (Y388) that the persulfidation of cysteine (C311) leads to the catalytic residue histidine (H16) being in place, preparing for the deprotonation of ABA, and then reorientates UPG and deprotonated ABA closer to the 'Michaelis' complex, facilitating the transfer of a glucose moiety. Ultimately, the persulfidation of UGT71C5 is in favor of ABA glycosylation. Our results provide insights into the molecular details of UGT71C5 recognizing substrates and insights concerning persulfidation as a possible mechanism for hydrogen sulfide (H2S) to modulate the content of ABA, which helps us understand how modulating ABA level strengthens plant tolerance.

Interactions at the Oviposition Scar: Molecular and Metabolic Insights into Elaeagnus angustifolia's Resistance Response to Anoplophora glabripennis.

The Russian olive (Elaeagnus angustifolia), which functions as a "dead-end trap tree" for the Asian long-horned beetle (Anoplophora glabripennis) in mixed plantations, can successfully attract Asian long-horned beetles for oviposition and subsequently kill the eggs by gum. This study aimed to investigate gum secretion differences by comparing molecular and metabolic features across three conditions-an oviposition scar, a mechanical scar, and a healthy branch-using high-performance liquid chromatography and high-throughput RNA sequencing methods. Our findings indicated that the gum mass secreted by an oviposition scar was 1.65 times greater than that secreted by a mechanical scar. Significant differences in gene expression and metabolism were observed among the three comparison groups. A Kyoto Encyclopedia of Genes and Genomes annotation and enrichment analysis showed that an oviposition scar significantly affected starch and sucrose metabolism, leading to the discovery of 52 differentially expressed genes and 7 differentially accumulated metabolites. A network interaction analysis of differentially expressed metabolites and genes showed that EaSUS1, EaYfcE1, and EaPGM1 regulate sucrose, uridine diphosphate glucose, α-D-glucose-1P, and D-glucose-6P. Although the polysaccharide content in the OSs was 2.22 times higher than that in the MSs, the sucrose content was lower. The results indicated that the Asian long-horned beetle causes Russian olive sucrose degradation and D-glucose-6P formation. Therefore, we hypothesized that damage caused by the Asian long-horned beetle could enhance tree gum secretions through hydrolyzed sucrose and stimulate the Russian olive's specific immune response. Our study focused on the first pair of a dead-end trap tree and an invasive borer pest in forestry, potentially offering valuable insights into the ecological self-regulation of Asian long-horned beetle outbreaks.

Glycolytic PFKFB3 and Glycogenic UGP2 Axis Regulates Perfusion Recovery in Experimental Hind Limb Ischemia.

Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle. Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model. Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in an increased perfusion recovery and higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 expression/levels in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-knock-down ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis). Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD.

Identification and functional characterization of a new flavonoid glycosyltransferase from Rheum palmatum

ObjectiveTo characterize a glycosyltransferase (RpUGT1) from Rheum palmatum and investigate its specificity toward flavonoid compounds. MethodsThe RpUGT1 was expressed in Escherichia coli and screened for catalytic activity against a range of flavonoid substrates using a high-throughput HPLC assay method. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) were used to determine the structure of the product. Homology modeling, molecular docking analyses and site-directed mutagenesis studies were conducted to identify key residues responsible for its function. ResultsThe recombinant RpUGT1 protein exhibited catalytic activity towards various flavonoids. Notably, RpUGT1 catalyzed the glycosylation of isorhamnetin to form 3-O-glucoside and kaempferol to form 7-O-glucoside, utilizing uridine diphosphate (UDP) glucose as the sugar donor. The homology modeling and molecular docking analyses identified key residues responsible for its activity. Subsequent site-directed mutagenesis studies highlighted the crucial role of K307 in catalysis. ConclusionThese discoveries offer valuable perspectives on the role of the UGT family and establish a groundwork for forthcoming research on the synthesis of flavonoids in plants.

Development of New Multi-Glycosylation Routes to Facilitate the Biosynthesis of Sweetener Mogrosides from Bitter Immature Siraitia Grosvenorii Using Engineered Escherichia coli.

Mogrosides, which have various pharmacological activities, are mainly extracted from Siraitia grosvenorii (Luo Han Guo) and are widely used as natural zero-calorie sweeteners. Unfortunately, the difficult cultivation and long maturation time of Luo Han Guo have contributed to a shortage of mogrosides. To overcome this obstacle, we developed a highly efficient biosynthetic method using engineered Escherichia coli to synthesize sweet mogrosides from bitter mogrosides. Three UDP-glycosyltransferase (UGT) genes with primary/branched glycosylation catalytic activity at the C3/C24 sites of mogrosides were screened and tested. Mutant M3, which could catalyze the glycosylation of nine types of mogrosides, was obtained through enhanced catalytic activity. This improvement in β-(1,6)-glycosidic bond formation was achieved through single nucleotide polymorphisms and direct evolution, guided by 3D structural analysis. A new multienzyme system combining three UGTs and UDP-glucose (UDPG) regeneration was developed to avoid the use of expensive UDPG. Finally, the content of sweet mogrosides in the immature Luo Han Guo extract increased significantly from 57% to 95%. This study not only established a new multienzyme system for the highly efficient production of sweet mogrosides from immature Luo Han Guo but also provided a guideline for the high-value utilization of rich bitter mogrosides from agricultural waste and residues.

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

BackgroundDue 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.ResultsBy 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.ConclusionsBy 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.

Highly Efficient Biosynthesis of Rebaudioside M8 through Structure-Guided Engineering of Glycosyltransferase UGT94E13.

Given the low-calorie, high-sweetness characteristics of steviol glycosides (SGs), developing SGs with improved taste profiles is a key focus. Rebaudioside M8 (Reb M8), a novel non-natural SG derivative obtained through glycosylation at the C-13 position of rebaudioside D (Reb D) using glycosyltransferase UGT94E13, holds promise for further development due to its enhanced sweetness. However, the low catalytic activity of UGT94E13 hampers further research and commercialization. This study aimed to improve the enzymatic activity of UGT94E13 through semirational design, and a variant UGT94E13-F169G/I185G was obtained with the catalytic activity improved by 13.90 times. A cascade reaction involving UGT94E13-F169G/I185G and sucrose synthase AtSuSy was established to recycle uridine diphosphate glucose, resulting in an efficient preparation of Reb M8 with a yield of 98%. Moreover, according to the analysis of the distances between the substrate Reb D and enzymes as well as between Reb D and the glucose donor through molecular dynamics simulations, it is found that the positive effect of shortening the distance on glycosylation reaction activity accounts for the improved catalytic activity of UGT94E13-F169G/I185G. Therefore, this study addresses the bottleneck in the efficient production of Reb M8 and provides a foundation for its widespread application in the food industry.

Abnormal metabolism in melanocytes participates in the activation of dendritic cell in halo nevus

A comprehensive analysis of spatial transcriptomics was carried out to better understand the progress of halo nevus. We found that halo nevus was characterized by overactive immune responses, triggered by chemokines and dendritic cells (DCs), T cells, and macrophages. Consequently, we observed abnormal cell death, such as apoptosis and disulfidptosis in halo nevus, some were closely related to immunity. Interestingly, we identified aberrant metabolites such as uridine diphosphate glucose (UDP-G) within the halo nevus. UDP-G, accompanied by the infiltration of DCs and T cells, exhibited correlations with certain forms of cell death. Subsequent experiments confirmed that UDP-G was increased in vitiligo serum and could activate DCs. We also confirmed that oxidative response is an inducer of UDP-G. In summary, the immune response in halo nevus, including DC activation, was accompanied by abnormal cell death and metabolites. Especially, melanocyte-derived UDP-G may play a crucial role in DC activation.

Rational Design for the Complete Synthesis of Stevioside in Saccharomyces cerevisiae.

Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high sweetness and low-calorie content. In this study, we constructed a Saccharomyces cerevisiae strain for the complete synthesis of stevioside using a metabolic engineering strategy. Firstly, the synthesis pathway of steviol was modularly constructed in S. cerevisiae BY4742, and the precursor pathway was strengthened. The yield of steviol was used as an indicator to investigate the expression effect of different sources of diterpene synthases under different combinations, and the strains with further improved steviol yield were screened. Secondly, glycosyltransferases were heterologously expressed in this strain to produce stevioside, the sequence of glycosyltransferase expression was optimized, and the uridine diphosphate-glucose (UDP-Glc) supply was enhanced. Finally, the results showed that the strain SST-302III-ST2 produced 164.89 mg/L of stevioside in a shake flask experiment, and the yield of stevioside reached 1104.49 mg/L in an experiment employing a 10 L bioreactor with batch feeding, which was the highest yield reported. We constructed strains with a high production of stevioside, thus laying the foundation for the production of other classes of steviol glycosides and holding good prospects for application and promotion.

Dark septate endophyte Anteaglonium sp. T010 promotes biomass accumulation in poplar by regulating sucrose metabolism and hormones.

Plant biomass is a highly promising renewable feedstock for the production of biofuels, chemicals and materials. Enhancing the content of plant biomass through endophyte symbiosis can effectively reduce economic and technological barriers in industrial production. In this study, we found that symbiosis with the dark septate endophyte (DSE) Anteaglonium sp. T010 significantly promoted the growth of poplar trees and increased plant biomass, including cellulose, lignin and starch. To further investigate whether plant biomass was related to sucrose metabolism, we analyzed the levels of relevant sugars and enzyme activities. During the symbiosis of Anteaglonium sp. T010, sucrose, fructose and glucose levels in the stem of poplar decreased, while the content of intermediates such as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P) and UDP-glucose (UDPG), and the activity of enzymes related to sucrose metabolism, including sucrose synthase (SUSY), cell wall invertase (CWINV), fructokinase (FRK) and hexokinase, increased. In addition, the contents of glucose, fructose, starch, and their intermediates G6P, F6P and UDPG, as well as the enzyme activities of SUSY, CWINV, neutral invertase and FRK in roots were increased, which ultimately led to the increase of root biomass. Besides that, during the symbiotic process of Anteaglonium sp. T010, there were significant changes in the expression levels of root-related hormones, which may promote changes in sucrose metabolism and consequently increase the plant biomass. Therefore, this study suggested that DSE fungi can increase the plant biomass synthesis capacity by regulating the carbohydrate allocation and sink strength in poplar.

CRISPR/Cas9-Based Functional Characterization of SfUGT50A15 Reveals Its Roles in the Resistance of Spodoptera frugiperda to Chlorantraniliprole, Emamectin Benzoate, and Benzoxazinoids.

UDP-glycosyltransferases (UGTs) are a diverse superfamily of enzymes. Insects utilize uridine diphosphate-glucose (UDP-glucose) as a glycosyl donor for glycosylation in vivo, involved in the glycosylation of lipophilic endosymbionts and xenobiotics, including phytotoxins. UGTs act as second-stage detoxification metabolizing enzymes, which are essential for the detoxification metabolism of insecticides and benzoxazine compounds. However, the UGT genes responsible for specific glycosylation functions in S. frugiperda are unclear at present. In this study, we utilized CRISPR/Cas9 to produce a SfUGT50A15-KO strain to explore its possible function in governing sensitivity to chemical insecticides or benzoxazinoids. The bioassay results suggested that the SfUGT50A15-KO strain was significantly more sensitive to chlorantraniliprole, emamectin benzoate, and benzoxazinoids than the wild-type strains. This finding suggests that the overexpression of the SfUGT50A15 gene may be linked to S. frugiperda resistance to pesticides (chlorantraniliprole and emamectin benzoate) as well as benzoxazinoids (BXDs).

Combinatorial metabolic engineering of Bacillus subtilis enables the efficient biosynthesis of isoquercitrin from quercetin

BackgroundIsoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards.ResultsHerein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three β-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor.ConclusionThe engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.