Flavonoid Glucosyltransferase Research Articles

Identification and Functional Verification of the Glycosyltransferase Gene Family Involved in Flavonoid Synthesis in Rubus chingii Hu.

Glycosylation is catalyzed by UDP-glycosyltransferase (UGT) and plays an important role in enriching the diversity of flavonoids. Rubus plants contain a lot of natural flavonoid glycosides, which are important plants with a homology of medicine and food. However, information about the Rubus UGT gene family is very limited. In this study, we carried out genome-wide analysis and identified the 172, 121, 130, 121 UGT genes in R. chingii, R. corchorifolius, R. idaeus, and R. occidentalis, respectively, and divided them into 18 groups. The analysis of the protein motif and gene structure showed that there were structural and functional conservations in the same group, but there were differences among different groups. Gene replication analysis showed that raspberry and dicotyledons had a higher homology. The expansion of the UGTs gene family was mainly driven by tandem replication events, and experienced purified selection during the long evolution of the raspberry. Cis-acting element analysis showed that they were related to plant growth and development, hormone regulation, and stress response. In addition, according to a comprehensive analysis of the co-expression network constructed by transcriptome data and phylogenetic homology, RchUGT169 was identified as a flavonoid glucosyltransferase. Through the transient expression in tobacco, it was verified that RchUGT169 could catalyze the conversion of kaempferol and quercetin to the corresponding flavonoid glycosides. In conclusion, this research enriched the understanding of the diversity of UGTs in Rubus and determined that RcUGT169 can catalyze flavonoids.

Adaptive response of flavonoids in Robinia pseudoacacia L. affected by the contamination of cadmium and elevated CO2 to arbuscular mycorrhizal symbiosis

As a key component in non-enzyme resistance system, flavonoids play a crucial role in the plant growth and defenses, which are significantly affected by biotic and abiotic factors such as fungi, bacteria, viruses, heavy metals, and atmospheric CO2. Arbuscular mycorrhizal fungi (AMF) play an important role in enhancing plant tolerance to adverse environments, which can significantly affect the synthesis of flavonoids by forming mycorrhizal symbionts with plant roots. However, few studies explored the combined effects of AMF, elevated CO2, and heavy metals on flavonoids in plants. Here, we investigated the adaptive response of flavonoids accumulation in Robinia pseudoacacia L. seedlings affected by the contamination of cadmium (Cd) and elevated CO2 to arbuscular mycorrhizal symbiosis. The results showed that G. mosseae decreased (p < 0.05) Cd content in leaves by 62.2% under elevated CO2. Moreover, G. mosseae colonization led to significant decreases in robinin, quercetin, kaempferol and acacetin by 17.4%, 11.1%, 15.5% and 23.1% under elevated CO2 + Cd, respectively. Additionally, G. mosseae down-regulated (p < 0.05) expression levels of phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes under elevated CO2 + Cd, and CHS and uridine diphosphate flavonoid glucosyltransferase (UFGT) activities decreased (p < 0.05). Quercetin, kaempferol and acacetin showed positive (p < 0.05) correlation with PAL and CHS genes expression and PAL, CHS, and UFGT activities. Cadmium, C/N ratio, carotenoids, leaf biomass, total chlorophyll, P, and starch in leaves and G. mosseae colonization rate in roots influenced (p < 0.05) flavonoids content. Overall, G. mosseae reduced flavonoids synthesis by down-regulating gene expression levels and activities of key enzymes under elevated CO2 + Cd. The results improved our understanding of the regulation of AMF on non-enzymatic resistance of plants grown in heavy metal-contaminated soils under increasing atmospheric CO2 scenarios.

Transcriptional Analysis of Tea Plants (Camellia sinensis) in Response to Salicylic Acid Treatment.

Salicylic acid (SA) is an important plant hormone and signal required for establishing resistance to diverse pathogens and plant diseases. The abundant polyphenols in tea plants also defend plants from biotic and abiotic stresses. However, whether exogenous SA would increase the resistance of tea plants to adversity and the relationship between SA and polyphenols are still poorly understood. Here, we carried out SA treatment on tea seedlings and performed transcriptome sequencing. SA treatment inhibited the phenylpropanoid and flavonoid metabolic pathways but promoted the lignin metabolic pathways. The increased accumulation of lignin in tea leaves after treating with SA indicated that lignin might coordinate SA, enhance, and improve plant defense and disease resistance. Simultaneously, an SA-inducible flavonoid glucosyltransferase (CsUGT0554) specifically involved in 7-OH site glycosylation was characterized in vitro. These results provided valuable information about the effects of SA on tea seedlings and the molecular basis for SA-mediated immune responses.

Integrated transcriptome and metabolome analysis reveals the anthocyanin biosynthesis mechanisms in blueberry (Vaccinium corymbosum L.) leaves under different light qualities.

Blueberry (Vaccinium corymbosum L.) is a popular fruit with an abundance of anthocyanins in its leaves and fruits. Light is one of the pivotal environmental elements that affects plant growth and development, but the regulatory mechanism between light quality and anthocyanin formation is poorly understood. An integrated transcriptome and metabolome analysis was performed to investigate the effects of white (control), blue (B), red (R), and red/blue (60R/40B) light on blueberry growth and reveal the potential pathway controlling anthocyanin biosynthesis in blueberry leaves. The anthocyanin content was significantly improved by the blue and red/blue light when compared with white light, whereas there was a significant reduction in the photosynthesis under the blue light, showing an inverse trend to that of anthocyanin accumulation. Transcriptomic analysis resulted in the assembly of 134,709 unigenes. Of these, 22 were differentially expressed genes (DEGs) that participate in the anthocyanin biosynthesis pathway, with the majority being significantly up-regulated under the blue light. Most of the photosynthesis-related genes that were down-regulated were expressed during anthocyanin accumulation. Targeted metabolome profiling identified 44 metabolites associated with anthocyanin biosynthesis. The contents of most of these metabolites were higher under blue light than the other light conditions, which was consistent with the transcriptome results. The integrated transcriptome and metabolome analysis suggested that, under blue light, leucoanthocyanidin dioxygenase (LDOX), O-methyltransferase (OMT), and UDP-glucose flavonoid glucosyltransferase (UFGT) were the most significantly expressed, and they promoted the synthesis of cyanidin (Cy), malvidin (Mv), and pelargonidin (Pg) anthocyanidins, respectively. The expression levels of dihydroflavonol 4-reductase (DFR) and OMT, as well as the accumulation of delphinidin (Dp), peonidin (Pn), and petunidin (Pt), were significantly increased by the red/blue light. The blue and red/blue lights promoted anthocyanin biosynthesis via inducing the expression of key structural genes and accumulation of metabolites involved in anthocyanin synthesis pathway. Moreover, there was a possible feedback regulating correlation between anthocyanin biosynthesis and photosynthesis under different light qualities in blueberry leaves. This study would provide a theoretical basis for elucidating the underlying regulatory mechanism of anthocyanin biosynthesis of V. corymbosum.

ABI5 regulates ABA-induced anthocyanin biosynthesis by modulating the MYB1-bHLH3 complex in apple.

Abscisic acid (ABA) induces anthocyanin biosynthesis in many plant species. However, the molecular mechanism of ABA-regulated anthocyanin biosynthesis remains unclear. As a crucial regulator of ABA signaling, ABSCISIC ACID-INSENSITIVE5 (ABI5) is involved in many aspects of plant growth and development, yet its regulation of anthocyanin biosynthesis has not been elucidated. In this study, we found that MdABI5, the apple homolog of Arabidopsis ABI5, positively regulated ABA-induced anthocyanin biosynthesis. A series of biochemical tests showed that MdABI5 specifically interacts with basic helix-loop-helix 3 (MdbHLH3), a positive regulator of anthocyanin biosynthesis. MdABI5 enhanced the binding of MdbHLH3 to its target genes dihydroflavonol 4-reductase (MdDFR) and UDP flavonoid glucosyl transferase (MdUF3GT). In addition, MdABI5 directly bound to the promoter of MdbHLH3 to activate its expression. Moreover, MdABI5 enhanced ABA-promoted interaction between MdMYB1 and MdbHLH3. Finally, antisense suppression of MdbHLH3 significantly reduced anthocyanin biosynthesis promoted by MdABI5, indicating that MdABI5-promoted anthocyanin biosynthesis was dependent on MdbHLH3. Taken together, our data suggest that MdABI5 plays a positive role in ABA-induced anthocyanin biosynthesis by modulating the MdbHLH3-MdMYB1 complex. Our work broadens the regulatory network of ABA-mediated anthocyanin biosynthesis, providing new insights to further study the transcriptional regulatory mechanisms behind this process.

Combined transcriptomics and proteomics analysis provides insight into metabolisms of sugars, organic acids and phenols in UV-C treated peaches during storage

3Ultraviolet-C (UV–C) irradiation is known for prolonging the shelf life of many fruit by regulating different pathways. To better understand the roles of UV-C treatment in regulating the metabolic pathways in peach fruit during cold storage, transcriptomics and proteomics approaches were applied to investigate changes in peaches treated with UV-C (1.5 kJ m−2). The results showed that most differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were largely matched to carbohydrates and secondary metabolites. Further analysis found that peaches treated with UV-C exhibited higher sucrose, citric acid, malic acid, phenols, flavonoids and anthocyanins compared with untreated peaches. Proteomics and transcriptomics together indicated that changes of sugars and acids were associated with the expressions of invertase, sucrose synthase, fructokinase, malate dehydrogenase and citrate synthase. UV-C irradiation promoted the synthesis of phenols, flavonoids and anthocyanins by up-regulating expressions of phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, chalcone synthase, dihydroflavonol 4-reductase and UDP-glucose:flavonoid glucosyltransferase. In summary, this research explained the general molecular mechanism of the changes of sugars, acids and phenols in peaches in response to UV-C.

Target-Genes Reveal Species and Genotypic Specificity of Anthocyanin Pigmentation in Citrus and Related Genera

Background: Anthocyanin pigmentation characterizes a number of tissues of Citrus and its relatives. The gain and loss of pigmentation is intriguing and is inherited variously among species. Methods: Citrus germplasm was used to investigate the anthocyanin pigmentation of tissues never before considered, including stamen, style and stigma, and of young leaves, petals, rind and flesh of 28 genotypes belonging to 14 species. Citrus genotypes encompassed citron, lemon, sweet orange, lime, and Citrus relatives included Microcitrus, Murraya, and Severinia. A relative qRT-PCR analysis was carried out on the structural and regulatory genes: phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3′-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), uridine diphosphate glucose flavonoid glucosyl-transferase (UFGT), glutathione S-transferase (GST), Ruby and Noemi. Image analysis and a genomic approach were employed to evaluate how the red pigmentation is inherited among tissues and species. Results: Pigmentation of young leaves and petals is specific to citron and its hybrids. Ruby controls the pigmentation of petals, but not of leaves. The red color of the rind and flesh is a trait that particularly characterizes a diversity of sweet oranges, citron hybrids and Citrus relatives. Color expression depends on external factors and also on developmental stage. The coloration of stamen and style is citron-specific, while a red stigma is exclusive to Moro orange and its hybrids. Conclusion: It is hypothesized that there is a relationship among Citrus species and genes controlling anthocyanin pigmentation.

Integrated metabolomic and transcriptomic analyses reveal differences in the biosynthetic pathway of anthocyanins in Fragaria nilgerrensis and Fragaria pentaphylla

Anthocyanins, which commonly exist in fruits, have substantial benefits for human health. Fragaria nilgerrensis and F. pentaphylla are two species of wild strawberry with white and red fruits, respectively. To understand the molecular mechanism that underlies the biosynthesis of anthocyanins in strawberry, combined analyses of transcriptomics and metabolomics were conducted between the two species of Fragaria to compare the anthocyanin component and the biosynthetic pathway. A total of 26 anthocyanins were obtained in both of the species. Cyanidin 3-O-glucoside chloride, cyanidin 3-galactoside, and cyanidin 3-glucoside were the major anthocyanins in F. nilgerrensis and F. pentaphylla. The content of most of the anthocyanin components in F. pentaphylla was higher than those in F. nilgerrensis. No glycoside pelargonidin was detected in the white fruits of F. nilgerrensis, revealing that the anthocyanins underlie the white mutation. Transcriptomic analysis revealed that the expression of almost all the anthocyanin structural genes and transcription factors genes MYB10 and MYB1 in the fruits of F. pentaphylla was up-regulated compared with those in the fruits of F. nilgerrensis. The results indicate that three structural genes, including chalcone synthase, dihydroflavonol reductase, and UDP-glucose: flavonol 3-O-glucosyltransferaseare likely to be the candidate genes related to anthocyanin biosynthesis in the two species of Fragaria. In addition, the lack of pelargonidin might be due to the block of transformation catalyzed by leucoanthocyanidin dioxygenase or UDP-glucose flavonoid glucosyl transferase.

Identification of a flavonoid 7-O-glucosyltransferase from Andrographis paniculata

Andrographis paniculata is an important traditional medicinal herb in which flavonoids are part of the primary specialized metabolites. A flavonoid glucosyltransferase with broad substrate spectrum (named ApUGT3) was successfully identified by screening homologous glycosyltransferase genes from A. paniculata. The enzyme displayed glycosylation activity toward multiple flavonoids in vitro, and the major products were identified as 7-O-glucosides. Phylogenetic analysis revealed that ApUGT3 is the first reported glycosyltransferase from the Acanthaceae family that belongs to cluster I, suggesting that ApUGT3 is a new flavonoid glycosyltransferase of this subcluster. This enzyme is potentially useful as powerful glycosylation catalysts to modify flavonoid-like compounds and improve their biological activities.

Identification of Differentially Up-regulated Genes in Apple with White Rot Disease.

Fuji, a major apple cultivar in Korea, is susceptible to white rot. Apple white rot disease appears on the stem and fruit; the development of which deteriorates fruit quality, resulting in decreases in farmers' income. Thus, it is necessary to characterize molecular markers related to apple white rot resistance. In this study, we screened for differentially expressed genes between uninfected apple fruits and those infected with Botryosphaeria dothidea, the fungal pathogen that causes white rot. Antimicrobial tests suggest that a gene expression involved in the synthesis of the substance inhibiting the growth of B. dothidea in apples was induced by pathogen infection. We identified seven transcripts induced by the infection. The seven transcripts were homologous to genes encoding a flavonoid glucosyltransferase, a metallothionein-like protein, a senescence-induced protein, a chitinase, a wound-induced protein, and proteins of unknown function. These genes have functions related to responses to environmental stresses, including pathogen infections. Our results can be useful for the development of molecular markers for early detection of the disease or for use in breeding white rotresistant cultivars.

Identification of Three (Iso)flavonoid Glucosyltransferases From Pueraria lobata.

(Iso)flavonoids are one of the largest groups of natural phenolic products conferring great value to the health of plants and humans. Pueraria lobata, a legume, has long been used in Chinese traditional medicine. (Iso)flavonoids mainly present as glycosyl-conjugates and accumulate in P. lobata roots. However, the molecular mechanism underlying the glycosylation processes in (iso)flavonoid biosynthesis are not fully understood. In the current study, three novel UDP-glycosyltransferases (PlUGT4, PlUGT15, and PlUGT57) were identified in P. lobata from RNA-seq data. Biochemical assays of these three recombinant PlUGTs showed all of them were able to glycosylate isoflavones (genistein and daidzein) at the 7-hydroxyl position in vitro. In comparison with the strict substrate specificity for PlUGT15 and PlUGT57, PlUGT4 displayed utilization of a broad range of sugar acceptors. Particularly, PlUGT15 exhibited a much higher catalytic efficiency toward isoflavones (genistein and daidzein) than any other identified 7-O-UGT from P. lobata. Moreover, the transcriptional expression patterns of these PlUGTs correlated with the accumulation of isoflavone glucosides in MeJA-treated P. lobata, suggesting their possible in vivo roles in the glycosylation process.

Characterization of a heat responsive UDP: Flavonoid glucosyltransferase gene in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) accumulates abundant flavonoid glycosides that are the major bioactive ingredients in tea. Biosynthesis of flavonoid glycosides are catalyzed by UDP-glucosyltransferases (UGTs) that are widely present in plants. Among one hundred and seventy-eight UGTs genes that we have previously identified in tea plant, few of them have been functionally characterized. In the present study, we further identified UGT73A17 gene that is responsible for the biosynthesis of a broad range of flavonoid glycosides. Sequence analysis revealed that the deduced UGT73A17 protein showed high identity with 7-O-glycosyltransferases at amino acid level and it was clustered into the clade containing several 7-O-glycosyltransferases from other plant species. Enzymatic assays revealed that the recombinant UGT73A17 protein (rUGT73A17) exhibited activity toward flavonols (kaempferol, quercetin, and myricetin), flavones (apigenin, luteolin, and tricetin), flavanone (naringenin), isoflavones (genistein) and epicatechin gallate, yielding 7-O-glucosides as the major in vitro products. In particular, rUGT73A17 displayed higher activity at high temperatures (eg. 50°C) than at low temperatures, which was consistent with its relatively high expression level at high temperatures. Two amino acid substitutions at I296L and V466A improved the enzymatic activity of rUGT73A17. Our study demonstrated that UGT73A17 is responsible for the biosynthesis of a broad range of flavonoid glucosides, which is also involved in heat response and quality of tea plant.

Functional Characterization of a Flavonoid Glycosyltransferase in Sweet Orange (Citrus sinensis).

Fruits of sweet orange (Citrus sinensis), a popular commercial Citrus species, contain high concentrations of flavonoids beneficial to human health. These fruits predominantly accumulate O-glycosylated flavonoids, in which the disaccharides [neohesperidose (rhamnosyl-α-1,2-glucose) or rutinose (rhamnosyl-α-1,6-glucose)] are linked to the flavonoid aglycones through the 3- or 7-hydroxyl sites. The biotransformation of the flavonoid aglycones into O-rutinosides or O-neohesperidosides in the Citrus plants usually consists of two glycosylation reactions involving a series of uridine diphosphate-sugar dependent glycosyltransferases (UGTs). Although several genes encoding flavonoid UGTs have been functionally characterized in the Citrus plants, full elucidation of the flavonoid glycosylation process remains elusive. Based on the available genomic and transcriptome data, we isolated a UGT with a high expression level in the sweet orange fruits that possibly encodes a flavonoid glucosyltransferase and/or rhamnosyltransferase. Biochemical analyses revealed that a broad range of flavonoid substrates could be glucosylated at their 3- and/or 7-hydrogen sites by the recombinant enzyme, including hesperetin, naringenin, diosmetin, quercetin, and kaempferol. Furthermore, overexpression of the gene could significantly increase the accumulations of quercetin 7-O-rhamnoside, quercetin 7-O-glucoside, and kaempferol 7-O-glucoside, implying that the enzyme has flavonoid 7-O-glucosyltransferase and 7-O-rhamnosyltransferase activities in vivo.

Recent advances in flower color variation and patterning of Japanese morning glory and petunia.

The Japanese morning glory (Ipomoea nil) and petunia (Petunia hybrida), locally called “Asagao” and “Tsukubane-asagao”, respectively, are popular garden plants. They have been utilized as model plants for studying the genetic basis of floricultural traits, especially anthocyanin pigmentation in flower petals. In their long history of genetic studies, many mutations affecting flower pigmentation have been characterized, and both structural and regulatory genes for the anthocyanin biosynthesis pathway have been identified. In this review, we will summarize recent advances in the understanding of flower pigmentation in the two species with respect to flower hue and color patterning. Regarding flower hue, we will describe a novel enhancer of flavonoid production that controls the intensity of flower pigmentation, new aspects related to a flavonoid glucosyltransferase that has been known for a long time, and the regulatory mechanisms of vacuolar pH being a key determinant of red and blue coloration. On color patterning, we describe particular flower patterns regulated by epigenetic and RNA-silencing mechanisms. As high-quality whole genome sequences of the Japanese morning glory and petunia wild parents (P. axillaris and P. inflata, respectively) were published in 2016, further study on flower pigmentation will be accelerated.

Characterization of UGT716A1 as a Multi-substrate UDP:Flavonoid Glucosyltransferase Gene in Ginkgo biloba.

Ginkgo biloba L., a “living fossil” and medicinal plant, is a well-known rich source of bioactive flavonoids. The molecular mechanism underlying the biosynthesis of flavonoid glucosides, the predominant flavonoids in G. biloba, remains unclear. To better understand flavonoid glucosylation in G. biloba, we generated a transcriptomic dataset of G. biloba leaf tissue by high-throughput RNA sequencing. We identified 25 putative UDP-glycosyltransferase (UGT) unigenes that are potentially involved in the flavonoid glycosylation. Among them, we successfully isolated and expressed eight UGT genes in Escherichia coli, and found that recombinant UGT716A1 protein was active toward broad range of flavonoid/phenylpropanoid substrates. In particular, we discovered the first recombinant UGT protein, UGT716A1 from G. biloba, possessing unique activity toward flavanol gallates that have been extensively documented to have significant bioactivity relating to human health. UGT716A1 expression level paralleled the flavonoid distribution pattern in G. biloba. Ectopic over-expression of UGT716A1 in Arabidopsis thaliana led to increased accumulation of several flavonol glucosides. Identification and comparison of the in vitro enzymatic activity of UGT716A1 homologs revealed a UGT from the primitive land species Physcomitrella patens also showed broader substrate spectrum than those from higher plants A. thaliana, Vitis vinifera, and Medicago truncatula. The characterization of UGT716A1 from G. biloba bridges a gap in the evolutionary history of UGTs in gymnosperms. We also discuss the implication of UGT716A1 for biosynthesis, evolution, and bioengineering of diverse glucosylated flavonoids.

Identification of a Flavonoid Glucosyltransferase Involved in 7-OH Site Glycosylation in Tea plants (Camellia sinensis)

Flavonol glycosides, which are often converted from aglycones in a process catalyzed by UDP-glycosyltransferases (UGTs), play an important role for the health of plants and animals. In the present study, a gene encoding a flavonoid 7-O-glycosyltransferase (CsUGT75L12) was identified in tea plants. Recombinant CsUGT75L12 protein displayed glycosyltransferase activity on the 7-OH position of multiple phenolic compounds. In relative comparison to wild-type seeds, the levels of flavonol-glucosides increased in Arabidopsis seeds overexpressing CsUGT75L12. In order to determine the key amino acid residues responsible for the catalytic activity of the protein, a series of site-directed mutagenesis and enzymatic assays were performed based on the 3D structural modeling and docking analyses. These results suggested that residue Q54 is a double binding site that functions as both a sugar receptor and donor. Residues H56 and T151, corresponding to the basic active residues H20 and D119 of VvGT1, were not irreplaceable for CsUGT75L12. In addition, residues Y182, S223, P238, T239, and F240 were demonstrated to be responsible for a ‘reversed’ sugar receptor binding model. The results of single and triple substitutions confirmed that the function of residues P238, T239, and F240 may substitute or compensate with each other for the flavonoid 7-O-glycosyltransferase activity.

Improved soluble bacterial expression and properties of the recombinant flavonoid glucosyltransferase UGT73G1 from Allium cepa

Glycosylation of quercetin using flavonol-specific glycosyltransferases offers an alternate method for isoquercitrin production. Obtaining sufficient quantities of bioactive enzymes is an important prerequisite for highly effective biocatalysis and biotransformation. In this study, a codon-optimized gene for the flavonoid glucosyltransferase UGT73G1 from Allium cepa was heterologously expressed in the preferred prokaryotic expression host Escherichia coli. By combining expression as a fusion protein with 6-histidine tags with coexpression with molecular chaperones, increased soluble expression of UGT73G1 was achieved in E. coli. Two-terminal 6-histidine tags contributed more to the expression than molecular chaperones, as demonstrated by comparison of specific activities in crude extracts obtained from the recombinant E. coli strains. Studies of the catalytic properties of purified UGT73G1 indicated that its activity was significantly promoted by Mn2+ and Mg2+, while it was strongly inhibited by Cu2+. These expression strategies enhanced the solubility and activity of the overexpressed protein and enabled characterization of this plant-derived glucosyltransferase expressed in a prokaryotic host.

Molecular cloning and characterization of a flavonoid glucosyltransferase from Byungkyool (Citrus platymamma hort. ex Tanaka)

Uridine diphosphate glucosyltransferase (UGT) attaches glucoside to proteins, various flavonoids, and phenolic compounds. The modification of flavonoid affects its water solubility, stability, and bioavailability of flavonoids. In this study, citrus genomic sequence database was searched for UGTs of citrus, and a UDP-glucosyltransferase (bGT173) was isolated from Byungkyool (Citrus platymamma hort. ex Tanaka). The cloned cDNA gene was 1365 bp in length and encoded 456 amino acids. Phylogenetic analysis suggested that bGT173 was a member of the flavonoid 3-O-glucosyltransferase group. mRNA expression of bGT173 was higher in leaves compared to flowers, stems, and fruits. The recombinant protein of bGT173 was expressed in Escherichia coli, and tested for its activity on seven flavonoids (apigenin, eriodictyol, hesperetin, kaempferol, luteolin, naringenin, and quercetin). Both kaempferol and quercetin were good substrates for bGT173, demonstrating that bGT173 preferentially glucosylated the 3-hydroxyl group of flavonols. Furthermore, quercetin 3-O-glucoside produced in E. coli showed the same anti-migration activity on pancreatic cancer cells similar to the standard chemical, suggesting that bGT173 is a good candidate for bioconversion of quercetin to quercetin 3-O-glucoside.

Engineering Saccharomyces cerevisiae with the deletion of endogenous glucosidases for the production of flavonoid glucosides.

BackgroundGlycosylation of flavonoids is a promising approach to improve the pharmacokinetic properties and biological activities of flavonoids. Recently, many efforts such as enzymatic biocatalysis and the engineered Escherichia coli biotransformation have increased the production of flavonoid glucosides. However, the low yield of flavonoid glucosides can not meet the increasing demand for human medical and dietary needs. Saccharomyces cerevisiae is a generally regarded as safe (GRAS) organism that has several attractive characteristics as a metabolic engineering platform for the production of flavonoid glucosides. However, endogenous glucosidases of S.cerevisiae as a whole-cell biocatalyst reversibly hydrolyse the glucosidic bond and hinder the biosynthesis of the desired products. In this study, a model flavonoid, scutellarein, was used to exploit how to enhance the production of flavonoid glucosides in the engineered S.cerevisiae.ResultsTo produce flavonoid glucosides, three flavonoid glucosyltransferases (SbGTs) from Scutellaria baicalensis Georgi were successfully expressed in E. coli, and their biochemical characterizations were identified. In addition, to synthesize the flavonoid glucosides in whole-cell S. cerevisiae, SbGT34 was selected for constructing the engineering yeast. Three glucosidase genes (EXG1, SPR1, YIR007W) were knocked out using homologous integration, and the EXG1 gene was determined to be the decisive gene of S. cerevisiae in the process of hydrolysing flavonoid glucosides. To further enhance the potential glycosylation activity of S. cerevisiae, two genes encoding phosphoglucomutase and UTP-glucose-1-phosphate uridylyltransferase involved in the synthetic system of uridine diphosphate glucose were over-expressed in S. cerevisiae. Consequently, approximately 4.8 g (1.2 g/L) of scutellarein 7-O-glucoside (S7G) was produced in 4 L of medium after 54 h of incubation in a 10-L fermenter while being supplied with ~3.5 g of scutellarein.ConclusionsThe engineered yeast harbouring SbGT with a deletion of glucosidases produced more flavonoid glucosides than strains without a deletion of glucosidases. This platform without glucosidase activity could be used to modify a wide range of valued plant secondary metabolites and to explore of their biological functions using whole-cell S. cerevisiae as a biocatalyst.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0535-2) contains supplementary material, which is available to authorized users.

A De novo Transcriptomic Approach to Identify Flavonoids and Anthocyanins "Switch-Off" in Olive (Olea europaea L.) Drupes at Different Stages of Maturation.

Highlights A de novo transcriptome reconstruction of olive drupes was performed in two genotypesGene expression was monitored during drupe development in two olive cultivarsTranscripts involved in flavonoid and anthocyanin pathways were analyzed in Cassanese and Leucocarpa cultivarsBoth cultivar and developmental stage impact gene expression in Olea europaea fruits.During ripening, the fruits of the olive tree (Olea europaea L.) undergo a progressive chromatic change characterized by the formation of a red-brown “spot” which gradually extends on the epidermis and in the innermost part of the mesocarp. This event finds an exception in the Leucocarpa cultivar, in which we observe a destabilized equilibrium between the metabolisms of chlorophyll and other pigments, particularly the anthocyanins whose switch-off during maturation promotes the white coloration of fruits. Despite its importance, genomic information on the olive tree is still lacking. Different RNA-seq libraries were generated from drupes of “Leucocarpa” and “Cassanese” olive genotypes, sampled at 100 and 130 days after flowering (DAF), and were used in order to identify transcripts involved in the main phenotypic changes of fruits during maturation and their corresponding expression patterns. A total of 103,359 transcripts were obtained and 3792 and 3064 were differentially expressed in “Leucocarpa” and “Cassanese” genotypes, respectively, during 100–130 DAF transition. Among them flavonoid and anthocyanin related transcripts such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol 3′-hydrogenase (F3′H), flavonol 3′5 ′-hydrogenase (F3′5′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose:anthocianidin: flavonoid glucosyltransferase (UFGT) were identified. These results contribute to reducing the current gap in information regarding metabolic processes, including those linked to fruit pigmentation in the olive.