The UDP-glycosyltransferase OsUGT706D2 positively regulates cold and submergence stress tolerance in rice

In a genome-wide association study,we identified a rice UDP-glycosyltransferase gene,OsUGT706D2,whose transcription was activated in response to cold and submergence stress and to exogenous abscisic acid(ABA).OsUGT706D2 positively regulated the biosynthesis of tricin-4’-O-(syringyl alcohol)ether-7-O-glucoside at both the transcriptional and metabolic levels.OsUGT706D2 mediated cold and submergence tolerance by modulating the expression of stress-responsive genes as well as the abscisic acid(ABA)signaling pathway.Gain of function of OsUGT706D2 increased cold and submergence tolerance and loss of function of OsUGT706D2 reduced cold tolerance.ABA positively regulated OsUGT706D2-mediated cold tolerance but reduced submergence tolerance.These findings suggest the potential use of OsUGT706D2 for improving abiotic stress tolerance in rice.

Enzymatic biosynthesis of novel neobavaisoflavone glucosides via Bacillus UDP-glycosyltransferase

The present study was designed to perform structural modifications of of neobavaisoflavone(NBIF), using an in vitro enzymatic glycosylation reaction, in order to improve its water-solubility. Two novel glucosides of NBIF were obtained from an enzymatic glycosylation by UDP-glycosyltransferase. The glycosylated products were elucidated by LC-MS, HR-ESI-MS, and NMR analysis. The HPLC peaks were integrated and the concentrations in sample solutions were calculated. The MTT assay was used to detect the cytotoxic activity of compounds in cancer cell lines. Based on the spectroscopic analyses, the two novel glucosides were identified as neobavaisoflavone-4′-O-β-D-glucopyranoside(1) and neobavaisoflavone-4′, 7-di-O-β-D-glucopyranoside(2). Additionally, the water-solubilities of compounds 1 and 2 were approximately 175.1-and 4 031.9-fold higher than that of the substrate, respectively. Among the test compounds, only NBIF exhibited weak cytotoxicity against four human cancer cell lines, with IC50 values ranging from 63.47 to 72.81 μmol·L^(-1). These results suggest that in vitro enzymatic glycosylation is a powerful approach to structural modification, improving water-solubility.

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

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.

A systematic identification of cold tolerance genes in peanut using yeast functional screening system

Peanut(Arachis hypogaea L.)is a thermophilic crop,and low temperature leads to a significant reduction in annual yields.Despite a few cold tolerant germplasms or cultivars have been discovered and developed,molecular mechanisms governing peanut cold tolerance is poorly understood.Identification of keys genes involved in cold tolerance is the first step to address the underlying mechanism.In this study,we isolated and characterized 157 genes with potentials to confer cold tolerance in peanut by using a yeast functional screening system.GO(Gene ontology)and KEGG(Kyoto encyclopedia of genes and genomes)enrichment analysis of these genes revealed that ribosome and photosynthesis proteins might play essential roles in peanut cold response.Transcriptome results indicated that 60 cold tolerance candidate genes were significantly induced or depressed by low temperature.qRT-PCR analysis demonstrated that several candidate genes could be also regulated by salt or drought stress.Individual overexpression of two UDP-glycosyltransferases(AhUGT2 and AhUGT268)in transgenic yeast cells could enhance their tolerance to multiple abiotic stress.In conclusion,this study advances our understanding of the mechanisms associated with the cold stress responses in peanut,and offers valuable gene resources for genetic improvement of abiotic stress tolerance in crops.

Applications of protein engineering in the microbial synthesis of plant triterpenoids

Triterpenoids are a class of natural products widely used in fields related to medicine and health due to their biological activities such as hepatoprotection,anti-inflammation,anti-viral,and anti-tumor.With the advancement in biotechnology,microorganisms have been used as cell factories to produce diverse natural products.Despite the significant progress that has been made in the construction of microbial cell factories for the heterogeneous biosynthesis of triterpenoids,the industrial production of triterpenoids employing microorganisms has been stymied due to the shortage of efficient enzymes as well as the low expression and low catalytic activity of heterologous proteins in microbes.Protein engineering has been demonstrated as an effective way for improving the specificity,catalytic activity,and stability of the enzyme,which can be employed to overcome these challenges.This review summarizes the current progress in the studies of Oxidosqualene cyclases(OSCs),cytochrome P450s(P450s),and UDP-glycosyltransferases(UGTs),the key enzymes in the triterpenoids synthetic pathway.The main obstacles restricting the efficient catalysis of these key enzymes are analyzed,the applications of protein engineering for the three key enzymes in the microbial synthesis of triterpenoids are systematically reviewed,and the challenges and prospects of protein engineering are also discussed.

Characterization of Panax ginseng UDP- Glycosyltransferases Catalyzing Protopanaxatriol and Biosyntheses of Bioactive Ginsenosides F1 and Rhl in Metabolically Engineered Yeasts

Ginsenosides, the main pharmacologically active natural compounds in ginseng （Panax ginseng）, are mostly the glycosylated products of protopanaxadiol （PPD） and protopanaxatriol （PPT）. No uridine diphosphate glycosyltransferase （UGT）, which catalyzes PPT to produce PPT-type ginsenosides, has yet been reported. Here, we show that UGTPgl, which has been demonstrated to regio-specifically glycosylate the C20-OH of PPD, also specifically glycosylates the C20-OH of PPT to produce bioactive ginsenoside FI. We report the characterization of four novel UGT genes isolated from P. ginseng, sharing high deduced amino acid identity （〉84%） with UGTPgl. We demonstrate that UGTPgl00 specifically glycosylates the C6-OH of PPT to produce bioactive ginsenoside Rhl, and UGTPgl01 catalyzes PPT to produce F1, followed by the generation of ginsenoside Rgl from FI. However, UGTPgl02 and UGTPgl03 were found to have no detectable activity on PPT. Through structural modeling and site-directed mutagenesis, we identified several key amino acids of these UGTs that may play important roles in determining their activities and substrate regio-specificities. Moreover, we constructed yeast recombinants to biosynthesize F1 and Rhl by introducing the genetically engineered PPT-producing pathway and UGTPgl or UGTPgl00. Our study reveals the possible biosynthetic pathways of PPT-type ginsenosides in Panax plants, and provides a sound manufacturing approach for bioactive PPT-type ginsenosides in yeast via synthetic biology strategies.

The expression of Spodoptera exigua P450 and UGT genes: tissue specificity and response to insecticides

Cytochrome P450 and UDP-glucosyltransferase (UGT) as phase I and phase II metabolism enzymes, respectively, play vital roles in the breakdown of endobiotics and xenobiotics. Insects can in crease the expression of detoxificatio n enzymes to cope with the stress from xenobiotics including insecticides. However, the molecular mechanisms for insecticide detoxification in Spodoptera exigua remain elusive, and the genes conferring insecticide metabolisms in this species are less well reported. In this study, 68 P450 and 32 UGT genes were identified. Phylogenetic analysis showed gene expansions in CYP3 and CYP4 clans of P450 genes and UGT33 family of this pest. P450 and UGT genes exhibited specific tissue expression patterns. Insecticide treatments in fat body cells of S. exigua revealed that the expression levels of P450 and UGT genes were significantly influenced by challenges of abamectin, lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb. Multiple genes for detoxification were affected in expression levels after insecticide exposures. The results demonstrated that lambda-cyhalothrin, chlorantraniliprole, metaflumizone and indoxacarb induced similar responses in the expression of P450 and UGT genes in fat body cells;eight P450 genes and four UGT genes were co-up-regulated significantly, and no or only a few CYP/UGT genes were down-regulated significantly by these four insecticides. However, abamectin triggered a distinct response for P450 and UGT gene expression;more P450 and UGT genes were down-regulated by abamectin than by the other four compounds. In con elusion, P450 and UGT genes from S. exigua were identified, and different responses to abamectin suggest a different mechanism for insecticide detoxification.

Glycosylation of N-hydroxy-pipecolic acid equilibrates between systemic acquired resistance response and plant growth

N-hydroxy-pipecolic acid(NHP)activates plant systemic acquired resistance(SAR).Enhanced defense responses are typically accompanied by deficiency in plant development and reproduction.Despite of extensive studies on SAR induction,the effects of NHPmetabolismon plant growth remain largely unclear.In this study,we discovered that NHP glycosylation is a critical factor that fine-tunes the tradeoff between SAR defense and plant growth.We demonstrated that a UDP-glycosyltransferase(UGT76B1)forming NHP glycoside(NHPG)controls the NHP to NHPG ratio.Consistently,the ugt76b1 mutant exhibits enhanced SAR response and an inhibitory effect on plant growth,while UGT76B1 overexpression attenuates SAR response,promotes growth,and delays senescence,indicating that NHP levels are dependent on UGT76B1 function in the course of SAR.Furthermore,our results suggested that,upon pathogen attack,UGT76B1-mediated NHP glycosylation forms a‘‘hand brake’’on NHP accumulation by attenuating the positive regulation of NHP biosynthetic pathway genes,highlighting the complexity of SAR-associated networks.In addition,we showed that UGT76B1-mediated NHP glycosylation in the local site is important for fine-tuning SAR response.Our results implicate that engineering plant immunity through manipulating the NHP/NHPG ratio is a promising method to balance growth and defense response in crops.

Integrative SMRT sequencing and ginsenoside profiling analysis provide insights into the biosynthesis of ginsenoside in Panax quinquefolium

Panax quinquefolium is one of the most common medicinal plants worldwide.Ginsenosides are the major pharmaceutical components in P.quinquefolium.The biosynthesis of ginsenosides in different tissues of P.quinquefolium remained largely unknown.In the current study,an integrative method of transcriptome and metabolome analysis was used to elucidate the ginsenosides biosynthesis pathways in different tissues of P.quinquefolium.Herein,22 ginsenosides in roots,leaves,and flower buds showed uneven distribution patterns.A comprehensive P.quinquefolium transcriptome was generated through single molecular real-time(SMRT)and second-generation sequencing(NGS)technologies,which revealed the ginsenoside pathway genes and UDP-glycosyltransferases(UGT)family genes explicitly expressed in roots,leaves,and flower buds.The weighted gene co-expression network analysis(WGCNA)of ginsenoside biosynthesis genes,UGT genes and ginsenoside contents indicated that three UGT genes were positively correlated to pseudoginsenoside F11,notoginsenoside R1,notoginsenoside R2 and pseudoginsenoside RT5.These results provide insights into ginsenoside biosynthesis in different tissues ofP.quinquefolium.

Metabolic engineering of yeasts for green and sustainable production of bioactive ginsenosides F2 and 3β,20S-Di-O-Glc-DM

Both natural ginsenoside F2 and unnatural ginsenoside 3β,20S-Di-O-Glc-DM were reported to exhibit anti-tumor activity.Traditional approaches for producing them rely on direct extraction from Panax ginseng,enzymatic catalysis or chemical synthesis,all of which result in low yield and high cost.Metabolic engineering of microbes has been recognized as a green and sustainable biotechnology to produce natural and unnatural products.Hence we engineered the complete biosynthetic pathways of F2 and 3β,20S-Di-OGlc-DM in Saccharomyces cerevisiae via the CRISPR/Cas9 system.The titers of F2 and 3β,20S-Di-O-GlcDM were increased from 1.2 to 21.0 mg/L and from 82.0 to 346.1 mg/L at shake flask level,respectively,by multistep metabolic engineering strategies.Additionally,pharmacological evaluation showed that both F2and 3β,20S-Di-O-Glc-DM exhibited anti-pancreatic cancer activity and the activity of 3β,20S-Di-O-GlcDM was even better.Furthermore,the titer of 3β,20S-Di-O-Glc-DM reached 2.6 g/L by fed-batch fermentation in a 3 L bioreactor.To our knowledge,this is the first report on demonstrating the anti-pancreatic cancer activity of F2 and 3β,20S-Di-O-Glc-DM,and achieving their de novo biosynthesis by the engineered yeasts.Our work presents an alternative approach to produce F2 and 3β,20S-Di-O-Glc-DM from renewable biomass,which lays a foundation for drug research and development.

Genome-wide analysis of UGT gene family identified key gene for the biosynthesis of bioactive flavonol glycosides in Epimedium pubescens Maxim

Epimedium pubescens Maxim.is a well-known traditional Chinese medicinal herb with flavonol glycosides as the major pharmaceutically active compounds.UDP-glycosyltransferases(UGTs)are a group of enzymes responsible for the glycosylation of flavonoid glycosides.In this study,a genome-wide analysis was performed to identify UGT family genes in E.pubescens.As a result,a total of 339 putative UGT genes were identified,which represents the largest UGT gene family known thus far,implying a significant expansion of the UGT gene family in E.pubescens.All EpUGTs were unevenly distributed across six chromosomes,and they were classified into 17 major groups.The expression profiles showed that UGT genes were differentially expressed in roots,leaves,flowers,shoots and fruits.In particular,several EpUGTs were highly induced by high light intensity,which was consistent with the accumulation level of bioactive flavonoids in E.pubescens.Six UGT79 genes that were preferentially expressed in roots or leaves were successfully expressed in E.coli,and only the recombinant EpGT60 protein was found to be active toward 8-prenylkaempferol and icaritin to produce the key bioactive compounds baohuoside II and baohuoside I.The optimal temperature,pH,k_(m) and V_(max) were determined for the recombinant EpGT60 protein.In addition,expression of recombinant EpGT60 in E.coli cell culture led to successful production of baohuoside II when fed 8-prenylkaempferol.Our study provides a foundation for further functional characterization of UGT genes in E.pubescens and provides key candidate genes for bioengineering bioactive flavonoids in E.pubescens.

BULKED SEGREGANT RNA SEQUENCING(BSR-SEQ)IDENTIFIES A NOVEL ALLELE ASSOCIATED WITH WEEPING TRAITS IN PRUNUS MUME

Weeping species are used both as ornamental plants and for breeding dwarf plant types.However,exploration of casual genes controlling weeping traits is rather limited.Here,we identified individuals with contrasting phenotypes from an Fx bi-parental mapping population of Prunus mume which was developed from a cross between the upright cultivar/Liuban,and the weeping cultivar'Fentai Chuizhi'.Bulked segregant RNA sequencing was used and five QTLs on chromosome 7 were identified.The Pm024074(PmilGT72B3)allele,belonging to the UDP-glycosyltransferase superfamily containing the coniferyl-alcohol glucosyltransferase domain,was identified in a genomic region overlapping with a previously identified QTL;and had a synonymous transition of T66(upright)to C(weeping)in the coding sequence and a 470-bp deletion in the promoter region.Pm024074 had exceptionally high expression in buds and stems of weeping P.mume.Weighted correlation network analysis indicates that genes neighboring Pm024074 were significantly associated with plant architecture.In addition,a reliable single nucleotide polymorphism marker was developed based on the variation in the Pm024074 gene,providing precise marker-assisted breeding for weeping traits.This study provides insights into the genetic mechanism governing the weeping trait in P.mume,and indicates potential applications for the manipulation of tree architecture.