A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is distinct from host homologues.

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.

Identification and expression analysis of the Xyloglucan transglycosylase/hydrolase (XTH) gene family under abiotic stress in oilseed (Brassica napus L.).

XTH genes are key genes that regulate the hydrolysis and recombination of XG components and plays role in the structure and composition of plant cell walls. Therefore, clarifying the changes that occur in XTHs during plant defense against abiotic stresses is informative for the study of the plant stress regulatory mechanism mediated by plant cell wall signals. XTH proteins in Arabidopsis thaliana was selected as the seed sequences in combination with its protein structural domains, 80 members of the BnXTH gene family were jointly identified from the whole genome of the Brassica napus ZS11, and analyzed for their encoded protein physicochemical properties, phylogenetic relationships, covariance relationships, and interoperating miRNAs. Based on the transcriptome data, the expression patterns of BnXTHs were analyzed in response to different abiotic stress treatments. The relative expression levels of some BnXTH genes under Al, alkali, salt, and drought treatments after 0, 6, 12 and 24 h were analyzed by using qRT-PCR to explore their roles in abiotic stress tolerance in B. napus. BnXTHs showed different expression patterns in response to different abiotic stress signals, indicating that the response mechanisms of oilseed rape against different abiotic stresses are also different. This paper provides a theoretical basis for clarifying the function and molecular genetic mechanism of the BnXTH gene family in abiotic stress tolerance in rapeseed.

Functional and structural dissection of glycosyltransferases underlying the glycodiversity of wolfberry-derived bioactive ingredients lycibarbarspermidines.

Lycibarbarspermidines are unusual phenolamide glycosides characterized by a dicaffeoylspermidine core with multiple glycosyl substitutions, and serve as a major class of bioactive ingredients in the wolfberry. So far, little is known about the enzymatic basis of the glycosylation of phenolamides including dicaffeoylspermidine. Here, we identify five lycibarbarspermidine glycosyltransferases, LbUGT1-5, which are the first phenolamide-type glycosyltransferases and catalyze regioselective glycosylation of dicaffeoylspermidines to form structurally diverse lycibarbarspermidines in wolfberry. Notably, LbUGT3 acts as a distinctive enzyme that catalyzes a tandem sugar transfer to the ortho-dihydroxy group on the caffeoyl moiety to form the unusual ortho-diglucosylated product, while LbUGT1 accurately discriminates caffeoyl and dihydrocaffeoyl groups to catalyze a site-selective sugar transfer. Crystal structure analysis of the complexes of LbUGT1 and LbUGT3 with UDP, combined with molecular dynamics simulations, revealed the structural basis of the difference in glycosylation selectivity between LbUGT1 and LbUGT3. Site-directed mutagenesis illuminates a conserved tyrosine residue (Y389 in LbUGT1 and Y390 in LbUGT3) in PSPG box that plays a crucial role in regulating the regioselectivity of LbUGT1 and LbUGT3. Our study thus sheds light on the enzymatic underpinnings of the chemical diversity of lycibarbarspermidines in wolfberry, and expands the repertoire of glycosyltransferases in nature.

Three-component systems represent a common pathway for extracytoplasmic addition of pentofuranose sugars into bacterial glycans.

Cell surface glycans are major drivers of antigenic diversity in bacteria. The biochemistry and molecular biology underpinning their synthesis are important in understanding host-pathogen interactions and for vaccine development with emerging chemoenzymatic and glycoengineering approaches. Structural diversity in glycostructures arises from the action of glycosyltransferases (GTs) that use an immense catalog of activated sugar donors to build the repeating unit and modifying enzymes that add further heterogeneity. Classical Leloir GTs incorporate α- or ß-linked sugars by inverting or retaining mechanisms, depending on the nucleotide sugar donor. In contrast, the mechanism of known ribofuranosyltransferases is confined to ß-linkages, so the existence of α-linked ribofuranose in some glycans dictates an alternative strategy. Here, we use Citrobacter youngae O1 and O2 lipopolysaccharide O antigens as prototypes to describe a widespread, versatile pathway for incorporating side-chain α-linked pentofuranoses by extracytoplasmic postpolymerization glycosylation. The pathway requires a polyprenyl phosphoribose synthase to generate a lipid-linked donor, a MATE-family flippase to transport the donor to the periplasm, and a GT-C type GT (founding the GT136 family) that performs the final glycosylation reaction. The characterized system shares similarities, but also fundamental differences, with both cell wall arabinan biosynthesis in mycobacteria, and periplasmic glucosylation of O antigens first discovered in Salmonella and Shigella. The participation of auxiliary epimerases allows the diversification of incorporated pentofuranoses. The results offer insight into a broad concept in microbial glycobiology and provide prototype systems and bioinformatic guides that facilitate discovery of further examples from diverse species, some in currently unknown glycans.

A UDP-glycosyltransferase gene PcUGT202A9 was associated with abamectin resistance in Panonychus citri (McGregor).

Panonychus citri (McGregor) strains have developed a high level of resistance to abamectin, but the underlying molecular mechanism is unknown. Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are critical for the removal of a variety of exogenous and endogenous substances. In this study, an enzyme activity assay revealed that UGTs potentially contribute to P. citri abamectin resistance. Spatiotemporal expression profiles showed that only PcUGT202A9 was significantly overexpressed in the abamectin-resistant strain (AbR) at all developmental stages. Moreover, UGT activity decreased significantly, whereas abamectin susceptibility increased significantly, in AbR after PcUGT202A9 was silenced. Three-dimensional modeling and molecular docking analyses revealed that PcUGT202A9 can bind stably to abamectin. Recombinant PcUGT202A9 activity was detected when α-naphthol was used, but the enzymatic activity was inhibited by abamectin (50 % inhibitory concentration: 803.3 ±â€¯14.20 µmol/L). High-performance liquid chromatography and mass spectrometry analyses indicated that recombinant PcUGT202A9 can effectively degrade abamectin and catalyze the conjugation of UDP-glucose to abamectin. These results imply PcUGT202A9 contributes to P. citri abamectin resistance.

Detailed structural characterization of five water-insoluble α-glucans produced by glucansucrases from Streptococcus spp.

Water-insoluble α-glucans synthesized from sucrose by glucansucrases from Streptococcus spp. are essential in dental plaque and caries formation. Because limited information is available on the fine structure of these biopolymers, we analyzed the structures of unmodified glucans produced by five recombinant Streptococcus (S.) mutans DSM 20523 and S. salivarius DSM 20560 glucansucrases in detail. A combination of methylation analysis, endo-dextranase and endo-mutanase hydrolyses, and HPSEC-RI was used. Furthermore, crystal-like regions were analyzed by using XRD and 13C MAS NMR spectroscopy. Our results showed that the glucan structures were highly diverse: Two glucans with 1,3- and 1,6-linkages were characterized in detail besides an almost exclusively 1,3-linked and a linear 1,6-linked glucan. Furthermore, one glucan contained 1,3-, 1,4-, and 1,6-linkages and thus had an unusual, not yet described structure. It was demonstrated that the glucans had a varying structural architecture by using partial enzymatic hydrolyses. Furthermore, crystal-like regions formed by 1,3-glucopyranose units were observed for the two 1,3- and 1,6-linked glucans and the linear 1,3-linked glucan. 1,6-linked regions were mobile and not involved in the crystal-like areas. Altogether, our results broaden the knowledge of the structure of water-insoluble α-glucans from Streptococcus spp.

Expression responses of XTH genes in tomato and potato to environmental mechanical forces: focus on behavior in response to rainfall, wind and touch.

Rainfall, wind and touch, as mechanical forces, were mimicked on 6-week-old soil-grown tomato and potato under controlled conditions. Expression level changes of xyloglucan endotransglucosylase/hydrolase genes (XTHs) of tomato (Solanum lycopersicum L. cv. Micro Tom; SlXTHs) and potato (Solanum tuberosum L. cv. Desirée; StXTHs) were analyzed in response to these mechanical forces. Transcription intensity of every SlXTHs of tomato was altered in response to rainfall, while the expression intensity of 72% and 64% of SlXTHs was modified by wind and touch, respectively. Ninety-one percent of StXTHs (32 out of 35) in potato responded to the rainfall, while 49% and 66% of the StXTHs were responsive to the wind and touch treatments, respectively. As previously demonstrated, all StXTHs were responsive to ultrasound treatment, and all were sensitive to one or more of the environmental mechanical factors examined in the current study. To our best knowledge, this is the first study to demonstrate that these ubiquitous mechanical environmental cues, such as rainfall, wind and touch, influence the transcription of most XTHs examined in both species.

A novel glycosyltransferase from Bacillus subtilis achieves zearalenone detoxification by diglycosylation modification.

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin produced by Fusarium spp., contaminates cereals and threatens human and animal health by inducing hepatotoxicity, immunotoxicity, and genotoxicity. In this study, a new Bacillus subtilis strain, YQ-1, with a strong ability to detoxify ZEN, was isolated from soil samples and characterized. YQ-1 was confirmed to degrade more than 46.26% of 20 µg mL-1 ZEN in Luria-Bertani broth and 98.36% in fermentation broth within 16 h at 37 °C; one of the two resulting products was ZEN-diglucoside. Under optimal reaction conditions (50 °C and pH 5.0-9.0), the reaction mixture generated by YQ-1 catalyzing ZEN significantly reduced the promoting effect of ZEN on MCF-7 cell proliferation, effectively eliminating the estrogenic toxicity of ZEN. In addition, a new glycosyltransferase gene (yqgt) from B. subtilis YQ-1 was cloned with 98% similarity to Bs-YjiC from B. subtilis 168 and over-expressed in E. coli BL21 (DE3). ZEN glycosylation activity converted 25.63% of ZEN (20 µg mL-1) to ZEN-diG after 48 h of reaction at 37 °C. The characterization of ZEN degradation by B. subtilis YQ-1 and the expression of YQGT provide a theoretical basis for analyzing the mechanism by which Bacillus spp. degrades ZEN.

Genome-wide investigation of UDP-Glycosyltransferase family in Tartary buckwheat (Fagopyrum tataricum).

BACKGROUND: Tartary buckwheat (Fagopyrum tataricum) belongs to Polygonaceae family and has attracted increasing attention owing to its high nutritional value. UDP-glycosyltransferases (UGTs) glycosylate a variety of plant secondary metabolites to control many metabolic processes during plant growth and development. However, there have been no systematic reports of UGT superfamily in F. tataricum. RESULTS: We identified 173 FtUGTs in F. tataricum based on their conserved UDPGT domain. Phylogenetic analysis of FtUGTs with 73 Arabidopsis UGTs clustered them into 21 families. FtUGTs from the same family usually had similar gene structure and motif compositions. Most of FtUGTs did not contain introns or had only one intron. Tandem repeats contributed more to FtUGTs amplification than segmental duplications. Expression analysis indicates that FtUGTs are widely expressed in various tissues and likely play important roles in plant growth and development. The gene expression analysis response to different abiotic stresses showed that some FtUGTs were involved in response to drought and cadmium stress. Our study provides useful information on the UGTs in F. tataricum, and will facilitate their further study to better understand their function. CONCLUSIONS: Our results provide a theoretical basis for further exploration of the functional characteristics of FtUGTs and for understanding the growth, development, and metabolic model in F. tataricum.

Integrated Full-Length Transcriptomics and Metabolomics Reveal Glycosyltransferase Involved in the Biosynthesis of Flavonol Glycosides in Laportea bulbifera.

Many species of the Urticaceae family are important cultivated fiber plants that are known for their economic and industrial values. However, their secondary metabolite profiles and associated biosynthetic mechanisms have not been well-studied. Using Laportea bulbifera as a model, we conducted widely targeted metabolomics, which revealed 523 secondary metabolites, including a unique accumulation of flavonol glycosides in bulblet. Through full-length transcriptomic and RNA-seq analyses, the related genes in the flavonoid biosynthesis pathway were identified. Finally, weighted gene correlation network analysis and functional characterization revealed four LbUGTs, including LbUGT78AE1, LbUGT72CT1, LbUGT71BX1, and LbUGT71BX2, can catalyze the glycosylation of flavonol aglycones (kaempferol, myricetin, gossypetin, and quercetagetin) using UDP-Gal and UDP-Glu as the sugar donors. LbUGT78AE1 and LbUGT72CT1 showed substrate promiscuity, whereas LbUGT71BX1 and LbUGT71BX2 exhibited different substrate and sugar donor selectivity. These results provide a genetic resource for studying Laportea in the Urticaceae family, as well as key enzymes responsible for the metabolism of valuable flavonoid glycosides.

BnXTH1 regulates cadmium tolerance by modulating vacuolar compartmentalization and the cadmium binding capacity of cell walls in ramie (Boehmeria nivea).

Xyloglucan endotransglucosylase/hydrolases (XTH) are cell wall-modifying enzymes important in plant response to abiotic stress. However, the role of XTH in cadmium (Cd) tolerance in ramie remains largely unknown. Here, we identified and cloned BnXTH1, a member of the XTH family, in response to Cd stress in ramie. The BnXTH1 promoter (BnXTH1p) demonstrated that MeJA induces the response of BnXTH1p to Cd stress. Moreover, overexpressing BnXTH1 in Boehmeria nivea increased Cd tolerance by significantly increasing the Cd content in the cell wall and decreasing Cd inside ramie cells. Cadmium stress induced BnXTH1-expression and consequently increased xyloglucan endotransglucosylase (XET) activity, leading to high xyloglucan contents and increased hemicellulose contents in ramie. The elevated hemicellulose content increased Cd chelation onto the cell walls and reduced the level of intracellular Cd. Interestingly, overexpressing BnXTH1 significantly increased the content of Cd in vacuoles of ramie and vacuolar compartmentalization genes. Altogether, these results evidence that Cd stress induced MeJA accumulation in ramie, thus, activating BnXTH1 expression and increasing the content of xyloglucan to enhance the hemicellulose binding capacity and increase Cd chelation onto cell walls. BnXTH1 also enhances the vacuolar Cd compartmentalization and reduces the level of Cd entering the organelles and soluble solution.

[Identification and expression of genome of uridine diphosphate glycosyltransferase (UGT) gene family from Chrysanthemum indicum].

Uridine diphosphate glycosyltransferase(UGT) is involved in the glycosylation of a variety of secondary metabolites in plants and plays an important role in plant growth and development and regulation of secondary metabolism. Based on the genome of a diploid Chrysanthemum indicum, the UGT gene family from Ch. indicum was identified by bioinformatics methods, and the physical and chemical properties, subcellular localization prediction, conserved motif, phylogeny, chromosome location, gene structure, and gene replication events of UGT protein were analyzed. Transcriptome and real-time fluorescence quantitative polymerase chain reaction(PCR) were used to analyze the expression pattern of the UGT gene in flowers and leaves of Ch. indicum. Quasi-targeted metabolomics was used to analyze the differential metabolites in flowers and leaves. The results showed that a total of 279 UGT genes were identified in the Ch. indicum genome. Phylogenetic analysis showed that these UGT genes were divided into 8 subfamilies. Members of the same subfamily were distributed in clusters on the chromosomes. Tandem duplications were the main driver of the expansion of the UGT gene family from Ch. indicum. Structural domain analysis showed that 262 UGT genes had complete plant secondary metabolism signal sequences(PSPG box). The analysis of cis-acting elements indicated that light-responsive elements were the most ubiquitous elements in the promoter regions of UGT gene family members. Quasi-targeted metabolome analysis of floral and leaf tissue revealed that most of the flavonoid metabolites, including luteolin-7-O-glucoside and kaempferol-7-O-glucoside, had higher accumulation in flowers. Comparative transcriptome analysis of flower and leaf tissue showed that there were 72 differentially expressed UGT genes, of which 29 genes were up-regulated in flowers, and 43 genes were up-regulated in leaves. Correlation network and phylogenetic analysis showed that CindChr9G00614970.1, CindChr2G00092510.1, and CindChr2G00092490.1 may be involved in the synthesis of 7-O-flavonoid glycosides in Ch. indicum, and real-time fluorescence quantitative PCR analysis further confirmed the reliability of transcriptome data. The results of this study are helpful to understand the function of the UGT gene family from Ch. indicum and provide data reference and theoretical basis for further study on the molecular regulation mechanism of flavonoid glycosides synthesis in Ch. indicum.

Unravelling and reconstructing the biosynthetic pathway of bergenin.

Bergenin, a rare C-glycoside of 4-O-methyl gallic acid with pharmacological properties of antitussive and expectorant, is widely used in clinics to treat chronic tracheitis in China. However, its low abundance in nature and structural specificity hampers the accessibility through traditional crop-based manufacturing or chemical synthesis. In the present work, we elucidate the biosynthetic pathway of bergenin in Ardisia japonica by identifying the highly regio- and/or stereoselective 2-C-glycosyltransferases and 4-O-methyltransferases. Then, in Escherichia coli, we reconstruct the de novo biosynthetic pathway of 4-O-methyl gallic acid 2-C-ß-D-glycoside, which is the direct precursor of bergenin and is conveniently esterified into bergenin by in situ acid treatment. Moreover, further metabolic engineering improves the production of bergenin to 1.41 g L-1 in a 3-L bioreactor. Our work provides a foundation for sustainable supply of bergenin and alleviates its resource shortage via a synthetic biology approach.

Mutations in Glycosyltransferases and Glycosidases: Implications for Associated Diseases.

Glycosylation, a crucial and the most common post-translational modification, coordinates a multitude of biological functions through the attachment of glycans to proteins and lipids. This process, predominantly governed by glycosyltransferases (GTs) and glycoside hydrolases (GHs), decides not only biomolecular functionality but also protein stability and solubility. Mutations in these enzymes have been implicated in a spectrum of diseases, prompting critical research into the structural and functional consequences of such genetic variations. This study compiles an extensive dataset from ClinVar and UniProt, providing a nuanced analysis of 2603 variants within 343 GT and GH genes. We conduct thorough MTR score analyses for the proteins with the most documented variants using MTR3D-AF2 via AlphaFold2 (AlphaFold v2.2.4) predicted protein structure, with the analyses indicating that pathogenic mutations frequently correlate with Beta Bridge secondary structures. Further, the calculation of the solvent accessibility score and variant visualisation show that pathogenic mutations exhibit reduced solvent accessibility, suggesting the mutated residues are likely buried and their localisation is within protein cores. We also find that pathogenic variants are often found proximal to active and binding sites, which may interfere with substrate interactions. We also incorporate computational predictions to assess the impact of these mutations on protein function, utilising tools such as mCSM to predict the destabilisation effect of variants. By identifying these critical regions that are prone to disease-associated mutations, our study opens avenues for designing small molecules or biologics that can modulate enzyme function or compensate for the loss of stability due to these mutations.

A kinetic model reveals the critical gating motifs for donor-substrate loading into Actinobacillus pleuropneumoniae N-glycosyltransferase.

Soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) catalyzes the glycosylation of asparagine residues, and represents one of the most encouraging biocatalysts for N-glycoprotein production. Since the sugar tolerance of ApNGT is restricted to limited monosaccharides (e.g., Glc, GlcN, Gal, Xyl, and Man), tremendous efforts are devoted to expanding the substrate scope of ApNGT via enzyme engineering. However, rational design of novel NGT variants suffers from an elusive understanding of the substrate-binding process from a dynamic point of view. Here, by employing extensive all-atom molecular dynamics (MD) simulations integrated with a kinetic model, we reveal, at the atomic level, the complete donor-substrate binding process from the bulk solvent to the ApNGT active-site, and the key intermediate states of UDP-Glc during its loading dynamics. We are able to determine the critical transition event that limits the overall binding rate, which guides us to pinpoint the key ApNGT residues dictating the donor-substrate entry. The functional roles of several identified gating residues were evaluated through site-directed mutagenesis and enzymatic assays. Two single-point mutations, N471A and S496A, could profoundly enhance the catalytic activity of ApNGT. Our work provides deep mechanistic insights into the structural dynamics of the donor-substrate loading process for ApNGT, which sets a rational basis for design of novel NGT variants with desired substrate specificity.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

UDP-glycosyltransferases act as key determinants of host plant range in generalist and specialist Spodoptera species.

Phytophagous insects have evolved sophisticated detoxification systems to overcome the antiherbivore chemical defenses produced by many plants. However, how these biotransformation systems differ in generalist and specialist insect species and their role in determining insect host plant range remains an open question. Here, we show that UDP-glucosyltransferases (UGTs) play a key role in determining the host range of insect species within the Spodoptera genus. Comparative genomic analyses of Spodoptera species that differ in host plant breadth identified a relatively conserved number of UGT genes in generalist species but high levels of UGT gene pseudogenization in the specialist Spodoptera picta. CRISPR-Cas9 knockouts of the three main UGT gene clusters of Spodoptera frugiperda revealed that UGT33 genes play an important role in allowing this species to utilize the poaceous plants maize, wheat, and rice, while UGT40 genes facilitate utilization of cotton. Further functional analyses in vivo and in vitro identified the UGT SfUGT33F32 as the key mechanism that allows generalist S. frugiperda to detoxify the benzoxazinoid DIMBOA (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), a potent insecticidal phytotoxin produced by poaceous plants. However, while this detoxification capacity is conserved in several generalist Spodoptera species, Spodoptera picta, which specializes on Crinum plants, is unable to detoxify DIMBOA due to a nonfunctionalizing mutation in SpUGT33F34. Collectively, these findings provide insight into the role of insect UGTs in host plant adaptation, the mechanistic basis of evolutionary transitions between generalism and specialism and offer molecular targets for controlling a group of notorious insect pests.

Genome-Wide Analysis and Identification of UDP Glycosyltransferases Responsive to Chinese Wheat Mosaic Virus Resistance in Nicotiana benthamiana.

Glycosylation, a dynamic modification prevalent in viruses and higher eukaryotes, is principally regulated by uridine diphosphate (UDP)-glycosyltransferases (UGTs) in plants. Although UGTs are involved in plant defense responses, their responses to most pathogens, especially plant viruses, remain unclear. Here, we aimed to identify UGTs in the whole genome of Nicotiana benthamiana (N. benthamiana) and to analyze their function in Chinese wheat mosaic virus (CWMV) infection. A total of 147 NbUGTs were identified in N. benthamiana. To conduct a phylogenetic analysis, the UGT protein sequences of N. benthamiana and Arabidopsis thaliana were aligned. The gene structure and conserved motifs of the UGTs were also analyzed. Additionally, the physicochemical properties and predictable subcellular localization were examined in detail. Analysis of cis-acting elements in the putative promoter revealed that NbUGTs were involved in temperature, defense, and hormone responses. The expression levels of 20 NbUGTs containing defense-related cis-acting elements were assessed in CWMV-infected N. benthamiana, revealing a significant upregulation of 8 NbUGTs. Subcellular localization analysis of three NbUGTs (NbUGT12, NbUGT16 and NbUGT17) revealed their predominant localization in the cytoplasm of N. benthamiana leaves, and NbUGT12 was also distributed in the chloroplasts. CWMV infection did not alter the subcellular localization of NbUGT12, NbUGT16, and NbUGT17. Transient overexpression of NbUGT12, NbUGT16, and NbUGT17 enhanced CWMV infection, whereas the knockdown of NbUGT12, NbUGT16 and NbUGT17 inhibited CWMV infection in N. benthamiana. These NbUGTs could serve as potential susceptibility genes to facilitate CWMV infection. Overall, the findings throw light on the evolution and function of NbUGTs.

Biosynthesis of Apigenin Glucosides in Engineered Corynebacterium glutamicum.

Glucosylation is a well-known approach to improve the solubility, pharmacological, and biological properties of flavonoids, making flavonoid glucosides a target for large-scale biosynthesis. However, the low yield of products coupled with the requirement of expensive UDP-sugars limits the application of enzymatic systems for large-scale. C. glutamicum is a Gram-positive and generally regarded as safe (GRAS) bacteria frequently employed for the large-scale production of amino acids and bio-fuels. Due to the versatility of its cell factory system and its non-endotoxin producing properties, it has become an attractive system for the industrial-scale biosynthesis of alternate products. Here, we explored the cell factory of C. glutamicum for efficient glucosylation of flavonoids using apigenin as a model flavonoid, with the heterologous expression of a promiscuous glycosyltransferase, YdhE from Bacillus licheniformis and the endogenous overexpression of C. glutamicum genes galU1 encoding UDP-glucose pyrophosphorylase and pgm encoding phosphoglucomutase involved in the synthesis of UDP-glucose to create a C. glutamicum cell factory system capable of efficiently glucosylation apigenin with a high yield of glucosides production. Consequently, the production of various apigenin glucosides was controlled under different temperatures yielding almost 4.2 mM of APG1(apigenin-4'-O-ß-glucoside) at 25°C, and 0.6 mM of APG2 (apigenin-7-O-ß-glucoside), 1.7 mM of APG3 (apigenin-4',7-O-ß-diglucoside) and 2.1 mM of APG4 (apigenin-4',5-O-ß-diglucoside) after 40 h of incubation with the supplementation of 5 mM of apigenin and 37°C. The cost-effective developed system could be used to modify a wide range of plant secondary metabolites with increased pharmacokinetic activities on a large scale without the use of expensive UDP-sugars.

Effects of priming glycosyltransferase genes cps 2E and cps 4E on the exopolysaccharide biosynthesis by Lactiplantibacillus plantarum YM-4-3 strain.

Microbial exopolysaccharides (EPS) have various physiological functions such as antioxidant, anti-tumor, cholesterol lowering, and immune regulation. However, improving traditional fermentation conditions to increase the production of EPS from Lactiplantibacillus plantarum (L. plantarum) is limited. In this study, we aimed to better improve EPS production and physiological functions of L. plantarum YM-4-3 strain by overexpressing and knocking out the priming glycosyltransferase genes cps 2E and cps 4E for the first time. As a result, the EPS production of the overexpression strain was 30.15 %, 26.84 % and 36.29 % higher than WT, respectively. The EPS production of the knockout strain was significantly lower than that of the WT. At the same time, transcriptome data showed that the gene expression levels of each experimental strain had changed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways found that the glycolysis/gluconeogenesis pathway had the highest gene enrichment in the metabolic pathway. The monosaccharide components of the EPS of each experimental strain were different from those of the WT and the EPS of the experimental strain showed stronger activity against oxidation. In conclusion, this study contributes to the efficient production and application of L. plantarum EPS and helps to understand the mechanism of EPS regulation in L. plantarum.