Uridine Diphosphate Glycosyltransferase Research Articles

GROWTH REGULATING FACTOR 7-mediated arbutin metabolism enhances rice salt tolerance.

Salt stress is an environmental factor that limits plant growth and crop production. With the rapid expansion of salinized arable land worldwide, investigating the molecular mechanisms underlying the salt stress response in plants is urgently needed. Here, we report that GROWTH REGULATING FACTOR 7 (OsGRF7) promotes salt tolerance by regulating arbutin (hydroquinone-β-D-glucopyranoside) metabolism in rice (Oryza sativa). Overexpression of OsGRF7 increased arbutin content, and exogenous arbutin application rescued the salt-sensitive phenotype of OsGRF7 knockdown and knockout plants. OsGRF7 directly promoted the expression of the arbutin biosynthesis genes URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASE 1 (OsUGT1) and OsUGT5, and knockout of OsUGT1 or OsUGT5 reduced rice arbutin content, salt tolerance, and grain size. Furthermore, OsGRF7 degradation through its interaction with F-BOX AND OTHER DOMAINS CONTAINING PROTEIN 13 (OsFBO13) reduced rice salinity tolerance and grain size. These findings highlight an underexplored role of OsGRF7 in modulating rice arbutin metabolism, salt stress response, and grain size, as well as its broad potential use in rice breeding.

Genome-wide analysis of UDP-glycosyltransferases family and identification of UGT genes involved in drought stress of Platycodon grandiflorus

IntroductionThe uridine diphosphate (UDP)-glycosyltransferase (UGT) family is the largest glycosyltransferase family, which is involved in the biosynthesis of natural plant products and response to abiotic stress. UGT has been studied in many medicinal plants, but there are few reports on Platycodon grandiflorus. This study is devoted to genome-wide analysis of UGT family and identification of UGT genes involved in drought stress of Platycodon grandiflorus (PgUGTs).MethodsThe genome data of Platycodon grandiflorus was used for genome-wide identification of PgUGTs, online website and bioinformatics analysis software was used to conduct bioinformatics analysis of PgUGT genes and the genes highly responsive to drought stress were screened out by qRT-PCR, these genes were cloned and conducted bioinformatics analysis.ResultsA total of 75 PgUGT genes were identified in P.grandiflorus genome and clustered into 14 subgroups. The PgUGTs were distributed on nine chromosomes, containing multiple cis-acting elements and 22 pairs of duplicate genes were identified. Protein-protein interaction analysis was performed to predict the interaction between PgUGT proteins. Additionally, six genes were upregulated after 3d under drought stress and three genes (PGrchr09G0563, PGrchr06G0523, PGrchr06G1266) responded significantly to drought stress, as confirmed by qRT-PCR. This was especially true for PGrchr06G1266, the expression of which increased 16.21-fold after 3d of treatment. We cloned and conducted bioinformatics analysis of three candidate genes, both of which contained conserved motifs and several cis-acting elements related to stress response, PGrchr06G1266 contained the most elements.DiscussionPgGT1 was confirmed to catalyze the C-3 position of platycodin D and only eight amino acids showed differences between gene PGr008G1527 and PgGT1, which means PGr008G1527 may be able to catalyze the C-3 position of platycodin D in the same manner as PgGT1. Seven genes were highly expressed in the roots, stems, and leaves, these genes may play important roles in the development of the roots, stems, and leaves of P. grandiflorus. Three genes were highly responsive to drought stress, among which the expression of PGrchr06G1266 was increased 16.21-fold after 3d of drought stress treatment, indicating that PGrchr06G1266 plays an important role in drought stress tolerance. To summarize, this study laied the foundation to better understand the molecular bases of responses to drought stress and the biosynthesis of platycodin.

Identification and characterization of camptothecin tailoring enzymes in Nothapodytes tomentosa.

Camptothecin is a complex monoterpenoid indole alkaloid with remarkable antitumor activity. Given that two C-10 modified camptothecin derivatives, topotecan and irinotecan, have been approved as potent anticancer agents, there is a critical need for methods to access other aromatic ring-functionalized congeners (e.g., C-9, C-10, etc.). However, contemporary methods for chemical oxidation are generally harsh and low-yielding when applied to the camptothecin scaffold, thereby limiting the development of modified derivatives. Reported herein, we have identified four tailoring enzymes responsible for C-9 modifications of camptothecin from Nothapodytes tomentosa, via metabolomic and transcriptomic analysis. These consist of a cytochrome P450 (NtCPT9H) which catalyzes the regioselective oxidation of camptothecin to 9-hydroxycamptothecin, as well as two methyltransferases (NtOMT1/2, converting 9-hydroxycamptothecin to 9-methoxycamptothecin), and a uridine diphosphate-glycosyltransferase (NtUGT5, decorating 9-hydroxycamptothecin to 9-β-D-glucosyloxycamptothecin). Importantly, the critical residues that contribute to the specific catalytic activity of NtCPT9H have been elucidated through molecular docking and mutagenesis experiments. This work provides a genetic basis for producing camptothecin derivatives through metabolic engineering. This will hasten the discovery of novel C-9 modified camptothecin derivatives, with profound implications for pharmaceutical manufacture.

Cloning, Expression, and Functional Characterization of Two Highly Efficient Flavonoid-di-O-glycosyltransferases ZmUGT84A1 and ZmUGT84A2 from Maize (Zea mays L.).

The maize (Zea mays L.) glycosyltransferase family 1 comprises many uridine diphosphate glycosyltransferase (UGT) members. However, UGT activities and biochemical functions have seldom been revealed. In this study, the genes of two flavonoid di-O-glycosyltransferases ZmUGT84A1 and ZmUGT84A2 were cloned from maize plant and expressed in Escherichia coli. Phylogenetic analysis showed that the two enzymes were homologous to AtUGT84A1 and AtUGT84A3. The two recombinant enzymes showed a high conversion rate of luteolin to its glucosides, mainly 4',7-di-O-glucoside and minorly 3',7-di-O-glucoside in two-step glycosylation reactions in vitro. Moreover, the recombinant ZmUGT84A1 and ZmUGT84A2 had a broad substrate spectrum, converting eriodictyol, naringenin, apigenin, quercetin, and kaempferol to monoglucosides and diglucosides. The highly efficient ZmUGT84A1 and ZmUGT84A2 may be used as a tool for the effective synthesis of various flavonoid O-glycosides and as markers for crop breeding to increase O-glycosyl flavonoid content in food.

Uridine Diphosphate-Glycosyltransferase RpUGT344D38 Contributes to λ-Cyhalothrin Resistance in Rhopalosiphum padi.

Uridine diphosphate-glycosyltransferase (UGT) is a key phase II enzyme in the insect detoxification system. Pyrethroids are commonly used to control the destructive wheat aphid Rhopalosiphum padi. In this study, we found a highly expressed UGT gene, RpUGT344D38, in both λ-cyhalothrin (LCR)- and bifenthrin (BTR)-resistant strains of R. padi. After exposure to λ-cyhalothrin and bifenthrin, the expression levels of RpUGT344D38 were significantly increased in the resistant strains. Knockdown of RpUGT344D38 did not affect the resistance of BTR, but it did significantly increase the susceptibility of LCR aphids to λ-cyhalothrin. Molecular docking analysis demonstrated that RpUGT344D38 had a stable binding interaction with both bifenthrin and λ-cyhalothrin. The recombinant RpUGT344D38 was able to metabolize 50% of λ-cyhalothrin. This study provides a comprehensive analysis of the role of RpUGT344D38 in the resistance of R. padi to bifenthrin and λ-cyhalothrin, contributing to a better understanding of aphid resistance to pyrethroids.

Spatial and Temporal Disparity Analyses of Glycosylated Benzaldehyde and Identification and Expression Pattern Analyses of Uridine Diphosphate Glycosyltransferase Genes in Prunus mume.

The species Prunus mume consists of uniquely aromatic woody perennials with large amounts of free aromatic substances in the flower cells. Uridine diphosphate glycosyltransferase (UGT) modifies these free aromatic substances into water-soluble glycoside-bound volatiles (GBVs) which play an important role in regulating the use of volatiles by plants for information exchange, defense, and stress tolerance. To investigate the changes in the glycosidic state of aromatic substances during the flowering period of P. mume and discern the location and expression of glycoside synthesis genes, we extracted and enzymatically hydrolyzed GBVs of P. mume and then utilized gas chromatography-mass spectrometry (GC-MS) to characterize and analyze the types and contents of GBV glycosides. Further, we identified and classified the members of the UGT gene family of P. mume using the bioinformatic method and analyzed the correlation between the expression of the UGT family genes in P. mume and the changes in glycosidic content. The results showed that the benzenoids were the main aromatic substance that was glycosylated during flowering in P. mume and that glycosidic benzaldehyde was the most prevalent compound in different flower parts and at different flowering stages. The titer of glycoside benzaldehyde gradually increased during the bud stage and reached the highest level at the big bud stage (999.6 μg·g-1). Significantly, titers of glycoside benzaldehyde significantly decreased and stabilized after flowering while the level of free benzaldehyde, in contrast, significantly increased and then reached a plateau after the flowering process was completed. A total of 155 UGT family genes were identified in the P. mume genome, which were divided into 13 subfamilies (A-E, G-N); according to the classification of Arabidopsis thaliana UGT gene subfamilies, the L subfamily contains 17 genes. The transcriptome analysis showed that PmUGTL9 and PmUGTL13 were highly expressed in the bud stage and were strongly correlated with the content of the glycosidic form of benzaldehyde at all stages of flowering. This study provides a theoretical basis to elucidate the function of UGT family genes in P. mume during flower development, to explore the mechanism of the storage and transportation of aromatic compounds in flower tissues, and to exploit industrial applications of aromatic products from P. mume.

Similarities in Structure and Function of UDP-Glycosyltransferase Homologs from Human and Plants.

The uridine diphosphate glycosyltransferase (UGT) superfamily plays a key role in the metabolism of xenobiotics and metabolic wastes, which is essential for detoxifying those species. Over the last several decades, a huge effort has been put into studying human and mammalian UGT homologs, but family members in other organisms have been explored much less. Potentially, other UGT homologs can have desirable substrate specificity and biological activities that can be harnessed for detoxification in various medical settings. In this review article, we take a plant UGT homology, UGT71G1, and compare its structural and biochemical properties with the human homologs. These comparisons suggest that even though mammalian and plant UGTs are functional in different environments, they may support similar biochemical activities based on their protein structure and function. The known biological functions of these homologs are discussed so as to provide insights into the use of UGT homologs from other organisms for addressing human diseases related to UGTs.

[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.

Sublethal effects of chlorantraniliprole on biological characteristics, detoxifying enzyme activity and gene expression profile in the Allium mongolicum Regel leaf beetle Galeruca daurica (Coleoptera: Chrysomelidae)

AbstractAllium mongolicum Regel leaf beetle, Galeruca daurica (Coleoptera: Chrysomelidae) is rampantly harmful in Inner Mongolia grassland. However, the current management strategies for this pest still heavily rely on chemical control using traditional insecticides or those with novel action. In the study, we conducted an indoor bioassay to evaluate the sublethal effects of chlorantraniliprole on the biological characteristics. Additionally, we assessed the activity of detoxification enzymes, specifically the primary enzymes carboxylesterase (CarE) and cytochrome P450, and insect's secondary metabolic enzymes, namely glutathione S‐transferase (GST) and uridine diphosphate glycosyltransferase (UGT), as well as its gene expression profile. The developmental period of the 3rd instar larvae of G. daurica was significantly prolonged after treatment with chlorantraniliprole, which negatively affected the hatching, pupation and diapause rates as well as their body weight. The larvae showed different dynamics of the enzyme activities within 24 h of chlorantraniliprole treatment. Different sublethal concentrations of chlorantraniliprole showed an inducing effect on the activities, while no significant difference in activity was observed for P450. Expression profiling of detoxifying enzyme genes screened by transcriptome data has revealed that 6 CarE, 2 GST, 7 UGT and 31 P450 genes were up‐regulated. In conclusion, chlorantraniliprole's sublethal effect on 3rd instar G. daurica larvae resulted in a deceleration of their growth and developmental processes and significantly increased the activity levels of CarE, GST and UGT. The detoxifying proteins encoded by up‐regulation genes may involve in the detoxification metabolism of chlorantraniliprole. Our results provide a basis for understanding the molecular mechanisms of chlorantraniliprole action and detoxification in this key grassland insect pest.

Long-day photoperiodic requirements for steviol glycosides and gibberellins biosynthesis and bio-sweetener levels optimization in Stevia rebaudiana Bertoni

Stevia rebaudiana, as a short-day perennial, produces steviol glycoside (StGlys) metabolites recognized as healthy natural sweeteners, ca. 300 times sweeter than sucrose, being stevioside (ST) and rebaudioside A (Reb-A), the main StGlys for the natural sweetener demanding industries. Nevertheless, discussions persist regarding the ideal light regimen and respective StGlys biosynthesis regulation in S. rebaudiana aimed at enhancing and optimizing the StGlys large-scale production for industrial purposes. The present work reports on the biosynthesis and the contents of StGlys in leaves and stems in different developmental stages of S. rebaudiana plants submitted to different photoperiods. The final contents resulted from the response of genes in the shared StGlys and gibberellins (GAs) biosynthesis pathway, notably showing differential gene expression patterns in leaves and stems at the different developmental stages. Plants developing under 16/8 h (light/dark) photoperiod increased StGlys contents resulting from the upregulation of S. rebaudiana kaurenoic acid 13-hydroxylase (SrKAH) and uridine diphosphate glycosyltransferase (SrUGTs) genes in association with the downregulation of GAs biosynthesis-related genes in leaves. In contrast, the positive overall regulation of GAs biosynthesis genes in stems appeared as a requirement of GAs for stem elongation and plant growth. Conclusively, the research reports a deeper understanding of transcriptional responses, biosynthesis, and ST and Reb-A yield mechanisms in S. rebaudiana plants under different photoperiodic conditions. Altogether, leading to the potential optimization of commercial production, extraction, and purification while highlighting photoperiodic-responsive StGlys/GAs genes as potential molecular targets for metabolic engineering and future development of superior S. rebaudiana genotypes aiming further large-scale StGlys production and yield to attend the continuous increasing commercial and industrial demand.

Multiple Clonostachys rosea UDP-Glycosyltransferases Contribute to the Production of 15-Acetyl-Deoxynivalenol-3-O-Glycoside When Confronted with Fusarium graminearum.

Mycotoxins, derived from toxigenic fungi such as Fusarium, Aspergillus, and Penicillium species have impacted the human food chain for thousands of years. Deoxynivalenol (DON), is a tetracyclic sesquiterpenoid type B trichothecene mycotoxin predominantly produced by F. culmorum and F. graminearum during the infection of corn, wheat, oats, barley, and rice. Glycosylation of DON is a protective detoxification mechanism employed by plants. More recently, DON glycosylating activity has also been detected in fungal microparasitic (biocontrol) fungal organisms. Here we follow up on the reported conversion of 15-acetyl-DON (15-ADON) into 15-ADON-3-O-glycoside (15-ADON-3G) in Clonostachys rosea. Based on the hypothesis that the reaction is likely being carried out by a uridine diphosphate glycosyl transferase (UDP-GTase), we applied a protein structural comparison strategy, leveraging the availability of the crystal structure of rice Os70 to identify a subset of potential C. rosea UDP-GTases that might have activity against 15-ADON. Using CRISPR/Cas9 technology, we knocked out several of the selected UDP-GTases in the C. rosea strain ACM941. Evaluation of the impact of knockouts on the production of 15-ADON-3G in confrontation assays with F. graminearum revealed multiple UDP-GTase enzymes, each contributing partial activities. The relationship between these positive hits and other UDP-GTases in fungal and plant species is discussed.

Proteomics Identified UDP-Glycosyltransferase Family Members as Pro-Viral Factors for Turnip Mosaic Virus Infection in Nicotiana benthamiana.

Viruses encounter numerous host factors that facilitate or suppress viral infection. Although some host factors manipulated by viruses were uncovered, we have limited knowledge of the pathways hijacked to promote viral replication and activate host defense responses. Turnip mosaic virus (TuMV) is one of the most prevalent viral pathogens in many regions of the world. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach to characterize cellular protein changes in the early stages of infection of Nicotiana benthamiana by wild type and replication-defective TuMV. A total of 225 differentially accumulated proteins (DAPs) were identified (182 increased and 43 decreased). Bioinformatics analysis showed that a few biological pathways were associated with TuMV infection. Four upregulated DAPs belonging to uridine diphosphate-glycosyltransferase (UGT) family members were validated by their mRNA expression profiles and their effects on TuMV infection. NbUGT91C1 or NbUGT74F1 knockdown impaired TuMV replication and increased reactive oxygen species production, whereas overexpression of either promoted TuMV replication. Overall, this comparative proteomics analysis delineates the cellular protein changes during early TuMV infection and provides new insights into the role of UGTs in the context of plant viral infection.

Identification of UGT85A glycosyltransferases associated with volatile conjugation in grapevine (Vitis vinifera × Vitislabrusca)

Glycosylated volatiles are considered as hidden aroma precursors that affect fruit flavor and are involved in plant defense response. Several uridine-diphosphate (UDP)-glycosyltransferase (UGT) members related to the glycosylation of volatiles have been identified from grapevine berries. Although grapevine leaves produced much higher levels of glycosylated volatiles than berries, UGTs responsible for the production of glycosylated volatiles in leaves have not yet been identified. Here, eight novel UGT genes were cloned, among these four members that belong to UGT85A family (named UGT85A24, UGT85A25, UGT85A26, and UGT85A27) were localized in the cytoplasm and their recombinant proteins showed activity toward volatiles detected in grapevine, with geraniol as the preferred substrate in vitro. Two UGT genes with 98.1% homology differ in R/S-linalool selection. The UGT85A26-catalyzed substrate showed enrichment in S-linalool, whereas UGT85A27 showed a strong preference for R-linalool. UGT85A24 and UGT85A25 were expressed mainly in berries, and UGT85A26 and UGT85A27 were expressed predominantly in grapevine mature leaves and correlated with the highest content of glycosylated volatiles in the mature leaves. Methyl jasmonate (MeJA) induced the expression of UGT85A26 and UGT85A27 as well as the production of linalyl-β-glucoside, citronellyl-β-glucoside and phenethyl-β-glucoside. Transient overexpression of UGT85A26 and UGT85A27 in tobacco leaves led to the accumulation of linalyl-β-glucoside and an increase in citronellyl-β-glucoside in vivo. These results indicate that the expression of UGT85A26 and UGT85A27 is a major regulator affecting the content of glycosylated volatiles in grapevine.

Characterization of UDP‐glycosyltranferase genes and their roles in chlorfenapyr tolerance in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

AbstractGlyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the main pest of mulberry leaves and causes great losses to the sericultural industry in China. Chlorfenapyr is a new type of arylpyrrole insecticide in mulberry fields. Uridine diphosphate–glycosyltransferase (UGT) is the major phase II metabolizing enzymes that play important roles in detoxifying the toxic substances in insects. However, the roles of UGT genes against chemical insecticides in lepidopteran pests still receive less attention. To better understand the mechanism of insecticide tolerance in G. pyloalis, a LC50 of chlorfenapyr to the fourth‐instar larvae was firstly determined. Subsequently, 24 UGT genes were identified from the previous constructed larvae transcriptome of G. pyloalis. Among these, 23 contained full‐length ORFs and shared high homology with Ostrinia furnacalis and Chilo suppressalis. qRT‐PCR validation revealed that four GpylUGTs (GpUGT33AQ1, GpUGT33AS1, GpUGT50A14, and GpUGT40AV1) were significantly upregulated after 24 h of chlorfenapyr treatment. Three GpylUGTs (GpUGT33AS1, GpUGT40AM4, and GpUGT40AK7) were upregulated after 48 h of chlorfenapyr treatment compared to the control groups. To further explore the functions of UGT genes, GpUGT33AS1 was selected and knocked down, which resulted in the decline of UGT expression and significantly increased the mortality of G. pyloalis larvae under LC30 chlorfenapyr exposure. This study revealed that UGTs in G. pyloalis contributed to the tolerance towards chlorfenapyr and provided basic insight into the insecticide detoxification mechanism in lepidopteran pests.

[Identification and expression of uridine diphosphate glycosyltransferase(UGT) gene family from Dendrobium officinale].

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception

Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl β-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl β-d-glucopyranoside arabinosylation to produce HexVic in planta.

Direct Yeast Surface Codisplay of Sequential Enzymes with Complementary Anchor Motifs: Enabling Enhanced Glycosylation of Natural Products.

Yeast surface display is an appealing technique for constructing multienzyme cascades. This technique is commonly achieved using a scaffold for the ordered arrangement of various enzymes. However, this method is typically complicated because scaffold use may engender extra metabolic burden on the cell host. Here, we established a direct yeast surface codisplay strategy by employing two complementary anchor motifs, Agα1 and Pir1. These motifs allow for the codisplay of sequential uridine diphosphate-glycosyltransferase (UGT) and sucrose synthase (SUS) on the surface of Pichia pastoris (syn. Komagataella phaffii) for the glycosylation of natural products. We manipulated the displayed stoichiometry, amount, and assembly order of UGT and SUS by coupling them with anchor motifs. Furthermore, their effect on enzyme activity was thoroughly investigated. The surface-codisplayed strain UGT-Pir-SUS-Agα exhibited greater thermostability than the single-displayed strains and their free counterparts. Moreover, the strain UGT-Pir-SUS-Agα was successfully applied to glycyrrhetinic acid (GA) glycosylation to produce GA-3-O-Glc, with sucrose being the sugar donor in this process. This generated 7.5- to 20- and 5.3-fold higher GA-3-O-Glc concentration compared with the free counterparts (enzyme mass loading of 20-fold in excess) and mixed single-displayed strains of UGT-Agα and SUS-Pir, respectively. This increase was due to the improved biochemical properties and substrate channeling effect of strain UGT-Pir-SUS-Agα. This controllable direct surface codisplay strategy, based on complementary anchor motifs, is readily extendable to other enzyme cascades.

Evaluation of pathways to the C-glycosyl isoflavone puerarin in roots of kudzu (Pueraria montana lobata).

Kudzu ( Pueraria montana lobata) is used as a traditional medicine in China and Southeast Asia but is a noxious weed in the Southeastern United States. It produces both O‐ and C‐glycosylated isoflavones, with puerarin (C‐glucosyl daidzein) as an important bioactive compound. Currently, the stage of the isoflavone pathway at which the C‐glycosyl unit is added remains unclear, with a recent report of direct C‐glycosylation of daidzein contradicting earlier labeling studies supporting C‐glycosylation at the level of chalcone. We have employed comparative mRNA sequencing of the roots from two Pueraria species, one of which produces puerarin (field collected P. montana lobata) and one of which does not (commercial Pueraria phaseoloides ), to identify candidate uridine diphosphate glycosyltransferase (UGT) enzymes involved in puerarin biosynthesis. Expression of recombinant UGTs in Escherichia coli and candidate C‐glycosyltransferases in Medicago truncatula were used to explore substrate specificities, and gene silencing of UGT and key isoflavone biosynthetic genes in kudzu hairy roots employed to test hypotheses concerning the substrate(s) for C‐glycosylation. Our results confirm UGT71T5 as a C‐glycosyltransferase of isoflavone biosynthesis in kudzu. Enzymatic, isotope labeling, and genetic analyses suggest that puerarin arises both from the direct action of UGT71T5 on daidzein and via a second route in which the C‐glycosidic linkage is introduced to the chalcone isoliquiritigenin.

PHARMACOGENETIC ASPECTS OF THE USE OF PERIOPERATIVE DRUGS IN PEDIATRICS. Review

Relevance. It is known that in children the frequency of perioperative critical events due to the introduction of drugs is more than 5%. For example fentanyl, which is used as an adjunct to surgical anesthesia, is a major factor in increasing the number of deaths from opioid overdoses. Among the areas of reducing the frequency of severe adverse reactions to perioperative drugs - taking into account the pharmacogenetic variations of the patient.
 Objective is to consider current data on pharmacogenetic aspects of pharmacokinetics and pharmacodynamics of perioperative drugs.
 Methods. Analysis of the data presented in PubMed by keywords "pharmacogenetics", "general anesthetics", "analgesics", "muscle relaxants", "children". Search depth – 7 years (2014-2020), with a retrospective deepening of some positions until 2002.
 Results. The pharmacogenetic aspects of the pharmacokinetics of perioperative drugs are related to the genes of the enzymes that metabolize them and their transporters. Current data on the prevalence of polymorphic alleles of CYP2C9 genes (provides metabolism of nonsteroidal anti-inflammatory and anticonvulsant drugs) and CYP2D6 (metabolizes opioids, antidepressants, antiemetics) in Europe and, in particular, in Ukraine were presented. Thus, the inactive allele CYP2C19 * 2 was found in 13%, while the allele of increased activity CYP2C19 * 17 - in 25% of the population of Ukraine; allele with lost CYP2D6 function (CYP2D6 * 4) - in 18.6% of Ukrainians. Homozygotes with CYP2C9 * 3 polymorphism metabolize nonsteroidal anti-inflammatory drugs much more slowly than wild-type carriers, which may lead to their accumulation and side effects. The analgesic effect of codeine occurs only after it is metabolized in the liver by CYP2D6 to morphine. Respiratory depression, apnea and death may occur in patients with excessive metabolic rate even after a single dose of codeine; however, was noted the ineffectiveness of ondansetron due to a decrease in its concentration in the blood in thesepatients. Concomitant use of midazolam and fentanyl may prolong the effect of fentanyl by  competing metabolism of midazolam by the hepatic enzyme CYP3A4, especially in patients with low metabolism. Plasma butyrylcholinesterase deficiency reduces succinylcholine inactivation in 1 in 1,500 people. Changing the genotype of uridine diphosphate glycosyltransferase causes increased glucuronidation of morphine, which may lead to a decrease in its effectiveness. As an example of pharmacogenetic changes in pharmacodynamics can be considered malignant hyperthermia, which occurs due to exposure to volatile anesthetics and depolarizing muscle relaxants in mutations in the RYR1 gene. Carriers of the minor allele (G) of the 5HT2A rs6313 gene require less propofol and 40% less time to start induction of anesthesia.
 Conclusions. The efficacy and safety of perioperative drugs are largely related to the pharmacogenetic aspects of their pharmacokinetics, especially mutations in genes of enzymes of the metabolism. Mutations in the genes of volatile anesthetic receptors and depolarizing muscle relaxants when their used cause a risk of malignant hyperthermia. The application of pharmacogenetics is the need to titrate drugs that have significant variability of action depending on the genotype. It is advisable to avoid the use of drugs with a high risk of pharmacogenetic reactions, if other alternative drugs are available.

Identification and Characterization of UDP-Glycosyltransferase Genes in a Cerambycid Beetle, Pharsalia antennata Gahan, 1894 (Coleoptera: Cerambycidae)

The cerambycid beetle, Pharsalia antennata Gahan, 1894 (Coleoptera: Cerambycidae), is a wood-boring pest that spends most of its life cycle in the trunks or under the bark of trees. These distinctive biological characteristics make it likely that this beetle will encounter a number of plant defensive compounds, coupled with a broad range of host plants, possibly resulting in the overexpression or expansion of uridine diphosphate (UDP)-glycosyltransferase (UGT) genes. Here, we identified and characterized the UGT gene family in P. antennata through transcriptome data, sequence and phylogenetic analyses, and PCR and homology modeling approaches. In total, 59 transcripts encoding UGTs were identified, 34 of which harbored full-length sequences and shared high conservation with the UGTs of Anoplophora glabripennis. Of the 34 PantUGTs, only 31.78% amino acid identity was observed on average, but catalytic and sugar binding residues were highly conserved. Phylogenetic analyses revealed four Cerambycidae-specific clades, including 30 members from P. antennata. Combining the transcriptome and PCR data showed that PantUGTs had a wide tissue expression, and the majority of the genes were presented mainly in antennae or abdomens, suggesting their putative roles in olfaction and detoxification. This study provides, for the first time, information on the molecular and genetic basis of P. antennata, greatly enhancing our knowledge of the detoxification-related UGT gene family.