Unravelling and reconstructing the biosynthetic pathway of bergenin.

Bergenin, a rare C-glycoside of 4-O-methyl gallic acid with pharmacological properties of antitussive and expectorant, is widely used in clinics to treat chronic tracheitis in China. However, its low abundance in nature and structural specificity hampers the accessibility through traditional crop-based manufacturing or chemical synthesis. In the present work, we elucidate the biosynthetic pathway of bergenin in Ardisia japonica by identifying the highly regio- and/or stereoselective 2-C-glycosyltransferases and 4-O-methyltransferases. Then, in Escherichia coli, we reconstruct the de novo biosynthetic pathway of 4-O-methyl gallic acid 2-C-ß-D-glycoside, which is the direct precursor of bergenin and is conveniently esterified into bergenin by in situ acid treatment. Moreover, further metabolic engineering improves the production of bergenin to 1.41 g L-1 in a 3-L bioreactor. Our work provides a foundation for sustainable supply of bergenin and alleviates its resource shortage via a synthetic biology approach.

Fisetin glycosides synthesized by cyclodextrin glycosyltransferase from Paenibacillus sp. RB01: characterization, molecular docking, and antioxidant activity.

Fisetin is a flavonoid that exhibits high antioxidant activity and is widely employed in the pharmacological industries. However, the application of fisetin is limited due to its low water solubility. In this study, glycoside derivatives of fisetin were synthesized by an enzymatic reaction using cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp. RB01 in order to improve the water solubility of fisetin. Under optimal conditions, CGTase was able to convert more than 400 mg/L of fisetin to its glycoside derivatives, which is significantly higher than the previous biosynthesis using engineered E. coli. Product characterization by HPLC and LC-MS/MS revealed that the transglycosylated products consisted of at least five fisetin glycoside derivatives, including fisetin mono-, di- and triglucosides, as well as their isomers. Enzymatic analysis by glucoamylase and α-glucosidase showed that these fisetin glycosides were formed by α-1,4-glycosidic linkages. Molecular docking demonstrated that there are two possible binding modes of fisetin in the enzyme active site containing CGTase-glysosyl intermediate, in which O7 and O4' atoms of fisetin positioned close to the C1 of glycoside donor, corresponding to the isomers of the obtained fisetin monoglucosides. In addition, the water solubility and the antioxidant activity of the fisetin monoglucosides were tested. It was found that their water solubility was increased at least 800 times when compared to that of their parent molecule while still maintaining the antioxidant activity. This study revealed the potential application of CGTase to improve the solubility of flavonoids.

Screening the Key Region of Sunlight Regulating the Flavonoid Profiles of Young Shoots in Tea Plants (Camellia sinensis L.) Based on a Field Experiment.

Both UV and blue light have been reported to regulate the biosynthesis of flavonoids in tea plants; however, the respective contributions of the corresponding regions of sunlight are unclear. Additionally, different tea cultivars may respond differently to altered light conditions. We investigated the responses of different cultivars ('Longjing 43', 'Zhongming 192', 'Wanghai 1', 'Jingning 1' and 'Zhonghuang 2') to the shade treatments (black and colored nets) regarding the biosynthesis of flavonoids. For all cultivars, flavonol glycosides showed higher sensitivity to light conditions compared with catechins. The levels of total flavonol glycosides in the young shoots of different tea cultivars decreased with the shade percentages of polyethylene nets increasing from 70% to 95%. Myricetin glycosides and quercetin glycosides were more sensitive to light conditions than kaempferol glycosides. The principal component analysis (PCA) result indicated that shade treatment greatly impacted the profiles of flavonoids in different tea samples based on the cultivar characteristics. UV is the crucial region of sunlight enhancing flavonol glycoside biosynthesis in tea shoots, which is also slight impacted by light quality according to the results of the weighted correlation network analysis (WGCNA). This study clarified the contributions of different wavelength regions of sunlight in a field experiment, providing a potential direction for slightly bitter and astringent tea cultivar breeding and instructive guidance for practical field production of premium teas based on light regimes.

Naturally Occurring Chromone Glycosides: Sources, Bioactivities, and Spectroscopic Features.

Chromone glycosides comprise an important group of secondary metabolites. They are widely distributed in plants and, to a lesser extent, in fungi and bacteria. Significant biological activities, including antiviral, anti-inflammatory, antitumor, antimicrobial, etc., have been discovered for chromone glycosides, suggesting their potential as drug leads. This review compiles 192 naturally occurring chromone glycosides along with their sources, classification, biological activities, and spectroscopic features. Detailed biosynthetic pathways and chemotaxonomic studies are also described. Extensive spectroscopic features for this class of compounds have been thoroughly discussed, and detailed 13C-NMR data of compounds 1-192, have been added, except for those that have no reported 13C-NMR data.

Daily dynamics of intermediate metabolite profiles lead to time-dependent phenylethanoid glycosides production in Scrophularia striata during the day/night cycle.

Phenylethanoid glycosides (PhGs) are important medicinal compounds found in Scrophularia striata, one of the plant species native to Iran. Since almost all aspects of plant life are controlled by night/light cycle, studying its relationship to valuable plant metabolites production will help us to determine the right time for their extraction. Therefore, the aim of this investigation is to figure out whether the diel light oscillations control PhGs production and how it relates to daily changes in upstream metabolic reactions and circadian clock in S. striata. For this, daily rhythms of metabolic pathways were examined every 4 h during a day/night cycle in 3 groups of control (16 h light/8 h dark), continuous light and darkness. The results showed that acteoside and echinacoside levels in each group peaked during the night and day, respectively. Thus, the PhGs production follows a rhythmic behavior in S. striata, which is probably controlled by circadian clock. Also, the levels of photosynthetic pigments, carbohydrates, amino acids, phenolic acids, phytohormones and phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) enzyme activities varied diel in a similar or different way among study groups. The observations revealed that light/dark cycle controls the carbon and energy flow from light reception to the production and consumption of starch, biosynthesis of phenylalanine, tyrosine, cinnamic acid and coumaric acid, activation of hormonal signaling pathways and enzymes involved in phenylpropanoid pathway. Overall, it can be concluded that PhGs accumulation time-dependent patterns is likely due to daily fluctuations in upstream metabolic reactions induced by light/dark cycle.

Discovery of Glycosyltransferases Involved in the Biosynthesis of Ligupurpuroside B.

In this study, we report the characterization of three glycosyltransferases involved in the biosynthesis of ligupurpuroside B, a complex acylated phenolic glycoside in Ligustrum robustum. UGT85AF8 catalyzed the formation of salidroside from tyrosol. UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase, and UGT79A19, an osmanthuside B 1,4-rhamnosyltransferase, sequentially converted osmanthuside A into ligupurpuroside B. Orthologs of UGT79G7 were also discovered from other plants producing verbascoside. These rhamnosyltransferases expand the toolbox for the biosynthesis of natural products with various sugar chains.

A Regulator Based "Semi-Targeted" Approach to Activate Silent Biosynthetic Gene Clusters.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel "semi-targeted" approach focusing on activating "silent" BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Controlled Production of Zearalenone-Glucopyranoside Standards with Cunninghamella Strains Using Sulphate-Depleted Media.

In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-ß-D-glucopyranoside (Z14G), zearalenone-16-ß-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, ß-zearalenol and other mycotoxins.

Engineering Thermotoga maritima ß-glucosidase for improved alkyl glycosides synthesis by site-directed mutagenesis.

Alkyl glycosides are well-characterized nonionic surfactants, and can be prepared by transglycosylation reactions with retaining GH1 glycosidases being normally used for this purpose. The produced alkyl glycosides can also be hydrolyzed by the glycosidase, and hence, the yields of alkyl glycosides can be too low for industrial use. To improve the transglycosylation-to-hydrolysis ratio for a ß-glucosidase from Thermotoga maritima (TmBglA) for the synthesis of alkyl glycoside, six mutants (N222F, N223C, N223Q, G224A, Y295F, and F414S) were produced. N222F, N223C, N223Q, G224A improved catalytic activity, F295Y and F414S are hydrolytically crippled with p-nitrophenol-ß-d-glucopyranoside (pNPG) as substrate with an 85 and 70-fold decrease in apparent kcat, respectively; N222F shows the highest kcat/km value for pNPG. The substrate selectivity altered from pNPG to pNP-ß-d-fucoside for N222F, F295Y, and F414S and from cellubiose to gentiobiose for N222F and F414S. Using pNPG (34 mM) and hexanol 80% (vol/vol), N222F, Y295F, and F414S synthesized hexyl-ß-glycoside (HG) yields of 84.7%, 50.9%, and 54.1%, respectively, HG increased from 14.49 (TmBglA) to 22.8 mM (N222F) at 2 hr by 57.42%. However, this higher transglycosylation effect depended on that three mutants creates an environment more suited for hexanol in the active site pocket, and consequently suppressed its HG hydrolysis.

Structural insight on assembly-line catalysis in terpene biosynthesis.

Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.

Structural basis for transglycosylation in glycoside hydrolase family GH116 glycosynthases.

Glycosynthases are glycoside hydrolase mutants that can synthesize oligosaccharides or glycosides from an inverted donor without hydrolysis of the products. Although glycosynthases have been characterized from a variety of glycoside hydrolase (GH) families, family GH116 glycosynthases have yet to be reported. We produced the Thermoanaerobacterium xylanolyticum TxGH116 nucleophile mutants E441D, E441G, E441Q and E441S and compared their glycosynthase activities to the previously generated E441A mutant. The TxGH116 E441G and E441S mutants exhibited highest glycosynthase activity to transfer glucose from α-fluoroglucoside (α-GlcF) to cellobiose acceptor, while E441D had low but significant activity as well. The E441G, E441S and E441A variants showed broad specificity for α-glycosyl fluoride donors and p-nitrophenyl glycoside acceptors. The structure of the TxGH116 E441A mutant with α-GlcF provided the donor substrate complex, while soaking of the TxGH116 E441G mutant with α-GlcF resulted in cellooligosaccharides extending from the +1 subsite out of the active site, with glycerol in the -1 subsite. Soaking of E441A or E441G with cellobiose or cellotriose gave similar acceptor substrate complexes with the nonreducing glucosyl residue in the +1 subsite. Combining structures with the ligands from the TxGH116 E441A with α-GlcF crystals with that of E441A or E441G with cellobiose provides a plausible structure of the catalytic ternary complex, which places the nonreducing glucosyl residue O4 2.5 Å from the anomeric carbon of α-GlcF, thereby explaining its apparent preference for production of ß-1,4-linked oligosaccharides. This functional and structural characterization provides the background for development of GH116 glycosynthases for synthesis of oligosaccharides and glycosides of interest.

Indolyl Septanoside Synthesis for In Vivo Screening of Bacterial Septanoside Hydrolases.

Building-up and breaking-down of carbohydrates are processes common to all forms of life. Glycoside hydrolases are a broad class of enzymes that play a central role in the cleavage of glycosidic bonds, which is fundamental to carbohydrate degradation. The large majority of substrates are five- and six-membered ring glycosides. Our interest in seven-membered ring septanose sugars has inspired the development of a way to search for septanoside hydrolase activity. Described here is a strategy for the discovery of septanoside hydrolases that uses synthetic indolyl septanosides as chromogenic substrates. Access to these tool compounds was enabled by a route where septanosyl halides act as glycosyl donors for the synthesis of the indolyl septanosides. The screening strategy leverages the known dimerization of 3-hydroxy-indoles to make colored dyes, as occurs when the ß-galactosidase substrate X-Gal is hydrolyzed. Because screens in bacterial cells would enable searches in organisms that utilize heptoses or from metagenomics libraries, we also demonstrate that septanosides are capable of entering E. coli cells through the use of a BODIPY-labeled septanoside. The modularity of the indolyl septanoside synthesis should allow the screening of a variety of substrates that mimic natural structures via this general approach.

Triterpene Glycosides from the Far Eastern Sea Cucumber Thyonidium (=Duasmodactyla) kurilensis (Levin): The Structures, Cytotoxicities, and Biogenesis of Kurilosides A3, D1, G, H, I, I1, J, K, and K1.

Nine new mono-, di-, and trisulfated triterpene penta- and hexaosides, kurilosides A3 (1), D1 (2), G (3), H (4), I (5), I1 (6), J (7), K (8), and K1 (9) and two desulfated derivatives, DS-kuriloside L (10), having a trisaccharide branched chain, and DS-kuriloside M (11), having hexa-nor-lanostane aglycone with a 7(8)-double bond, have been isolated from the Far-Eastern deep-water sea cucumber Thyonidium (=Duasmodactyla) kurilensis (Levin) and their structures were elucidated based on 2D NMR spectroscopy and HR-ESI mass-spectrometry. Five earlier unknown carbohydrate chains and two aglycones (having a 16ß,(20S)-dihydroxy-fragment and a 16ß-acetoxy,(20S)-hydroxy fragment) were found in these glycosides. All the glycosides 1-9 have a sulfate group at C-6 Glc, attached to C-4 Xyl1, while the positions of the other sulfate groups vary in different groups of kurilosides. The analysis of the structural features of the aglycones and the carbohydrate chains of all the glycosides of T. kurilensis showed their biogenetic relationships. Cytotoxic activities of the compounds 1-9 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells, and erythrocytes were studied. The highest cytotoxicity in the series was demonstrated by trisulfated hexaoside kuriloside H (4), having acetoxy-groups at C(16) and C(20), the latter one obviously compensated the absence of a side chain, essential for the membranolytic action of the glycosides. Kuriloside I1 (6), differing from 4 in the lacking of a terminal glucose residue in the bottom semi-chain, was slightly less active. The compounds 1-3, 5, and 8 did not demonstrate cytotoxic activity due to the presence of hydroxyl groups in their aglycones.

[Application of immobilized glycosidase in the synthesis of glycoside compounds].

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.

Engineering CGTase to improve synthesis of alkyl glycosides.

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.

A willow UDP-glycosyltransferase involved in salicinoid biosynthesis.

The salicinoids are phenolic glycosides that are characteristic secondary metabolites of the Salicaceae, particularly willows and poplars. Despite the well-known pharmacology of salicin, that led to the development of aspirin >100 years ago, the biosynthetic pathways leading to salicinoids have yet to be defined. Here, we describe the identification, cloning, and biochemical characterization of SpUGT71L2 and SpUGT71L3-isozymic glycosyltransferases from Salix purpurea-that function in the glucosylation of ortho-substituted phenols. The best substrate in vitro was salicyl-7-benzoate. Its product, salicyl-7-benzoate glucoside, was shown to be endogenous in poplar and willow. Together they are inferred to be early intermediates in the biosynthesis of salicortin and related metabolites in planta. The role of this UDP-glycosyltransferase was confirmed via the metabolomic analysis of transgenic plants produced by RNAi knockdown of the poplar orthologue (UGT71L1) in the hybrid clone Populus tremula×P. alba, INRA 717-1B4.

Application of immobilized glycosidase in the synthesis of glycoside compounds / 生物工程学报

Glycoside compounds are widely used in medicine, food, surfactant, and cosmetics. The glycosidase-catalyzed synthesis of glycoside can be operated at mild reaction conditions with low material cost. The glycosidase-catalyzed processes include reverse hydrolysis and transglycosylation, appropriately reducing the water activity in both processes may effectively improve the catalytic efficiency of glucosidase. However, glucosidase is prone to be deactivated at low water activity. Thus, glucosidase was immobilized to maintain its activity in the low water activity environment, and even in neat organic solvent system. This article summarizes the advances in glycosidase immobilization in the past 30 years, including single or comprehensive immobilization techniques, and immobilization techniques combined with genetic engineering, with the aim to provide a reference for the synthesis of glycosides using immobilized glycosidases.

Dissection of the general two-step di-C-glycosylation pathway for the biosynthesis of (iso)schaftosides in higher plants.

Schaftoside and isoschaftoside are bioactive natural products widely distributed in higher plants including cereal crops and medicinal herbs. Their biosynthesis may be related with plant defense. However, little is known on the glycosylation biosynthetic pathway of these flavonoid di-C-glycosides with different sugar residues. Herein, we report that the biosynthesis of (iso)schaftosides is sequentially catalyzed by two C-glycosyltransferases (CGTs), i.e., CGTa for C-glucosylation of the 2-hydroxyflavanone aglycone and CGTb for C-arabinosylation of the mono-C-glucoside. The two enzymes of the same plant exhibit high homology but remarkably different sugar acceptor and donor selectivities. A total of 14 CGTa and CGTb enzymes were cloned and characterized from seven dicot and monocot plants, including Scutellaria baicalensis, Glycyrrhiza uralensis, Oryza sativa ssp. japonica, and Zea mays, and the in vivo functions for three enzymes were verified by RNA interference and overexpression. Through transcriptome analysis, we found homologous genes in 119 other plants, indicating this pathway is general for the biosynthesis of (iso)schaftosides. Furthermore, we resolved the crystal structures of five CGTs and realized the functional switch of SbCGTb to SbCGTa by structural analysis and mutagenesis of key amino acids. The CGT enzymes discovered in this paper allow efficient synthesis of (iso)schaftosides, and the general glycosylation pathway presents a platform to study the chemical defense mechanisms of higher plants.

Exploring and applying the substrate promiscuity of a C-glycosyltransferase in the chemo-enzymatic synthesis of bioactive C-glycosides.

Bioactive natural C-glycosides are rare and chemical C-glycosylation faces challenges while enzymatic C-glycosylation catalyzed by C-glycosyltransferases provides an alternative way. However, only a small number of C-glycosyltransferases have been found, and most of the discovered C-glycosyltransferases prefer to glycosylate phenols with an acyl side chain. Here, a promiscuous C-glycosyltransferase, AbCGT, which is capable of C-glycosylating scaffolds lacking acyl groups, is identified from Aloe barbadensis. Based on the substrate promiscuity of AbCGT, 16 C-glycosides with inhibitory activity against sodium-dependent glucose transporters 2 are chemo-enzymatically synthesized. The C-glycoside 46a shows hypoglycemic activity in diabetic mice and is biosynthesized with a cumulative yield on the 3.95 g L‒1 scale. In addition, the key residues involved in the catalytic selectivity of AbCGT are explored. These findings suggest that AbCGT is a powerful tool in the synthesis of lead compounds for drug discovery and an example for engineering the catalytic selectivity of C-glycosyltransferases.

Biosynthesis of oxygenated brasilane terpene glycosides involves a promiscuous N-acetylglucosamine transferase.

Investigation of the metabolome of the ascomycete Annulohypoxylon truncatum led to the identification of novel oxygenated brasilane glycosides and the revision of the stereochemistry of the brasilane A octahydro-1H-indene core scaffold to trans. The bra biosynthetic gene cluster containing five genes (braA-braE) was identified and verified by heterologous expression experiments in Aspergillus oryzae demonstrating that BraC is a multifunctional P450 monooxygenase. In vitro studies of BraB revealed it to be a very rare fungal UDP-GlcNAc dependent N-acetylglucosamine transferase. UDP-glucose is also accepted as a donor, and a broad acceptor substrate tolerance for various primary and secondary alcohols was observed.