UDP-glycosyltransferases Research Articles

Construction and Optimization of a Yeast Cell Factory for Producing Active Unnatural Ginsenoside 3β-O-Glc2-DM.

Ginsenosides are major active components of Panax ginseng, which are generally glycosylated at C3-OH and/or C20-OH of protopanaxadiol (PPD) and C6-OH and/or C20-OH of protopanaxatriol. However, the glucosides of dammarenediol-II (DM), which is the direct precursor of PPD, have scarcely been separated from P. ginseng. Because different positions and numbers of the hydroxyl and glycosyl groups lead to a diversity of structure and function of the ginsenosides, it can be inferred that DM glucosides may have different pharmacological activities compared with natural ginsenosides. Herein, we first constructed the cell factory for de novo biosynthesis of 3-O-(β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl)-dammar-24-ene-3β,20S-diol (3β-O-Glc2-DM) by introducing the codon-optimized genes encoding dammarenediol-II synthase, two UDP-glycosyltransferases (UGTs) including UGT74AC1-M7 from Siraitia grosvenorii and UGTPg29 from P. ginseng in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titer of 3β-O-Glc2-DM was then increased from 18.9 to 148.0 mg/L by several metabolic engineering strategies including overexpressing the rate-limiting enzymes of triterpenoid biosynthesis, balancing carbon flux of biosynthetic pathways of triterpenoid and ergosterol, and engineering endoplasmic reticulum. Furthermore, the 3β-O-Glc2-DM titer of 766.3 mg/L was achieved through fed-batch fermentation in a 3-L bioreactor. Finally, in vitro assays demonstrated that 3β-O-Glc2-DM exhibited a protective effect on H/R-induced cardiomyocyte damage. This work provides a feasible approach for production of 3β-O-Glc2-DM as a potential cardioprotective drug candidate.

Identification of putative genes for caffeoylated flavonoid glycoside biosynthesis in Pseudognaphalium affine

Pseudognaphalium affine (D. Don) Anderberg, commonly found in East Asia, has extensive applications as both a traditional medicine and a vegetable in China. The caffeoylated flavonoid glycosides produced by P. affine exhibit remarkable anti-complement activities. Although these compounds have potential therapeutic value, the biosynthetic pathway responsible for their production remains largely unknown. To elucidate the key catalytic steps involved in caffeoylated flavonoid glycoside biosynthesis, we conducted a comprehensive analysis of the full-length transcriptome of P. affine. Further phylogenetic tree analysis predicted potential UDP glycosyltransferase (UGT) and BAHD acyltransferase (BAHD-AT) related with caffeoylated flavonoid glycoside biosynthesis. Subsequently, enzyme assay led to the discovery of PaUGT23 as a key enzyme responsible for the glycosylation of hydroxy groups in flavonoids, resulting in the formation of luteolin-4′-O-glucoside, luteolin-7-O-glucoside, quercetin-4′-O-glucoside, quercetin-7-O-glucoside, and apigenin-7-O-glucoside, while PaBAHD21 was found to catalyze the caffeoylation of flavonoid glycosides, resulting in the formation of luteolin 4′-O-β-D-(6″-E-caffeoyl)-glucopyranoside, quercetin 4′-O-β-D-(6″-E-caffeoyl)-glucopyranoside, apigenin 4′-O-β-D-(6″-E-caffeoyl)-glucopyranoside and apigenin 7-O-β-D-(6″-E-caffeoyl)-glucopyranoside. Moreover, their catalytic activities were verified in vivo by transient transfection experiment. This study presents the first comprehensive analysis of the full-length transcriptome in P. affine, providing significant insights into the biosynthesis and accumulation mechanisms of bioactive caffeoylated flavonoid glycosides.

Chemical and Transcriptomic Analyses Provide New Insights into Key Genes for Ginsenoside Biosynthesis in the Rhizome of Panax japonicus C. A. Meyer.

Panax japonicus C. A. Meyer is renowned for its significant therapeutic effects and is commonly used worldwide. Its active ingredients, triterpenoid saponins, show variation in content among different tissues. The tissue-specific distribution of saponins is potentially related to the expression of vital genes in the biosynthesis pathway. In this study, the contents of five saponins (ginsenoside Ro, chikusetsusaponin IV, chikusetsusaponin IVa, ginsenoside Rg1, and ginsenoside Rb1) in three different tissues were determined by HPLC. Transcriptome sequencing analysis identified differentially expressed genes (DEGs) involved in triterpenoid saponin biosynthesis, highlighting significant correlations between saponin contents and the expression levels of 10 cytochrome p450 monooxygenase (CYP) and 3 UDP-glycosyltransferase (UGT) genes. Cloning, sequencing, and prokaryotic expression of UGT genes confirmed the molecular weights of UGT proteins. Gene sequence alignment and phylogenetic analysis provided preliminary insights into UGT gene functions. Meanwhile, the function of one UGT gene was characterized in the yeast. These findings advance our understanding of the triterpenoid saponin biosynthesis in P. japonicus and support future research in traditional Chinese medicine (TCM) and synthetic biology.

Engineering the Substrate Specificity of UDP-Glycosyltransferases for Synthesizing Triterpenoid Glycosides with a Linear Trisaccharide as Aided by Ancestral Sequence Reconstruction.

Triterpenoids have wide applications in the pharmaceutical and agricultural industries. The glycosylation of triterpenoids catalyzed by UDP-glycosyltransferases (UGTs) is a crucial method for producing valuable derivatives with enhanced functions. However, only a few UDP-glucosyltransferases have been reported to synthesize the rare triterpenoids with linear-chain trisaccharide at C3-OH. This study revealed that the UGT91H subfamily primarily contributed to the 2"-O-glycosylation of triterpenoids with high regioselectivity, then the substrate scope was further expanded by ancestral sequence reconstruction (ASR). With ancestral enzyme UGT91H_A1 as a model, the sequence-structure-function relationship was explored. A RTAS loop (R212/T213/A214/S215) was identified to affect the substrate specificity of UGT91H_A1. Transferring this RTAS loop to the corresponding position of UGT91H enzymes successfully expanded their substrate spectra. The functional role of RTAS loop was further elucidated by molecular dynamics simulation and quantum mechanical computation. UGT91H_A1 was applied to the low-cost synthesis of terpenoid rhamnosides with a linear trisaccharide in combining with a self-sufficient UDP-rhamnose regeneration system. Finally, we developed a phylogeny-based platform to efficiently mining new UGT91Hs from plant genomic data. This study provided robust biocatalysts for synthesizing various triterpenoid glycosides with a linear trisaccharide and demonstrated ASR as an efficient tool in engineering the function of UDP-glycosyltransferases.

Transcriptional responses of detoxification genes to coumaphos in a nontarget species, Galleria mellonella (greater wax moth) (Lepidoptera: Pyralidae), in the beehive environment

The greater wax moth Galleria mellonella is a cosmopolitan pest of hives of the western honey bee Apis mellifera, where it remains exposed to varroicides applied by beekeepers in past decades as pest management chemicals for control of Varroa destructor, a devastating ectoparasite of bees. The prolonged presence of coumaphos residues, an organophosphate varroicide, in beeswax may be responsible for current levels of tolerance exhibited by G. mellonella, a non-target species that infests beehives. In this study, a field-collected strain of waxworms exhibited a higher LC50 value for coumaphos than that of a laboratory strain that had not been continuously exposed to coumaphos residues at field concentrations. Despite its higher tolerance for coumaphos, the field strain experienced growth inhibition at ecologically relevant concentration of coumaphos. Moreover, at low environmental concentrations that did not alter growth, detoxification gene expression levels were substantially altered. RNA-Seq analysis revealed 1181 and 658 differentially expressed genes in fat body and midgut, respectively, with 378 and 186 of those genes upregulated. This large-scale upregulation encompassed 21 genes encoding cytochrome P450 monooxygenases (CYPs), 13 encoding UDP-glycosyltransferases (UGTs), 5 encoding glutathione-S-transferases (GSTs), 2 encoding carboxylesterases (COEs), and 2 encoding ABC transporters (ABCs) in either tissue. Expression analysis of 13 selected candidate detoxification genes by RT-qPCR was consistent with their expression from RNA-Seq data. In sum, our results indicate that long-lasting pesticide residues in beeswax from past Varroa mite management may continue to act as selective agents on detoxification systems of hive residents other than the initial target species and that multiple resistance mechanisms to these chemicals may coexist within the beehive fauna.

Genome-wide analysis of UDP-glycosyltransferase family in Citrus sinensis and characterization of a UGT gene encoding flavonoid 1–2 rhamnosyltransferase

UDP-glycosyltransferases (UGTs) play a crucial role in the glycosylation of secondary metabolites in plants, which is of significant importance for growth and response to biotic or abiotic stress. Despite the wide identification of UGT family members in various species, limited information is available regarding this family in citrus. In this study, we identified 87 UGT genes from the Citrus sinensis genome and classified them into 14 groups. We characterized their gene structures and motif compositions, providing insights into the molecular basis underlying discrepant functions of UGT genes within each evolutionary branch. Tandem duplication events were found to be the main driving force behind UGT gene expansion. Additionally, we identified numerous cis-acting elements in the promoter region of UGT genes, including those responsive to light, growth factors, phytohormones, and stress conditions. Notably, light-responsive elements were found with a frequency of 100 %. We elucidated the expression pattern of UGTs during fruit development in Citrus aurantium using RNA-seq and quantitative real-time PCR (qRT-PCR), revealing that 10 key UGT genes are closely associated with biosynthesis of bitter flavanone neohesperidosides (FNHs). Furthermore, we identified Ca1,2RhaT as a flavonoid 1–2 rhamnosyltransferase (1,2RhaT) involved in FNHs biosynthesis for the first time. Isolation and functional characterization of the gene Ca1,2RhaT from Citrus aurantium in vitro and in vivo indicated that Ca1,2RhaT encoded a citrus 1,2RhaT and possessed rhamnosyl transfer activities. This work provides comprehensive information on the UGT family while offering new insights into understanding molecular mechanisms regulating specific accumulation patterns of FNHs or non-bitter flavanone rutinosides (FRTs) in citrus.

High-quality haplotype-resolved chromosome assembly provides evolutionary insights and targeted steviol glycosides (SGs) biosynthesis in Stevia rebaudiana Bertoni.

Stevia rebaudiana Bertoni is popular source of plant-derived low/no-calorie natural sweeteners (LNCSs), collectively known as steviol glycosides (SGs). Nevertheless, genetic predisposition for targeted biosynthesis of SGs is complex due to multi-substrate functionality of key uridine diphosphate glycosyltransferases (UGTs). Here, we created a high-quality monoploid assembly of 1.34 Gb with N50 value of 110 Mb, 55 551 predicted protein-coding genes, and ~80% repetitive regions in Rebaudioside-A (Reb-A) enriched cultivar of S. rebaudiana. Additionally, a haplotype-based chromosome assembly consisting of haplotype A and haplotype B with an overall genome size of 2.33Gb was resolved, harbouring 639 634 variants including single nucleotide polymorphisms (SNPs), indels and structural variants (SVs). Furthermore, a lineage-specific whole genome duplication analysis revealed that gene families encoding UGTs and Cytochrome-P450 (CYPs) were tandemly duplicated. Additionally, expression analysis revealed five tandemly duplicated gene copies of UGT76G1 having significant correlations with Reb-A content, and identified key residue (leu200val) in the glycosylation of Reb-A. Furthermore, missense variations identified in the acceptor region of UGT76G1 in haplotype resolve genome, transcriptional and molecular docking analysis were confirmed with resequencing of 10 diverse stevia genotypes (~25X). Gene regulatory network analysis identified key transcription factors (MYB, bHLH, bZIP and AP2-ERF) as potential regulators of SG biosynthesis. Overall, this study provides haplotype-resolved chromosome-level genome assembly for genome editing and enhancing breeding efforts for targeted biosynthesis of SGs in S. rebaudiana.

Knocking Out of UDP-Glycosyltransferase Gene UGT2B10 via CRISPR/Cas9 in Helicoverpa armigera Reveals Its Function in Detoxification of Insecticides.

The role of insect UDP-glycosyltransferases (UGTs) in the detoxification of insecticides has rarely been reported. A UGT gene UGT2B10 was previously found overexpressed in a fenvalerate-resistant strain of Helicoverpa armigera. Herein, UGT2B10 was cloned, and its involvement in insecticide detoxification was investigated. UGT2B10 was highly expressed in the larvae, mainly in the fat body and midgut. Treatment with UGT inhibitors 5-nitrouracil and sulfinpyrazone significantly enhanced the fenvalerate toxicity. Knocking down UGT2B10 by RNAi significantly increased the larvae mortality by 17.89%. UGT2B10 was further knocked out by CRISPR/Cas9, and a homozygous strain (HD-dUGT2B10) with a C-base deletion at exon 2 was obtained. The sensitivity of HD-dUGT2B10 to fenvalerate, deltamethrin, cyantraniliprole, acetamiprid, and lufenuron increased significantly, with sensitivity index increased 2.523-, 2.544-, 2.250-, 2.473-, and 3.556-fold, respectively. These results suggested that UGT2B10 was involved in the detoxification of H. armigera to insecticides mentioned above, shedding light upon further understanding of the detoxification mechanisms of insecticides by insect UGTs.

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

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

Comparative genomics uncovers the evolutionary dynamics of detoxification and insecticide target genes across 11 phlebotomine sand flies.

Sand flies infect more than one million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: Cytochrome P450s (CYPs), Glutathione-S-Transferases (GSTs), UDP-Glycosyltransferases (UGTs), Carboxyl/Cholinesterases (CCEs) and ATP-Binding Cassette (ABC) transporters. Using this comparative approach we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of a) CYP4G genes involved in cuticular hydrocarbon biosynthesis, b) ABCB genes involved in xenobiotic toxicity and c) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and Voltage Gated Sodium Channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are associated with insecticide resistance in the major malaria vectors Anopheles gambiae s.l. and Anopheles funestus

Malaria remains one of the highest causes of morbidity and mortality, with 249 million cases and over 608,000 deaths in 2022. Insecticides, which target the Anopheles mosquito vector, are the primary method to control malaria. The widespread nature of resistance to the most important insecticide class, the pyrethroids, threatens the control of this disease. To reverse the stall in malaria control there is urgent need for new vector control tools, which necessitates understanding the molecular basis of pyrethroid resistance. In this study we utilised multi-omics data to identify uridine-diphosphate (UDP)-glycosyltransferases (UGTs) potentially involved in resistance across multiple Anopheles species. Phylogenetic analysis identifies sequence similarities between Anopheline UGTs and those involved in agricultural pesticide resistance to pyrethroids, pyrroles and spinosyns. Expression of five UGTs was characterised in An. gambiae and An. coluzzii to determine constitutive over-expression, induction, and tissue specificity. Furthermore, a UGT inhibitor, sulfinpyrazone, restored susceptibility to pyrethroids and DDT in An. gambiae, An. coluzzii, An. arabiensis and An. funestus, the major African malaria vectors. Taken together, this study provides clear association of UGTs with pyrethroid resistance as well as highlighting the potential use of sulfinpyrazone as a novel synergist for vector control.

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

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

Characterization and protein engineering of a novel UDP-glycosyltransferase involved in pseudoginsenoside Rt5 biosynthesis from Panax japonicus

As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

RpUGT344J7 is involved in the reproduction switch of Rhopalosiphum padi with holocyclic life cycle.

Many aphid species exhibit both cyclical parthenogenesis (CP) and the obligate parthenogenesis (OP) life history, which are genetically determined. In CP aphid lineages, the parthenogenetic individuals can switch from asexual to sexual reproduction quickly in response to environmental factors such as changes in photoperiod and temperature. However, the OP aphid lineages do not undergo sexual reproduction under any conditions. So far, mechanisms underlying the reproduction switch in CP aphids have not been fully elucidated. Rhopalosiphum padi, a serious worldwide insect pest of wheat, has both CP and OP lineages. Uridine diphosphate-glycosyltransferases (UGTs) are enzymes that participate in the metabolic detoxification of xenobiotics. Here, we identified 43 RpUGT genes from R. padi genome and transcriptome sequences, and found that: (1) the UGT content of the CP lineage was significantly higher than that in the OP lineage at the key time points when CP lineage mainly produce virginoparae, gynoparae, and males under inducing condition, while there were no significant difference under normal conditions; (2) RpUGT344J7 gene was highly expressed during the time points when CP lineages produce gynopara and males; (3) the critical time points for CP lineages to produce virginoparaee, gynoparae, and males were affected when the CP lineages were injected with dsRpUGT344J7; (4) the knockdown of RpUGT344J7 caused a significant reduction in the total number of virginoparae, gynoparae, and males in the offspring under inducing condition. The findings contribute to our understanding of the molecular mechanisms underlying the quick shift from asexual to sexual reproduction in aphid species.

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.

Expression and Functional Analysis of Two Cytochrome P450 Monooxygenase Genes and a UDP-Glycosyltransferase Gene Linked with Thiamethoxam Resistance in the Colorado Potato Beetle.

Cytochrome P450 monooxygenases (P450s) and UDP-glycosyltransferases (UGTs) are involved in the evolution of insecticide resistance. Leptinotarsa decemlineata (Say), the Colorado potato beetle (CPB), is a notorious insect that has developed resistance to various insecticides including neonicotinoids. This study investigated whether the differentially expressed P450 genes CYP9Z140 and CYP9AY1 and UGT gene UGT321AP1, found in our transcriptome results, conferred resistance to thiamethoxam in L. decemlineata. Resistance monitoring showed that the sampled field populations of L. decemlineata adults collected from Urumqi City and Qapqal, Jimsar, and Mulei Counties of Xinjiang in 2021-2023 developed low levels of resistance to thiamethoxam with resistance ratios ranging from 6.66- to 9.52-fold. Expression analyses indicated that CYP9Z140, CYP9AY1, and UGT321AP1 were significantly upregulated in thiamethoxam-resistant populations compared with susceptible populations. The expression of all three genes also increased significantly after thiamethoxam treatment compared with the control. Spatiotemporal expression patterns showed that the highest expression of CYP9Z140 and CYP9AY1 occurred in pupae and the midgut, whereas UGT321AP1 was highly expressed in adults and Malpighian tubules. Knocking down all three genes individually or simultaneously using RNA interference increased the sensitivity of adult L. decemlineata to thiamethoxam. These results suggest that overexpression of CYP9Z140, CYP9AY1, and UGT321AP1 contributes to the development of thiamethoxam resistance in L. decemlineata and provides a scientific basis for improving new resistance management of CPB.

The UDP-glycosyltransferase gene OsUGT706E2 negatively regulates rice tolerance to blast disease and abiotic stresses

Despite the crucial role of UDP-glycosyltransferases (UGTs) in various stress responses in plants, their biological functions in rice (Oryza sativa L.) remain poorly understood. In this study, we introduce a novel gene, OsUGT706E2, which is expressed at significantly higher levels in wild rice compared to cultivated rice. OsUGT706E2 is active in multiple tissues and localizes to both the nucleus and cytoplasm. Its transcription is notably up-regulated in response to cold stress, blast disease, and several hormone treatments. Overexpression of OsUGT706E2 markedly reduces seedling tolerance to blast disease, cold, and osmotic stress, whereas knocking out OsUGT706E2 enhances seedling tolerance to blast disease and osmotic stress. The expression levels of stress-related genes in OsUGT706E2 overexpressing plants were significantly lower compared to wild-type plants following stress treatment. Conversely, OsUGT706E2 knockout plants exhibited markedly higher expression levels of these genes than the control plants after stress treatment. Metabolome analysis further indicated that OsUGT760E2 influences metabolite content in rice. Specifically, OsUGT760E2-overexpressing seedlings had higher amino acid content and lower lipid content compared to wild-type seedlings, while OsUGT760E2-knockout seedlings showed lower amino acid content and higher lipid content than control seedlings. These findings suggest that OsUGT760E2 negatively regulates resistance to blast disease as well as tolerance to cold and osmotic stress in rice. As a result, OsUGT760E2 represents a promising target for enhancing rice stress tolerance through gene editing approaches.

Advances and Challenges in Biomanufacturing of Glycosylation of Natural Products

Glycosylation is one of the most common and important modifications in natural products (NPs), which can alter the biological activities and properties of NPs, effectively increase structural diversity, and improve pharmacological activities. The biosynthesis of glycosylation in natural products involves multiple complex biological processes, which are coordinated by many enzymes. UDP-glycosyltransferases (UGTs) play a crucial role in glycosylation modification, and have attracted long-term and widespread research attention. UGTs can catalyze the O-, C-, S-, and N-glycosylation of different substrates, producing a variety of glycosides with broad biological activity, while improving the solubility, stability, bioavailability, pharmacological activity, and other functions of NPs. In recent years, the rapid development of synthetic biology and advanced manufacturing technologies, especially the widespread application of artificial intelligence in the field of synthetic biology, has led to a series of new discoveries in the biosynthesis of NP glycosides by UGT. This work summarizes the latest progress and challenges in the field of NP glycosylation, covering the research results and potential applications of glycosylated derivatives of terpenes, flavonoids, polyphenols, aromatic compounds, and other compounds in terms of biogenesis. Looking to the future, research may leverage artificial intelligence-driven synthetic biology techniques to decipher genes related to the synthetic pathway, which is expected to further promote the large-scale synthesis and application of glycosylated NPs, and increase the diversity of NPs in the pharmaceutical, functional food, and cosmetic industries.

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.

Characterization of the UDP-glycosyltransferase UGT33D1 in silkworm Bombyx mori.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT-interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms.