Eugenol functions as a signal mediating cold and drought tolerance via UGT71A59-mediated glucosylation in tea plants.

Cold and drought stress are the most critical stresses encountered by crops and occur simultaneously under field conditions. However, it is unclear whether volatiles contribute to both cold and drought tolerance, and if so, by what mechanisms they act. Here, we show that airborne eugenol can be taken up by the tea (Camellia sinensis) plant and metabolized into glycosides, thus enhancing cold and drought tolerance of tea plants. A uridine diphosphate (UDP)-glucosyltransferase, UGT71A59, was discovered, whose expression is strongly induced by multiple abiotic stresses. UGT71A59 specifically catalyzes glucosylation of eugenol glucoside in vitro and in vivo. Suppression of UGT71A59 expression in tea reduced the accumulation of eugenol glucoside, lowered reactive oxygen species (ROS) scavenging capacity, and ultimately impaired cold and drought stress tolerance. Exposure to airborne eugenol triggered a marked increase in UGT71A59 expression, eugenol glucoside accumulation, and cold tolerance by modulating ROS accumulation and CBF1 expression. It also promoted drought tolerance by altering abscisic acid homeostasis and stomatal closure. CBF1 and CBF3 play positive roles in eugenol-induced cold tolerance and CBF2 may be a negative regulator of eugenol-induced cold tolerance in tea plants. These results provide evidence that eugenol functions as a signal in cold and drought tolerance regulation and shed new light on the biological functions of volatiles in the response to multiple abiotic stresses in plants.

Enzymatic glucosylation of polyphenols using glucansucrases and branching sucrases of glycoside hydrolase family 70.

Polyphenols exhibit various beneficial biological activities and represent very promising candidates as active compounds for food industry. However, the low solubility, poor stability and low bioavailability of polyphenols have severely limited their industrial applications. Enzymatic glycosylation is an effective way to improve the physicochemical properties of polyphenols. As efficient transglucosidases, glycoside hydrolase family 70 (GH70) glucansucrases naturally catalyze the synthesis of polysaccharides and oligosaccharides from sucrose. Notably, GH70 glucansucrases show broad acceptor substrate promiscuity and catalyze the glucosylation of a wide range of non-carbohydrate hydroxyl group-containing molecules, including benzenediol, phenolic acids, flavonoids and steviol glycosides. Branching sucrase enzymes, a newly established subfamily of GH70, are shown to possess a broader acceptor substrate binding pocket that acts efficiently for glucosylation of larger size polyphenols such as flavonoids. Here we present a comprehensive review of glucosylation of polyphenols using GH70 glucansucrase and branching sucrases. Their catalytic efficiency, the regioselectivity of glucosylation and the structure of generated products are described for these reactions. Moreover, enzyme engineering is effective for improving their catalytic efficiency and product specificity. The combined information provides novel insights on the glucosylation of polyphenols by GH70 glucansucrases and branching sucrases, and may promote their applications.

Novel cerebrospinal fluid biomarkers of glucose transporter type 1 deficiency syndrome: Implications beyond the brain's energy deficit.

We used next-generation metabolic screening to identify new biomarkers for improved diagnosis and pathophysiological understanding of glucose transporter type 1 deficiency syndrome (GLUT1DS), comparing metabolic cerebrospinal fluid (CSF) profiles from 12 patients to those of 116 controls. This confirmed decreased CSF glucose and lactate levels in patients with GLUT1DS and increased glutamine at group level. We identified three novel biomarkers significantly decreased in patients, namely gluconic + galactonic acid, xylose-α1-3-glucose, and xylose-α1-3-xylose-α1-3-glucose, of which the latter two have not previously been identified in body fluids. CSF concentrations of gluconic + galactonic acid may be reduced as these metabolites could serve as alternative substrates for the pentose phosphate pathway. Xylose-α1-3-glucose and xylose-α1-3-xylose-α1-3-glucose may originate from glycosylated proteins; their decreased levels are hypothetically the consequence of insufficient glucose, one of two substrates for O-glucosylation. Since many proteins are O-glucosylated, this deficiency may affect cellular processes and thus contribute to GLUT1DS pathophysiology. The novel CSF biomarkers have the potential to improve the biochemical diagnosis of GLUT1DS. Our findings imply that brain glucose deficiency in GLUT1DS may cause disruptions at the cellular level that go beyond energy metabolism, underlining the importance of developing treatment strategies that directly target cerebral glucose uptake.

Glucosylation of Isoeugenol and Monoterpenes in Corynebacterium glutamicum by YdhE from Bacillus lichenformis.

Corynebacterium glutamicum has been regarded as a food-grade microorganism. In recent years, the research to improve the activities of beneficial therapeutics and pharmaceutical substances has resulted in the engineering of the therapeutically favorable cell factory system of C. glutamicum. In this study, we successfully glucosylated isoeugenol and other monoterpene derivatives in C. glutamicum using a promiscuous YdhE, which is a glycosyltransferase from Bacillus lichenformis. For efficient glucosylation, cultivation conditions such as the production time, substrate concentration, carbon source, and culture medium were optimized. Our system successfully converted about 93% of the isoeugenol to glucosylated compounds in the culture. The glucoside compounds were then purified, analyzed, and identified as isoeugenol-1-O-ß-d-glucoside and isoeugenol-1-O-ß-d-(2″-acetyl)-glucoside.

Recent Progress in 1,2-cis glycosylation for Glucan Synthesis.

Controlling the stereoselectivity of 1,2-cis glycosylation is one of the most challenging tasks in the chemical synthesis of glycans. There are various 1,2-cis glycosides in nature, such as α-glucoside and ß-mannoside in glycoproteins, glycolipids, proteoglycans, microbial polysaccharides, and bioactive natural products. In the structure of polysaccharides such as α-glucan, 1,2-cis α-glucosides were found to be the major linkage between the glucopyranosides. Various regioisomeric linkages, 1→3, 1→4, and 1→6 for the backbone structure, and 1→2/3/4/6 for branching in the polysaccharide as well as in the oligosaccharides were identified. To achieve highly stereoselective 1,2-cis glycosylation, including α-glucosylation, a number of strategies using inter- and intra-molecular methodologies have been explored. Recently, Zn salt-mediated cis glycosylation has been developed and applied to the synthesis of various 1,2-cis linkages, such as α-glucoside and ß-mannoside, via the 1,2-cis glycosylation pathway and ß-galactoside 1,4/6-cis induction. Furthermore, the synthesis of various structures of α-glucans has been achieved using the recent progressive stereoselective 1,2-cis glycosylation reactions. In this review, recent advances in stereoselective 1,2-cis glycosylation, particularly focused on α-glucosylation, and their applications in the construction of linear and branched α-glucans are summarized.

Enzymatic Synthesis of α-Glucosyl-Baicalin through Transglucosylation via Cyclodextrin Glucanotransferase in Water.

Baicalin is a biologically active flavone glucuronide with poor water solubility that can be enhanced via glucosylation. In this study, the transglucosylation of baicalin was successfully achieved with CGTases from Thermoanaerobacter sp. and Bacillus macerans using α-cyclodextrin as a glucosyl donor. The synthesis of baicalin glucosides was optimized with CGTase from Thermoanaerobacter sp. Enzymatically modified baicalin derivatives were α-glucosylated with 1 to 17 glucose moieties. The two main glucosides were identified as Baicalein-7-O-α-D-Glucuronidyl-(1→4')-O-α-D-Glucopyranoside (BG1) and Baicalein-7-O-α-D-Glucuronidyl-(1→4')-O-α-D-Maltoside (BG2), thereby confirming recent findings reporting that glucuronyl groups are acceptors of this CGTase. Optimized conditions allowed for the attainment of yields above 85% (with a total glucoside content higher than 30 mM). BG1 and BG2 were purified via centrifugal partition chromatography after an enrichment through deglucosylation with amyloglucosidase. Transglucosylation increased the water solubility of BG1 by a factor of 188 in comparison to that of baicalin (molar concentrations), while the same value for BG2 was increased by a factor of 320. Finally, BG1 and BG2 were evaluated using antioxidant and anti-glycation assays. Both glucosides presented antioxidant and anti-glycation properties in the same order of magnitude as that of baicalin, thereby indicating their potential biological activity.

O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins.

The serine-rich repeat (SRR) glycoproteins of gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec system. Although all accessory Sec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin gordonii surface protein B (GspB). Because these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues and that O-acetylation prevented Glc deposition. Whereas streptococci expressing nonacetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to WT levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, because O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

Characterization of the (Engineered) Branching Sucrase GtfZ-CD2 from Apilactobacillus kunkeei for Efficient Glucosylation of Benzenediol Compounds.

Branching sucrases, a subfamily of Glycoside Hydrolase family (GH70), display transglycosidase activity using sucrose as donor substrate to catalyze glucosylation reaction in the presence of suitable acceptor substrates. In this study, the (α1→3) branching sucrase GtfZ-CD2 from Apilactobacillus kunkeei DSM 12361 was demonstrated to glucosylate benzenediol compounds (i.e., catechol, resorcinol, and hydroquinone) to form monoglucoside and diglucoside products. The production and yield of catechol glucosylated products were significantly higher than that of resorcinol and hydroquinone, revealing a preference for adjacent aromatic hydroxyl groups in glucosylation. Amino residues around acceptor substrate binding subsite +1 were targeted for semirational mutagenesis, yielding GtfZ-CD2 variants with improved resorcinol and hydroquinone glucosylation. Mutant L1560Y with improved hydroquinone mono-glucosylated product synthesis allowed enzymatic conversion of hydroquinone into α-arbutin. This study thus revealed the high potential of GH70 branching sucrases for glucosylating noncarbohydrate molecules. IMPORTANCE Glycosylation represents one of the most important ways to expand the diversity of natural products and improve their physico-chemical properties. Aromatic polyphenol compounds widely found in plants are reported to exhibit various remarkable biological activities; however, they generally suffer from low solubility and stability, which can be improved by glycosylation. Our present study on the glucosylation of benzenediol compounds by GH70 branching sucrase GtfZ-CD2 and its semirational engineering to improve the glucosylation efficiency provides insight into the mechanism of acceptor substrates binding and its glucosylation selectivity. The results demonstrate the potential of using branching sucrase as an effective enzymatic glucosylation tool.

Clostridium difficile Toxin Biology.

Clostridium difficile is the cause of antibiotics-associated diarrhea and pseudomembranous colitis. The pathogen produces three protein toxins: C. difficile toxins A (TcdA) and B (TcdB), and C. difficile transferase toxin (CDT). The single-chain toxins TcdA and TcdB are the main virulence factors. They bind to cell membrane receptors and are internalized. The N-terminal glucosyltransferase and autoprotease domains of the toxins translocate from low-pH endosomes into the cytosol. After activation by inositol hexakisphosphate (InsP6), the autoprotease cleaves and releases the glucosyltransferase domain into the cytosol, where GTP-binding proteins of the Rho/Ras family are mono-O-glucosylated and, thereby, inactivated. Inactivation of Rho proteins disturbs the organization of the cytoskeleton and affects multiple Rho-dependent cellular processes, including loss of epithelial barrier functions, induction of apoptosis, and inflammation. CDT, the third C. difficile toxin, is a binary actin-ADP-ribosylating toxin that causes depolymerization of actin, thereby inducing formation of the microtubule-based protrusions. Recent progress in understanding of the toxins' actions include insights into the toxin structures, their interaction with host cells, and functional consequences of their actions.

Human neutrophils are resistant to Clostridioides difficile toxin B.

OBJECTIVE: The main objective of this study was to evaluate the glucosyltransferase activity of C. difficile TcdB on the activity of human PMNs. METHODS: To better understand the interaction between PMNs and TcdB, PMNs were treated with sub-lethal concentrations of TcdB. We evaluated: (i) the glucosylation of GTPases, (ii) the phagocytic and bactericidal activity, and (iii) PMNs activation (through quantification of TNF-α, IL-8, and expression of CD11b cell surface activation marker). RESULTS: We found that TcdB did not glucosylate RhoA and Rac1 GTPases and did not affect the phagocytic or bactericidal capacity of PMNs. Moreover, TcdB did not increase the production of TNF-α, IL-8, or the expression of activation marker CD11b. The only significant effect of TcdB on PMNs was the partial inhibition of TNF-α and IL-8 production and the diminished expression of CD11b induced by E. coli-LPS. CONCLUSION: Our results show that human PMNs are resistant to TcdB GTPase glucosyltransferase activity against RhoA and Rac1.

N-Glucosylation in Corynebacterium glutamicum with YdhE from Bacillus lichenformis.

Corynebacterium glutamicum is traditionally known as a food-grade microorganism due to its high ability to produce amino acids and its endotoxin-free recombinant protein expression factory. In recent years, studies to improve the activities of useful therapeutics and pharmaceutical compounds have led to the engineering of the therapeutically advantageous C. glutamicum cell factory system. One of the well-studied ways to improve the activities of useful compounds is glucosylation with glycosyltransferases. In this study, we successfully and efficiently glycosylated therapeutic butyl-4-aminobenzoate and other N-linked compounds in C. glutamicum using a promiscuous YdhE, which is a glycosyltransferase from Bacillus lichenformis. For efficient glucosylation, components, such as promoter, codons sequence, expression temperatures, and substrate and glucose concentrations were optimized. With glucose as the sole carbon source, we achieved a conversion rate of almost 96% of the glycosylated products in the culture medium. The glycosylated product of high concentration was successfully purified by a simple purification method, and subjected to further analysis. This is a report of the in vivo cultivation and glucosylation of N-linked compounds in C. glutamicum.

Cholesterol glucosylation-based survival strategy in Helicobacter pylori.

Helicobacter pylori is a major chronic health problem, infecting more than half of the population worldwide. H. pylori infection is linked with various clinical complications ranging from gastritis to gastric cancer. The resolution of gastritis and peptic ulcer appears to be linked with the eradication of H. pylori. However, resistance to antibiotics and eradication failure rates are reaching alarmingly high levels. This calls for urgent action in finding alternate methods for H. pylori eradication. Here, we discuss the recently identified mechanism of H. pylori known as cholesterol glucosylation, mediated by the enzyme cholesterol-α-glucosyltransferase, encoded by the gene cgt. Cholesterol glucosylation serves several functions that include promoting immune evasion, enhancing antibiotic resistance, maintaining the native helical morphology, and supporting functions of prominent virulence factors such as CagA and VacA. Consequently, strategies aiming at inhibition of the cholesterol glucosylation process have the potential to attenuate the potency of H. pylori infection and abrogate H. pylori immune evasion capabilities. Knockout of H. pylori cgt results in unsuccessful colonization and elimination by the host immune responses. Moreover, blocking cholesterol glucosylation can reverse antibiotic susceptibility in H. pylori. In this work, we review the main roles of cholesterol glucosylation in H. pylori and evaluate whether this mechanism can be targeted for the development of alternate methods for eradication of H. pylori infection.

pH-promoted O-α-glucosylation of flavonoids using an engineered α-glucosidase mutant.

Retaining glycosidase mutants lacking its general acid/base catalytic residue are originally termed thioglycoligases which synthesize thio-linked disaccharides using sugar acceptor bearing a nucleophilic thiol group. A few thioglycoligases derived from retaining α-glycosidases have been classified into a new class of catalysts, O-glycoligases which transfer sugar moiety to a hydroxy group of sugar acceptors, resulting in the formation of O-linked glycosides or oligosaccharides. In this study, an efficient O-α-glucosylation of flavonoids was developed using an O-α-glycoligase derived from a thermostable α-glucosidase from Sulfolobus solfataricus (MalA-D416A). The O-glycoligase exhibited efficient transglycosylation activity with a broad substrate spectrum for all kinds of tested flavonoids including flavone, flavonol, flavanone, flavanonol, flavanol and isoflavone classes in yields of higher than 90%. The glucosylation by MalA-D416A preferred alkaline conditions, suggesting that pH-promoted deprotonation of hydroxyl groups of the flavonoids would accelerate turnover of covalent enzyme intermediate via transglucosylation. More importantly, the glucosylation of flavonoids by MalA-D416A was exclusively regioselective, resulting in the synthesis of flavonoid 7-O-α-glucosides as the sole product. Kinetic analysis and molecular dynamics simulations provided insights into the acceptor specificity and the regiospecificity of O-α-glucosylation by MalA-D416A. This pH promoted transglycosylation using O-α-glycoligases may prove to be a general synthesis route to flavonoid O-α-glycosides.

Efficient production of l-menthyl α-glucopyranoside from l-menthol via whole-cell biotransformation using recombinant Escherichia coli.

l-Menthyl α-D-glucopyranoside (α-MenG) is a glycoside derivative of l-menthol with improved water-solubility and new flavor property as a food additive. α-MenG can be synthesized through biotransformation, but its scale-up production was rarely reported. In this study, the properties of an α-glucosidase from Xanthomonas campestris pv. campestris 8004 (Agl-2) in catalyzing the glucosylation of menthol was investigated. Agl-2 can almost completely glycosylate l-menthol (> 99%) when using 1.2 M maltose as glycosyl donor. Accumulated glucose resulted from maltose hydrolysis and transglycosylation caused the inhibition of the glucosylation rate (40% reduction of the glucosylation rate in the presence of 1.2 M glucose) which can be avoided through whole-cell catalysis with recombinant E. coli. Interestingly, in spite of the poor solubility of menthol, the productivity of α-MenG reached 24.7 g/(L·h) in a 2 L catalyzing system, indicating industrialization of the reported approach.

4-O-Glucosylation of Trichothecenes by Fusarium Species: A Phase II Xenobiotic Metabolism for t-Type Trichothecene Producers.

The t-type trichothecene producers Fusarium sporotrichioides and Fusarium graminearum protect themselves against their own mycotoxins by acetylating the C-3 hydroxy group with Tri101p acetylase. To understand the mechanism by which they deal with exogenously added d-type trichothecenes, the Δtri5 mutants expressing all but the first trichothecene pathway enzymes were fed with trichodermol (TDmol), trichothecolone (TCC), 8-deoxytrichothecin, and trichothecin. LC-MS/MS and NMR analyses showed that these C-3 unoxygenated trichothecenes were conjugated with glucose at C-4 by α-glucosidic linkage. As t-type trichothecenes are readily incorporated into the biosynthetic pathway following the C-3 acetylation, the mycotoxins were fed to the ΔFgtri5ΔFgtri101 mutant to examine their fate. LC-MS/MS and NMR analyses demonstrated that the mutant conjugated glucose at C-4 of HT-2 toxin (HT-2) by α-glucosidic linkage, while the ΔFgtri5 mutant metabolized HT-2 to 3-acetyl HT-2 toxin and T-2 toxin. The 4-O-glucosylation of exogenously added t-type trichothecenes appears to be a general response of the ΔFgtri5ΔFgtri101 mutant, as nivalenol and its acetylated derivatives appeared to be conjugated with hexose to some extent. The toxicities of 4-O-glucosides of TDmol, TCC, and HT-2 were much weaker than their corresponding aglycons, suggesting that 4-O-glucosylation serves as a phase II xenobiotic metabolism for t-type trichothecene producers.

Influence of Shigella flexneri 2a O Antigen Acetylation on Its Bacteriophage Sf6 Receptor Activity and Bacterial Interaction with Human Cells.

Shigella flexneri is a major causative agent of bacillary dysentery in developing countries, where serotype 2a2 is the prevalent strain. To date, approximately 30 serotypes have been identified for S. flexneri, and the major contribution to the emergence of new serotypes is chemical modifications of the lipopolysaccharide (LPS) component O antigen (Oag). Glucosylation, O-acetylation, and phosphoethanolamine (PEtN) modifications increase the Oag diversity, providing benefits to S. flexneri LPS Oag acts as a primary receptor for bacteriophage Sf6, which infects only a limited range of S. flexneri serotypes (Y and X). It uses its tailspike protein (Sf6TSP) to establish initial interaction with LPS Oags that it then hydrolyzes. Currently, there is a lack of comprehensive study on the parent and serotype variant strains from the same genetic background and an understanding of the importance of LPS Oag O-acetylations. Therefore, a set of isogenic strains (based on S. flexneri 2457T [2a2]) with deletions of different Oag modification genes (oacB, oacD, and gtrII) that resemble different naturally occurring serotype Y and 2a strains was created. The impacts of these Oag modifications on S. flexneri sensitivity to Sf6 and the pathogenesis-related properties were then compared. We found that Sf6TSP can hydrolyze serotype 2a LPS Oag, identified that 3/4-O-acetylation is essential for resistance of serotype 2a strains to Sf6, and showed that serotype 2a strains have better invasion ability. Lastly, we revealed two new serotype conversions for S. flexneri, thereby contributing to understanding the evolution of this important human pathogen.IMPORTANCE The emergence of antibiotic-resistant strains and lack of efficient vaccines have made Shigella a priority organism for the World Health Organization (1). Therefore, bacteriophage therapy has received increasing attention as an alternative therapeutic approach. LPS Oag is the most variable part of LPS due to chemical modifications and is the target of bacteriophage Sf6 (S. flexneri specific). We dissected the evolution of S. flexneri serotype Y to 2a2, which revealed a new role for a gene acquired during serotype conversion and furthermore identified new specific forms of LPS receptor for Sf6. Collectively, these results unfold the importance of the acquisition of those Oag modification genes and further our understanding of the relationship between Sf6 and S. flexneri.

Crystal structure of α-glucosyl transfer enzyme XgtA from Xanthomonas campestris WU-9701.

The α-glucosyl transfer enzyme XgtA is a novel type α-Glucosidase (EC 3.2.1.20) produced by Xanthomonas campestris WU-9701. One of the unique properties of XgtA is that it shows extremely high α-glucosylation activity toward alcoholic and phenolic -OH groups in compounds using maltose as an α-glucosyl donor and allows for the synthesis of various useful α-glucosides with high yields. XgtA shows no hydrolytic activity toward sucrose and no α-glucosylation activity toward saccharides to produce oligosaccharides. In this report, the crystal structure of XgtA was solved at 1.72 Å resolution. The crystal belonged to space group P22121, with unit-cell parameters a = 73.07, b = 83.48, and c = 180.79 Å. The ß→α loop 4 of XgtA, which is proximal to the catalytic center, formed a unique structure that is not observed in XgtA homologs. Furthermore, XgtA was found to contain unique amino acid residues around its catalytic center. The unique structure of XgtA provides an insight into the mechanism for the regulation of substrate specificity in this enzyme.

OleD Loki as a Catalyst for Hydroxamate Glycosylation.

Herein we describe the ability of the permissive glycosyltransferase (GT) OleD Loki to convert a diverse set of >15 histone deacetylase (HDAC) inhibitors (HDACis) into their corresponding hydroxamate glycosyl esters. Representative glycosyl esters were subsequently evaluated in assays for cancer cell line cytotoxicity, chemical and enzymatic stability, and axolotl embryo tail regeneration. Computational substrate docking models were predictive of enzyme-catalyzed turnover and suggest certain HDACis may form unproductive, potentially inhibitory, complexes with GTs.

Microbial Transformation of Prenylquercetins by Mucor hiemalis.

Quercetin, one of the most widely distributed flavonoids, has been found to show various biological activities including antioxidant, anticancer, and anti-inflammatory effects. It has been reported that bioactivity enhancement of flavonoids has often been closely associated with nuclear prenylation, as shown in 8-prenylquercetin and 5'-prenylquercetin. It has also been revealed in many studies that the biological activities of flavonoids could be improved after glucosylation. Three prenylated quercetins were prepared in this study, and microbial transformation was carried out in order to identify derivatives of prenylquercetins with increased water solubility and improved bioavailability. The fungus M. hiemalis was proved to be capable of converting prenylquercetins into more polar metabolites and was selected for preparative fermentation. Six novel glucosylated metabolites were obtained and their chemical structures were elucidated by NMR and mass spectrometric analyses. All the microbial metabolites showed improvement in water solubility.

OcUGT1-Catalyzing Glycodiversification of Steroids through Glucosylation and Transglucosylation Actions.

Steroidal glycosides are important sources of innovative drugs. The increased diversification of steroidal glycosides will expand the probability of discovering active molecules. It is an efficient approach to diversify steroidal glycosides by using steroidal glycosyltransferases. OcUGT1, a uridine diphosphate-d-glucose (UDP-Glc)-dependent glycosyltransferase from Ornithogalum caudatum, is a multifunctional enzyme, and its glycodiversification potential towards steroids has never been fully explored. Herein, the glycodiversification capability of OcUGT1 towards 25 steroids through glucosylation and transglucosylation reactions were explored. Firstly, each of 25 compounds was glucosylated with UDP-Glc. Under the action of OcUGT1, five steroids (testosterone, deoxycorticosterone, hydrocortisone, estradiol, and 4-androstenediol) were glucosylated to form corresponding mono-glucosides and biosides. Next, OcUGT1-mediated transglucosylation activity of these compounds with another sugar donor ortho-nitrophenyl-ß-d-glucopyranoside (oNPGlc) was investigated. Results revealed that the same five steroids could be glucosylated to generate mono-glucosides and biosides by OcUGT1 through transglucosylation reactions. These data indicated that OcUGT1-assisted glycodiversification of steroids could be achieved through glucosylation and transglucosylation reactions. These results provide a way to diversify steroidal glycosides, which lays the foundation for the increase of the probability of obtaining active lead compounds.