Search

Glycolipid biosurfactant, sophorolipids (SLs) and rhamnolipids (RLs) can be widely used in agriculture, food and chemical industries. The different physicochemical properties of SLs and RLs, such as hydrophilic lipophilic value (HLB) and critical micelle concentration (CMC), determine they have different application focus. Researchers are still hoping to obtain new glycolipid surfactants with unique surface activities. In this study, we successfully transformed two rhamnosyltransferase genes rhlA and rhlB from Pseudomonas aeruginosa to the sophorolipid-producing Starmerella bombicola CGMGG 1576 to obtain a recombinant strain was SbrhlAB. Two novel components with molecular weight of 554 (C26H50O12) and 536 (C26H48O11) were identified with the ASB C18 column from the fermentation broth of SbrhlAB, the former was a non-acetylated acidic C14:0 glycolipid containing one glucose and one rhamnose, and the latter was an acidic C14:1 glycolipid containing two rhamnoses. With the Venusil MP C18 column, one new glycolipid component was identified as an acidic C18:3 glycolipid with one rhamnose (C24H40O7), which has not been reported before. Our present study demonstrated that novel glycolipids can be synthesized in vivo by reasonable genetic engineering. The results will be helpful to engineer sophorolipid-producing yeast to produce some specific SLs or their derivatives in more rational and controllable way. [ABSTRACT FROM AUTHOR]

Abstract Glycolipid biosurfactant, sophorolipids (SLs) and rhamnolipids (RLs) can be widely used in agriculture, food and chemical industries. The different physicochemical properties of SLs and RLs, such as hydrophilic lipophilic value (HLB) and critical micelle concentration (CMC), determine they have different application focus. Researchers are still hoping to obtain new glycolipid surfactants with unique surface activities. In this study, we successfully transformed two rhamnosyltransferase genes rhlA and rhlB from Pseudomonas aeruginosa to the sophorolipid-producing Starmerella bombicola CGMGG 1576 to obtain a recombinant strain was Sb rhlAB . Two novel components with molecular weight of 554 (C26H50O12) and 536 (C26H48O11) were identified with the ASB C18 column from the fermentation broth of Sb rhlAB , the former was a non-acetylated acidic C14:0 glycolipid containing one glucose and one rhamnose, and the latter was an acidic C14:1 glycolipid containing two rhamnoses. With the Venusil MP C18 column, one new glycolipid component was identified as an acidic C18:3 glycolipid with one rhamnose (C24H40O7), which has not been reported before. Our present study demonstrated that novel glycolipids can be synthesized in vivo by reasonable genetic engineering. The results will be helpful to engineer sophorolipid-producing yeast to produce some specific SLs or their derivatives in more rational and controllable way.

Anthraquinone and its derivatives show remarkable biological properties such as anticancer, antibacterial, antifungal, and antiviral activities. Hence, anthraquinones derivatives have been of prime interest in drug development. This study developed a recombinant Escherichia coli strain to modify chrysazin to chrysazin-8-O-α-l-rhamnoside (CR) and chrysazin-8-O-α-l-2′-O-methylrhamnoside (CRM) using rhamnosyl transferase and sugar-O-methyltransferase. Biosynthesized CR and CRM were structurally characterized using HPLC, high-resolution mass spectrometry, and various nuclear magnetic resonance analyses. Antimicrobial effects of chrysazin, CR, and CRM against 18 superbugs, including 14 Gram-positive and 4 Gram-negative pathogens, were investigated. CR and CRM exhibited antimicrobial activities against nine pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in a disk diffusion assay at a concentration of 40 µg per disk. There were MIC and MBC values of 7.81–31.25 µg/mL for CR and CRM against methicillin-sensitive S. aureus CCARM 0205 (MSSA) for which the parent chrysazin is more than >1000 µg/mL. Furthermore, the anti-proliferative properties of chrysazin, CR, and CRM were assayed using AGS, Huh7, HL60, and HaCaT cell lines. CR and CRM showed higher antibacterial and anticancer properties than chrysazin. [ABSTRACT FROM AUTHOR]

Rhamnose-associated molecules are attracting attention because they are present in bacteria but not mammals, making them potentially useful as antibacterial agents. Additionally, they are also valuable for tumor immunotherapy. Thus, studies on the functions and biosynthetic pathways of rhamnose-containing compounds are in progress. In this paper, studies on the biosynthetic pathways of three rhamnose donors, i.e., deoxythymidinediphosphate-L-rhamnose (dTDP-Rha), uridine diphosphate-rhamnose (UDP-Rha), and guanosine diphosphate rhamnose (GDP-Rha), are firstly reviewed, together with the functions and crystal structures of those associated enzymes. Among them, dTDP-Rha is the most common rhamnose donor, and four enzymes, including glucose-1-phosphate thymidylyltransferase RmlA, dTDP-Glc-4,6-dehydratase RmlB, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC, and dTDP-4-keto-Rha reductase RmlD, are involved in its biosynthesis. Secondly, several known rhamnosyltransferases from Geobacillus stearothermophilus, Saccharopolyspora spinosa, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae are discussed. In these studies, however, the functions of rhamnosyltransferases were verified by employing gene knockout and radiolabeled substrates, which were almost impossible to obtain and characterize the products of enzymatic reactions. Finally, the application of rhamnose-containing compounds in disease treatments is briefly described. [ABSTRACT FROM AUTHOR]

The fungal cell wall is an attractive structure to look for new antifungal drug targets and for understanding the host-fungus interaction. Sporothrix schenckii is one of the main causative agents of both human and animal sporotrichosis and currently is the species most studied of the Sporothrix genus. The cell wall of this organism has been previously analyzed, and rhamnoconjugates are signature molecules found on the surface of both mycelia and yeast-like cells. Similar to other reactions where sugars are covalently linked to other sugars, lipids, or proteins, the rhamnosylation process in this organism is expected to involve glycosyltransferases with the ability to transfer rhamnose from a sugar donor to the acceptor molecule, i.e., rhamnosyltransferases. However, no obvious rhamnosyltransferase has thus far been identified within the S. schenckii proteome or genome. Here, using a Hidden Markov Model profile strategy, we found within the S. schenckii genome five putative genes encoding for rhamnosyltransferases. Expression analyses indicated that only two of them, named RHT1 and RHT2, were significantly expressed in yeast-like cells and during interaction with the host. These two genes were heterologously expressed in Escherichia coli, and the purified recombinant proteins showed rhamnosyltransferase activity, dependent on the presence of UDP-rhamnose as a sugar donor. To the best of our knowledge, this is the first report about rhamnosyltransferases in S. schenckii. [ABSTRACT FROM AUTHOR]

Rhamnose-associated molecules are attracting attention because they are present in bacteria but not mammals, making them potentially useful as antibacterial agents. Additionally, they are also valuable for tumor immunotherapy. Thus, studies on the functions and biosynthetic pathways of rhamnose-containing compounds are in progress. In this paper, studies on the biosynthetic pathways of three rhamnose donors, i.e., deoxythymidinediphosphate-L-rhamnose (dTDP-Rha), uridine diphosphate-rhamnose (UDP-Rha), and guanosine diphosphate rhamnose (GDP-Rha), are firstly reviewed, together with the functions and crystal structures of those associated enzymes. Among them, dTDP-Rha is the most common rhamnose donor, and four enzymes, including glucose-1-phosphate thymidylyltransferase RmlA, dTDP-Glc-4,6-dehydratase RmlB, dTDP-4-keto-6-deoxy-Glc-3,5-epimerase RmlC, and dTDP-4-keto-Rha reductase RmlD, are involved in its biosynthesis. Secondly, several known rhamnosyltransferases from Geobacillus stearothermophilus, Saccharopolyspora spinosa, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Streptococcus pneumoniae are discussed. In these studies, however, the functions of rhamnosyltransferases were verified by employing gene knockout and radiolabeled substrates, which were almost impossible to obtain and characterize the products of enzymatic reactions. Finally, the application of rhamnose-containing compounds in disease treatments is briefly described.

Anthraquinone and its derivatives show remarkable biological properties such as anticancer, antibacterial, antifungal, and antiviral activities. Hence, anthraquinones derivatives have been of prime interest in drug development. This study developed a recombinant Escherichia coli strain to modify chrysazin to chrysazin-8-O-α-l-rhamnoside (CR) and chrysazin-8-O-α-l-2′-O-methylrhamnoside (CRM) using rhamnosyl transferase and sugar-O-methyltransferase. Biosynthesized CR and CRM were structurally characterized using HPLC, high-resolution mass spectrometry, and various nuclear magnetic resonance analyses. Antimicrobial effects of chrysazin, CR, and CRM against 18 superbugs, including 14 Gram-positive and 4 Gram-negative pathogens, were investigated. CR and CRM exhibited antimicrobial activities against nine pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) in a disk diffusion assay at a concentration of 40 µg per disk. There were MIC and MBC values of 7.81–31.25 µg/mL for CR and CRM against methicillin-sensitive S. aureus CCARM 0205 (MSSA) for which the parent chrysazin is more than >1000 µg/mL. Furthermore, the anti-proliferative properties of chrysazin, CR, and CRM were assayed using AGS, Huh7, HL60, and HaCaT cell lines. CR and CRM showed higher antibacterial and anticancer properties than chrysazin.

The fungal cell wall is an attractive structure to look for new antifungal drug targets and for understanding the host-fungus interaction. Sporothrix schenckii is one of the main causative agents of both human and animal sporotrichosis and currently is the species most studied of the Sporothrix genus. The cell wall of this organism has been previously analyzed, and rhamnoconjugates are signature molecules found on the surface of both mycelia and yeast-like cells. Similar to other reactions where sugars are covalently linked to other sugars, lipids, or proteins, the rhamnosylation process in this organism is expected to involve glycosyltransferases with the ability to transfer rhamnose from a sugar donor to the acceptor molecule, i.e., rhamnosyltransferases. However, no obvious rhamnosyltransferase has thus far been identified within the S. schenckii proteome or genome. Here, using a Hidden Markov Model profile strategy, we found within the S. schenckii genome five putative genes encoding for rhamnosyltransferases. Expression analyses indicated that only two of them, named RHT1 and RHT2, were significantly expressed in yeast-like cells and during interaction with the host. These two genes were heterologously expressed in Escherichia coli, and the purified recombinant proteins showed rhamnosyltransferase activity, dependent on the presence of UDP-rhamnose as a sugar donor. To the best of our knowledge, this is the first report about rhamnosyltransferases in S. schenckii.

KL55, KL74, and KL85 capsular polysaccharide (CPS) biosynthesis loci in Acinetobacter baumannii BAL_204, BAL_309, and LUH5543 genomes, respectively, are related and each contains genes for l -Rha p and d -Glc p A synthesis. The CPSs were isolated and studied by sugar analysis, Smith degradation, and 1H and 13C NMR spectroscopy. The K55 and K74 CPSs are built up of branched octasaccharide repeats (K units) containing one residue each of d -Glc p A and d -Glc p NAc and six residues of l -Rha p. The K55 unit differs from the K74 unit in the linkage between D-Glc p A and an l -Rha p residue in the K unit (1 → 3 versus 1 → 2) and linkage between K units. However, most K units in the isolated K74 CPS were modified by β-elimination of a side-chain α- l -Rha p -(1 → 3)-α- l -Rha p disaccharide from position 4 of GlcA to give 4-deoxy- l - threo -hex-4-enuronic acid (1:~3 ratio of intact and modified units). The K85 CPS has a branched heptasaccharide K unit similar to the K74 unit but with one fewer α- l -Rha p residue in the side chain. In contrast to previous findings on A. baumannii CPSs, each K locus includes fewer glycosyltransferase (Gtr) genes than the number required to form all linkages in the K units. Hence, one Gtr appears to be multifunctional catalysing formation of two 1 → 2 and one 1 → 3 linkages between the l -Rha residues. [ABSTRACT FROM AUTHOR]

Tartary buckwheat is highly valued for its abundant rutin (quercetin 3- O -rutinoside). As a flavonoid glycoside, rutin is synthesized with the crucial involvement of UDP-dependent glycosyltransferases (UGTs). However, the functions and transcriptional regulation of the UGT-encoded genes remain poorly understood. This study identified a key gene, FtUFGT163 , potentially encoding flavonol 3- O -glucoside (1 → 6) rhamnosyltransferase in Tartary buckwheat through omics analysis and molecular docking methods. The recombinant FtUFGT163 expressed in Escherichia coli demonstrated the capacity to glycosylate isoquercetin into rutin. Overexpression of FtUFGT163 significantly enhanced the rutin content in Tartary buckwheat. Further investigation identified a novel bZIP transcription factor, FtGBF1, that enhances FtUFGT163 expression by binding to the G-box element within its promoter, thereby augmenting rutin biosynthesis. Additional molecular biology experiments indicated that the specific positive regulator of rutin, FtMYB5/6, could directly activate the FtGBF1 promoter. Collectively, this study elucidates a novel regulatory module, termed "FtMYB5/6-FtGBF1- FtUFGT163 ", which effectively coordinates the biosynthesis of rutin in Tartary buckwheat, offering insights into the genetic enhancement of nutraceutical components in crops.

Rhamnogalacturonan I (RG-I) comprises approximately one quarter of the pectin molecules in land plants, and the backbone of RG-I consists of a repeating sequence of [2)-α-L-Rha(1-4)-α-D-GalUA(1-] disaccharide. Four Arabidopsis thaliana genes encoding RG-I rhamnosyltransferases (AtRRT1 to AtRRT4), which synthesize the disaccharide repeats, have been identified in the glycosyltransferase family (GT106). However, the functional role of RG-I in plant cell walls and the evolutional history of RRTs remains to be clarified. Here, we characterized the sole ortholog of AtRRT1–AtRRT4 in liverwort, Marchantia polymorpha, namely, MpRRT1. MpRRT1 had RRT activity and genetically complemented the AtRRT1-deficient mutant phenotype in A. thaliana. However, the MpRRT1-deficient M. polymorpha mutants showed no prominent morphological changes and only an approximate 20% reduction in rhamnose content in the cell wall fraction compared to that in wild-type plants, suggesting the existence of other RRT gene(s) in the M. polymorpha genome. As expected, we detected RRT activities in other GT106 family proteins such as those encoded by MpRRT3 in M. polymorpha and FRB1/AtRRT8 in A. thaliana, the deficient mutant of which affects cell adhesion. Our results show that RRT genes are more redundant and diverse in GT106 than previously thought.

Rhamnogalacturonan I (RG-I) comprises approximately one quarter of the pectin molecules in land plants, and the backbone of RG-I consists of a repeating sequence of [2)-α-L-Rha(1-4)-α-D-GalUA(1-] disaccharide. Four Arabidopsis thaliana genes encoding RG-I rhamnosyltransferases (AtRRT1 to AtRRT4), which synthesize the disaccharide repeats, have been identified in the glycosyltransferase family (GT106). However, the functional role of RG-I in plant cell walls and the evolutional history of RRTs remains to be clarified. Here, we characterized the sole ortholog of AtRRT1–AtRRT4 in liverwort, Marchantia polymorpha , namely, MpRRT1. MpRRT1 had RRT activity and genetically complemented the At RRT1 -deficient mutant phenotype in A. thaliana. However, the Mp RRT1 -deficient M. polymorpha mutants showed no prominent morphological changes and only an approximate 20% reduction in rhamnose content in the cell wall fraction compared to that in wild-type plants, suggesting the existence of other RRT gene(s) in the M. polymorpha genome. As expected, we detected RRT activities in other GT106 family proteins such as those encoded by Mp RRT3 in M. polymorpha and FRB1/ At RRT8 in A. thaliana , the deficient mutant of which affects cell adhesion. Our results show that RRT genes are more redundant and diverse in GT106 than previously thought. [ABSTRACT FROM AUTHOR]

Summary: Glycosylation is a key modification for most molecules including plant natural products, for example, flavonoids and isoflavonoids, and can enhance the bioactivity and bioavailability of the natural products. The crystal structure of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana was determined, and the structures of UGT89C1 in complexes with UDP‐β‐l‐rhamnose and acceptor quercetin revealed the detailed interactions between the enzyme and its substrates. Structural and mutational analysis indicated that Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP‐β‐l‐rhamnose. The mutant H357Q exhibited activity with both UDP‐β‐l‐rhamnose and UDP‐glucose. Structural comparison and mutagenesis confirmed that His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis independently as UGT89C1 lacks the other catalytic Asp that is highly conserved in other reported UGTs and forms a hydrogen bond with the catalytic base His. Ser124 is located in the corresponding position of the catalytic Asp in other UGTs and is not able to form a hydrogen bond with His21. Mutagenesis further showed that Ser124 may not be important in its catalysis, suggesting that His21 and acceptor may form an acceptor‐His dyad and UGT89C1 utilizes a catalytic dyad in catalysis instead of catalytic triad. The information of structure and mutagenesis provides structural insights into rhamnosyltransferase substrate specificity and rhamnosylation mechanism. Significance Statement: The crystal structures of plant rhamnosyltransferase UGT89C1 from Arabidopsis thaliana revealed the key structural features and residues for sugar donor recognition and specificity for UDP‐β‐l‐rhamnose. UGT89C1 utilizes His21 as the only catalytic residue for its catalysis as it lacks the other catalytic residue Asp, which is highly conserved in other reported UGTs, suggesting that His21 forms a catalytic dyad with acceptor to facilitate the UGT89C1‐mediated rhamnosylation. [ABSTRACT FROM AUTHOR]

Abstract The uridine diphosphate glycosyltransferase (UGT) plays the central role in glycosylation of small molecules by transferring sugars to various acceptors including bioactive natural products in plants. UGT89C1 from Arabidopsis thaliana is a novel UGT, a rhamnosyltransferase, specifically recognizes UDP- l -rhamnose as donor. To provide an insight into the sugar specificity for UDP- l -rhamnose and interactions between UGT89C1 and its substrates, the UGT89C1 was expressed in Escherichia coli and purified toward biochemical and structural studies. Enzyme activity assay was performed, and the recombinant UGT89C1 recognized UDP- l -rhamnose and rhamnosylated kaempferol. Crystals of At UGT89C1 were obtained, they diffracted to 2.7 Å resolution and belonged to space group I 4 1. At UGT89C1 was also co-crystallized with UDP. Interestingly, two crystal forms were obtained in the same crystallization condition, including the previous I 4 1 crystal form, and the new crystal form that diffracted to 3.0 Å resolution and belonged to space group P 2 1. Highlights • Expression in Escherichia coli and purification of the recombinant UGT89C1 have been established. • The recombinant UGT89C1 specifically recognized UDP- l -rhamnose and rhamnosylated kaempferol. • UGT89C1 was crystallized without and with UDP, and two crystal forms were obtained with UDP. • The X-ray crystallographic analysis of UGT89C1 was performed and discussed. [ABSTRACT FROM AUTHOR]

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat. [ABSTRACT FROM AUTHOR]

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 μM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.

The pectin in plant cell walls consists of three domains: homogalacturonan, rhamnogalacturonan (RG)-I, and RG-II. It is predicted that around 50 different glycosyltransferases are required for their biosynthesis. Among these, the activities of only a few glycosyltransferases have been detected because pectic oligosaccharides are not readily available for use as substrates. In this study, fluorogenic pyridylaminated RG-I-backbone oligosaccharides (PA-RGs) with 3–14 degrees of polymerization (DP) were prepared. Using these oligosaccharides, the activity of RG-I:rhamnosyltransferase (RRT), involved in the biosynthesis of the RG-I backbone diglycosyl repeating units (-4GalUAα1-2Rhaα1-), was detected from the microsomes of azuki bean epicotyls. RRT was found to prefer longer acceptor substrates, PA-RGs with a DP > 7, and it does not require any metal ions for its activity. RRT is located in the Golgi and endoplasmic reticulum. The activity of RRT coincided with epicotyl growth, suggesting that RG-I biosynthesis is involved in plant growth. [ABSTRACT FROM AUTHOR]

In this study, we analyze 31 transgenic lines and their respective untransformed background lines to determine the transgene effects on targeted structures including the pectin components rhamnogalacturonan I (RG-I) and homogalacturonan (HG), neutral side chains (galactan/arabinangalactan), acetylation of pectin, and cellulose level. Modification arising from the pectin backbone- or pectin side chain transgenic lines either increased or decreased the HG:RG-I ratio, side chain length, and methyl esterification of pectin in the tuber cell wall. The pectin esterification transgenic line exhibited only limited side effects. The cellulose level-targeting transgenic lines yielded an unexpectedly high HG:RG-I ratio and longer pectic side chains. These results clearly demonstrate that in effects of a transgene are not restricted to the direct activity of the targeted enzyme but have consequences for the structure of the cell wall matrix. Analysis of whole cell wall structure is therefore necessary to assess the complete effect, direct and indirect, of a transgene. [ABSTRACT FROM AUTHOR]

Because structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue ( AB) of L. erinus is blue-flowered and accumulates delphinidin 3- O- p-coumaroylrutinoside-5- O-malonylglucoside-3′5′- O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender ( AL) is mauve-flowered, and LC- MS analyses showed that AL accumulated delphinidin 3- O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes ( RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers. [ABSTRACT FROM AUTHOR]

The type and amount of anthocyanins in raspberries, and other small fruits, has recently received increased attention. Black raspberry (Rubus occidentalis L.), in particular, has long been recognized as a rich source of anthocyanins and has been the focus of many recent studies examining their potential health benefits. In this study, we characterized a novel anthocyanin profile found in seedlings of two wild black raspberry populations collected from South Dakota, USA. Seedlings from these populations lack pigments glycosylated with rutinoside in their fruit, have elevated levels of cyanidin-3-sambubioside, and contain a small but significant amount of pelargonidin-3-glucoside, a pigment reported only once previously in black raspberry. Affected fruit also have lower than typical total anthocyanins (77.5–134.4 mg 100 mL−1). Based on the available evidence, we believe the plants have a mutation in the gene encoding anthocyanidin-3-glycoside rhamnosyltransferase (3RT), providing a unique opportunity to identify and study one of the major genes in the anthocyanin pathway and its effect on fruit anthocyanins and color.

Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo ( Citrus maxima) and orange ( Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 ( AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7- O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3- O-glucoside in addition to naringenin-7- O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7- O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1, 2RhaT or the Cs1, 6RhaT genes as a whole-cell-biocatalyst. [ABSTRACT FROM AUTHOR]

Abstract: The type and amount of anthocyanins in raspberries, and other small fruits, has recently received increased attention. Black raspberry (Rubus occidentalis L.), in particular, has long been recognized as a rich source of anthocyanins and has been the focus of many recent studies examining their potential health benefits. In this study, we characterized a novel anthocyanin profile found in seedlings of two wild black raspberry populations collected from South Dakota, USA. Seedlings from these populations lack pigments glycosylated with rutinoside in their fruit, have elevated levels of cyanidin-3-sambubioside, and contain a small but significant amount of pelargonidin-3-glucoside, a pigment reported only once previously in black raspberry. Affected fruit also have lower than typical total anthocyanins (77.5–134.4mg100mL−1). Based on the available evidence, we believe the plants have a mutation in the gene encoding anthocyanidin-3-glycoside rhamnosyltransferase (3RT), providing a unique opportunity to identify and study one of the major genes in the anthocyanin pathway and its effect on fruit anthocyanins and color. [Copyright &y& Elsevier]

Aims: Ste15 and ste22 present in the Ebosin biosynthesis gene cluster ( ste) were previously shown to function in Ebosin biosynthesis and both of the protein products are predicted to be glycosyltransferases. In this study, their biochemical activities were confirmed. Methods and Results: ste15 and ste22 were cloned and expressed in Escherichia coli. With a continuous coupled spectrophotometric assay and using the purified proteins, we now demonstrated that the protein Ste15 has the ability of catalysing the transfer of glucose specifically from UDP-glucose to an Ebosin precursor that lacks glucose, the lipid carrier located in the cytoplasmic membrane of the gene ste15 disrupt mutant Streptomyces sp. 139 ( ste15 −). The protein Ste22 can catalyse the transfer of rhamnose specifically from TDP-rhamnose to an Ebosin precursor that lacks rhamnose, a lipophilic carrier in the cytoplasmic membrane of the gene ste22 disrupt mutant Streptomyces sp. 139 ( ste22 −). Conclusions: The gene product of ste15 was identified to be a glucosyltransferase, and the protein encoded by ste22 was found to be a rhamnosyltransferase. Significance and Impact of the Study: Both of two enzymes play essential roles in the formation of repeating units of sugars during Ebosin biosynthesis. These are the first glucosyltransferase and rhamnosyltransferase in the biosynthesis of a Streptomyces exopolysaccharide to be characterized. [ABSTRACT FROM AUTHOR]

Abstract: Carbohydrate polymers are medically and industrially important. The S-layer of many Gram-positive organisms comprises protein and carbohydrate polymers and forms an almost paracrystalline array on the cell surface. Not only is this array important for the bacteria but it has potential application in the manufacture of commercially important polysaccharides and glycoconjugates as well. The S-layer glycoprotein glycan from Geobacillus stearothermophilus NRS 2004/3a is mainly composed of repeating units of three rhamnose sugars linked by α-1,3-, α-1,2-, and β-1,2-linkages. The formation of the β-1,2-linkage is catalysed by the enzyme WsaF. The rational use of this system is hampered by the fact that WsaF and other enzymes in the pathway share very little homology to other enzymes. We report the structural and biochemical characterisation of WsaF, the first such rhamnosyltransferase to be characterised. Structural work was aided by the surface entropy reduction method. The enzyme has two domains, the N-terminal domain, which binds the acceptor (the growing rhamnan chain), and the C-terminal domain, which binds the substrate (dTDP-β-l-rhamnose). The structure of WsaF bound to dTDP and dTDP-β-l-rhamnose coupled to biochemical analysis identifies the residues that underlie catalysis and substrate recognition. We have constructed and tested by site-directed mutagenesis a model for acceptor recognition. [Copyright &y& Elsevier]

Abstract: A new bacterial strain isolated from activated sludge, identified as Pseudomonas aeruginosa EMS1, produced a biosurfactant when grown on acidified soybean oil as the sole carbon source. An optimum biosurfactant production of 5g/L was obtained with the following medium composition: 2% acidified soybean oil, 0.3% NH4NO3, 0.03% KH2PO4, 0.03% K2HPO4, 0.02% MgSO4 ·7H2O and 0.025% CaCl2 ·2H2O, with shaking at 200rpm for an incubation period of 100h at 30°C. The production of the biosurfactant was found to be a function of cell growth, with maximum production occurring during the exponential phase. Hemolysis of erythrocytes and thin-layer chromatography studies revealed that the secreted biosurfactant was rhamnolipid. To overcome the complex environmental regulation with respect to rhamnolipid biosynthesis, and to replace the opportunistic pathogen P. aeruginosa with a safe industrial strain, attempts were made to achieve rhamnolipid production in a heterologous host, Pseudomonas putida, using molecular cloning of rhlAB rhamnosyltransferase genes with the rhlRI quorum sensing system, assuming that a functional rhamnosyltransferase would catalyze the formation of rhamnosyl-6-hydroxydecanoyl-6-hydroxydecanoate (mono-rhamnolipid) in P. putida. It was shown that rhamnolipid can be produced in the heterologous strain, P. putida, when provided with the rhamnosyltransferase genes. [Copyright &y& Elsevier]

Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone-7-O-neohesperidosides responsible, in part, for their characteristic taste. Non-bitter species, such as mandarin and orange, accumulate only tasteless flavanone-7-O-rutinosides. The key flavor-determining step of citrus flavanone-glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7-O-neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non-bitter species, was not cross-reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone-neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaTon a separate gene cluster together with the only other functionally characterized flavonoid-glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides. [ABSTRACT FROM AUTHOR]

Rhamnose is a naturally occurring deoxysugar present as a glycogenic component of plant and microbial natural products. A recombinant mutant Escherichia coli strain was developed by overexpressing genes involved in the TDP-L-rhamnose biosynthesis pathway of different bacterial strains and Saccharothrix espanaensis rhamnosyl transferase to conjugate intrinsic cytosolic TDP-L-rhamnose with anthraquinones supplemented exogenously. Among the five anthraquinones (alizarin, emodin, chrysazin, anthrarufin, and quinizarin) tested, quinizarin was biotransformed into a rhamoside derivative with the highest conversion ratio by whole cells of engineered E. coli . The quinizarin glycoside was identified by various chromatographic and spectroscopic analyses. The anti-proliferative property of the newly synthesized rhamnoside, quinizarin-4-O-α-L-rhamnoside, was assayed in various cancer cells.

Flavonoids are widely distributed in mulberry leaves and have been recognized for their beneficial physiological effects on the human health. Here, we analyzed variations in 44 flavonoid compounds among 91 mulberry resources. Metabolic profiling revealed that O -rhamnosylated flavonols and malonylated flavonol glycosides, including rutin and quercetin 3- O -(6″- O -malonylglucoside) (Q3MG), were absent from Morus notabilis and multiple mulberry ( Morus alba L.) resources. Transcriptome and phylogenetic analyses of flavonoid-related UDP-glycosyltransferases (UGTs) suggested that the flavonol 3- O -glucoside- O -rhamnosyltransferase (FGRT) KT324624 is a key enzyme involved in rutin synthesis. A recombinant FGRT protein was able to convert kaempferol/quercetin 3- O -glucoside to kaempferol 3- O -rutinoside (K3G6″Rha) and rutin. The recombinant FGRT was able to use 3- O -glucosylated flavonols but not flavonoid aglycones or 7- O -glycosylated flavonoids as substrates. The enzyme preferentially used UDP-rhamnose as the sugar donor, indicating that it was a flavonol 3- O -glucoside: 6″- O -rhamnosyltransferase. This study provided insights into the biosynthesis of rutin in mulberry.

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 μM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids. [ABSTRACT FROM AUTHOR]

Group A carbohydrate (GAC) is a bacterial peptidoglycan-anchored surface rhamnose polysaccharide (RhaPS) that is essential for growth of Streptococcus pyogenes and contributes to its ability to infect the human host. In this study, using molecular and synthetic biology approaches, biochemistry, radiolabeling techniques, and NMR and MS analyses, we examined the role of GacB, encoded in the S. pyogenes GAC gene cluster, in the GAC biosynthesis pathway. We demonstrate that GacB is the first characterized α-d-GlcNAc-β-1,4-l-rhamnosyltransferase that synthesizes the committed step in the biosynthesis of the GAC virulence determinant. Importantly, the substitution of S. pyogenes gacB with the homologous gene from Streptococcus agalactiae (Group B Streptococcus ), Streptococcus equi subsp. zooepidemicus (Group C Streptococcus ), Streptococcus dysgalactiae subsp. equisimilis (Group G Streptococcus ), or Streptococcus mutans complemented the GAC biosynthesis pathway. These results, combined with those from extensive in silico studies, reveal a common phylogenetic origin of the genes required for this priming step in >40 pathogenic species of the Streptococcus genus, including members from the Lancefield Groups B, C, D, E, G, and H. Importantly, this priming step appears to be unique to streptococcal ABC transporter-dependent RhaPS biosynthesis, whereas the Wzx/Wzy-dependent streptococcal capsular polysaccharide pathways instead require an α-d-Glc-β-1,4-l-rhamnosyltransferase. The insights into the RhaPS priming step obtained here open the door to targeting the early steps of the group carbohydrate biosynthesis pathways in species of the Streptococcus genus of high clinical and veterinary importance., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Zorzoli et al.)

Serine-rich repeat glycoproteins (SRRPs) are conserved in Gram-positive bacteria. They are crucial for modulating biofilm formation and bacterial-host interactions. Glycosylation of SRRPs plays a pivotal role in the process; thus understanding the glycosyltransferases involved is key to identifying new therapeutic drug targets. The glycosylation of Fap1, an SRRP of Streptococcus parasanguinis, is mediated by a gene cluster consisting of six genes: gtf1, gtf2, gly, gtf3, dGT1, and galT2 Mature Fap1 glycan possesses the sequence of Rha1-3Glc1-(Glc1-3GlcNAc1)-2,6-Glc1-6GlcNAc. Gtf12, Gtf3, and dGT1 are responsible for the first four steps of the Fap1 glycosylation, catalyzing the transfer of GlcNAc, Glc, Glc, and GlcNAc residues to the protein backbone sequentially. The role of GalT2 and Gly in the Fap1 glycosylation is unknown. In the present study, we synthesized the fully modified Fap1 glycan in Escherichia coli by incorporating all six genes from the cluster. This study represents the first reconstitution of an exogenous stepwise O-glycosylation synthetic pathway in E. coli In addition, we have determined that GalT2 mediates the fifth step of the Fap1 glycosylation by adding a rhamnose residue, and Gly mediates the final glycosylation step by transferring glucosyl residues. Furthermore, inactivation of each glycosyltransferase gene resulted in differentially impaired biofilms of S. parasanguinis, demonstrating the importance of Fap1 glycosylation in the biofilm formation. The Fap1 glycosylation system offers an excellent model to engineer glycans using different permutations of glycosyltransferases and to investigate biosynthetic pathways of SRRPs because SRRP genetic loci are highly conserved., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)