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Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3- O- glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species ( Glycyrrhiza uralensis, Glycine max , and Lotus japonicus ) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae . CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane–localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins. Saponins such as glycyrrhizin, a natural sweetener found in licorice root, are a class of triterpenoids synthesized that are characterized by a glucoronic acid moiety at the C-3 position. Here the authors show that saponin glucuronosylation is catalyzed by cellulose-synthase like enzymes and reconstitute glycyrrhizin synthesisin yeast.

The objective was to determine the effects of dietary fibre with bulking, viscous and gel-forming properties on satiation, and to identify the underlying mechanisms. We conducted a randomised crossover study with 121 men and women. Subjects were healthy, non-restrained eaters, aged 18–50 years and with normal BMI (18·5–25 kg/m 2 ). Test products were cookies containing either: no added fibre (control), cellulose (bulking, 5 g/100 g), guar gum (viscous, 1·25 g/100 g and 2·5 g/100 g) or alginate (gel forming, 2·5 g/100 g and 5 g/100 g). Physico-chemical properties of the test products were confirmed in simulated upper gastrointestinal conditions. In a cinema setting, ad libitum intake of the test products was measured concurrently with oral exposure time per cookie by video recording. In a separate study with ten subjects, 4 h gastric emptying rate of a fixed amount of test products was assessed by 13 C breath tests. Ad libitum energy intake was 22 % lower for the product with 5 g/100 g alginate (3·1 ( sd 1·6) MJ) compared to control (4·0 ( sd 2·2) MJ, P < 0·001). Intake of the other four products did not differ from control. Oral exposure time for the product with 5 g/100 g alginate (2·3 ( sd 1·9) min) was 48 % longer than for control (1·6 ( sd 0·9) min, P = 0·01). Gastric emptying of the 5 g/100 g alginate product was faster compared to control ( P < 0·05). We concluded that the addition of 5 g/100 g alginate (i.e. gel-forming fibre) to a low-fibre cookie results in earlier satiation. This effect might be due to an increased oral exposure time.

Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5'-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the ¹⁴C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [¹⁴C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD).

Prospecting macroalgae (seaweeds) as feedstocks for bioconversion into biofuels and commodity chemical compounds is limited primarily by the availability of tractable microorganisms that can metabolize alginate polysaccharides. Here, we present the discovery of a 36—kilo—base pair DNA fragment from Vibrio splendidus encoding enzymes for alginate transport and metabolism. The genomic integration of this ensemble, together with an engineered system for extracellular alginate depolymerization, generated a microbial platform that can simultaneously degrade, uptake, and metabolize alginate. When further engineered for ethanol synthesis, this platform enables bioethanol production directly from macroalgae via a consolidated process, achieving a titer of 4.7% volume/volume and a yield of 0.281 weight ethanol/weight dry macroalgae (equivalent to ~80% of the maximum theoretical yield from the sugar composition in macroalgae).

Humans express seven heparan sulfate (HS) 3-O-sulfotransferases that differ in substrate specificity and tissue expression. Although genetic studies have indicated that 3-O-sulfated HS modulates many biological processes, ligand requirements for proteins engaging with HS modified by 3-O-sulfate (3-OS) have been difficult to determine. In particular, the context in which the 3-OS group needs to be presented for binding is largely unknown. We describe herein a modular synthetic approach that can provide structurally diverse HS oligosaccharides with and without 3-OS. The methodology was employed to prepare 27 hexasaccharides that were printed as a glycan microarray to examine ligand requirements of a wide range of HS-binding proteins. The binding selectivity of antithrombin-III (AT-III) compared well with anti-Factor Xa activity supporting robustness of the array technology. Many of the other examined HS-binding proteins required an IdoA2S-GlcNS3S6S sequon for binding but exhibited variable dependence for the 2-OS and 6-OS moieties, and a GlcA or IdoA2S residue neighboring the central GlcNS3S. The HS oligosaccharides were also examined as inhibitors of cell entry by herpes simplex virus type 1, which, surprisingly, showed a lack of dependence of 3-OS, indicating that, instead of glycoprotein D (gD), they competitively bind to gB and gC. The compounds were also used to examine substrate specificities of heparin lyases, which are enzymes used for depolymerization of HS/heparin for sequence determination and production of therapeutic heparins. It was found that cleavage by lyase II is influenced by 3-OS, while digestion by lyase I is only affected by 2-OS. Lyase III exhibited sensitivity to both 3-OS and 2-OS.

In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes. Malignant phenotypes in the mammary epithelium have been correlated to increases in extracellular matrix stiffness. It is now shown that the effect of matrix stiffness in normal mammary epithelial cells can be offset by an increase in basement-membrane ligands and that both the stiffness and composition of the matrix are sensed by the β4 integrin. The results suggest that the relationship between matrix stiffness and composition is a more relevant predictor of breast-cancer progression.

Cancer metastasis is the primary cause of morbidity and mortality, and accounts for up to 95% of cancer-related deaths 1 . Cancer cells often reprogram their metabolism to efficiently support cell proliferation and survival 2 , 3 . However, whether and how those metabolic alterations contribute to the migration of tumour cells remain largely unknown. UDP-glucose 6-dehydrogenase (UGDH) is a key enzyme in the uronic acid pathway, and converts UDP-glucose to UDP-glucuronic acid 4 . Here we show that, after activation of EGFR, UGDH is phosphorylated at tyrosine 473 in human lung cancer cells. Phosphorylated UGDH interacts with Hu antigen R (HuR) and converts UDP-glucose to UDP-glucuronic acid, which attenuates the UDP-glucose-mediated inhibition of the association of HuR with SNAI1 mRNA and therefore enhances the stability of SNAI1 mRNA. Increased production of SNAIL initiates the epithelial–mesenchymal transition, thus promoting the migration of tumour cells and lung cancer metastasis. In addition, phosphorylation of UGDH at tyrosine 473 correlates with metastatic recurrence and poor prognosis of patients with lung cancer. Our findings reveal a tumour-suppressive role of UDP-glucose in lung cancer metastasis and uncover a mechanism by which UGDH promotes tumour metastasis by increasing the stability of SNAI1 mRNA. UDP-glucose has a tumour-suppressive role by inhibiting the association between HuR and SNAI1 mRNA, whereas UGDH-mediated metabolism of UDP-glucose leads to increased SNAI1 mRNA stability and expression, thereby promoting tumour cell migration and lung cancer metastasis.

Saccharomyces cerevisiae bearing engineered alginate and mannitol catabolic pathways can ferment sugars from brown macroalgae to produce ethanol, potentially allowing the use of brown macroalgae as a viable feedstock for the production of biofuels and renewable chemicals. Brown algae as a biofuel feedstock Brown macroalgae are seen as a viable feedstock for the production of biofuels, with the advantage that they can be farmed in coastal waters without using valuable arable land. However, the most abundant sugars in brown macroalgae are alginate, mannitol and glucan, and the full potential of this feedstock cannot be realized without extensive re-engineering of the alginate and mannitol catabolic pathways in Saccharomyces cerevisiae . In this paper the authors identify a 4-deoxy-L-erythro-5-hexoseulose uronate transporter in Asteromyces cruciatus brown algae and use it to develop a S. cerevisiae strain that can use the unique sugars in brown macroalgae for high-efficiency ethanol fermentation. With appropriate genetic modifications, this synthetic biology platform can be used to produce many other biofuels and renewable chemicals. The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways 1 , 2 , 3 in the standard industrial microbe Saccharomyces cerevisiae . Here we present the discovery of an alginate monomer (4-deoxy- l -erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus 4 . The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l −1 ) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Abstract The Gram-negative pathogen Pseudomonas aeruginosa is found ubiquitously within the environment and is recognised as an opportunistic human pathogen that commonly infects burn wounds and immunocompromised individuals, or patients suffering from the autosomal recessive disorder cystic fibrosis (CF). During chronic infection, P. aeruginosa is thought to form structured aggregates known as biofilms characterised by a self-produced matrix which encases the bacteria, protecting them from antimicrobial attack and the host immune response. In many cases, antibiotics are ineffective at eradicating P. aeruginosa from chronically infected CF airways. Cyclic-di-GMP has been identified as a key regulator of biofilm formation; however, the way in which its effector proteins elicit a change in biofilm formation remains unclear. Identifying regulators of biofilm formation is a key theme of current research and understanding the factors that activate biofilm formation may help to expose potential new drug targets that slow the onset of chronic infection. This minireview outlines the contribution made by exopolysaccharides to biofilm formation, and describes the current understanding of biofilm regulation in P. aeruginosa with a particular focus on CF airway-associated infections. Here, we review our understanding of what controls production of the sticky ‘glue’ that holds bacterial communities together.

This review examines the recent models describing the mode of action of various xylanolytic enzymes and how these enzymes can be applied (sequentially or simultaneously) with their distinctive roles in mind to achieve efficient xylan degradation. With respect to homeosynergy, synergism appears to be as a result of β-xylanase and/or oligosaccharide reducing-end β-xylanase liberating xylo-oligomers (XOS) that are preferred substrates of the processive β-xylosidase. With regards to hetero-synergism, two cross relationships appear to exist and seem to be the reason for synergism between the enzymes during xylan degradation. These cross relations are the debranching enzymes such as α-glucuronidase or side-chain cleaving enzymes such as carbohydrate esterases (CE) removing decorations that would have hindered back-bone-cleaving enzymes, while backbone-cleaving-enzymes liberate XOS that are preferred substrates of the debranching and side-chain-cleaving enzymes. This interaction is demonstrated by high yields in co-production of xylan substituents such as arabinose, glucuronic acid and ferulic acid, and XOS. Finally, lytic polysaccharide monooxygenases (LPMO) have also been implicated in boosting whole lignocellulosic biomass or insoluble xylan degradation by glycoside hydrolases (GH) by possibly disrupting entangled xylan residues. Since it has been observed that the same enzyme (same Enzyme Commission, EC, classification) from different GH or CE and/or AA families can display different synergistic interactions with other enzymes due to different substrate specificities and properties, in this review, we propose an approach of enzyme selection (and mode of application thereof) during xylan degradation, as this can improve the economic viability of the degradation of xylan for producing precursors of value added products.