Sugar Precursors Research Articles

Immobilization of UDP‐Galactose on an Amphiphilic Resin

Nucleotide sugars are activated sugar precursors used by glycosyltransferases to form glycans. Here we report the first examples of resin‐bound nucleotide sugars: fluorinated UDP‐galactose and UDP‐galactose. They were conjugated to a polyethylene glycol resin through the C6 position with an SMCC linker in good to excellent yields. This synthesis represents an efficient method to access resin‐bound nucleotide sugars in a modular approach that enables modifications of the sugar or nucleotide before conjugation. The system has been conceived as a new chemistry‐based screening system for enzymes that use UDP‐galactose.

The Small RNA ErsA of Pseudomonas aeruginosa Contributes to Biofilm Development and Motility through Post-transcriptional Modulation of AmrZ.

The small RNA ErsA of Pseudomonas aeruginosa was previously suggested to be involved in biofilm formation via negative post-transcriptional regulation of the algC gene that encodes the virulence-associated enzyme AlgC, which provides sugar precursors for the synthesis of several polysaccharides. In this study, we show that a knock-out ersA mutant strain forms a flat and uniform biofilm, not characterized by mushroom-multicellular structures typical of a mature biofilm. Conversely, the knock-out mutant strain showed enhanced swarming and twitching motilities. To assess the influence of ErsA on the P. aeruginosa transcriptome, we performed RNA-seq experiments comparing the knock-out mutant with the wild-type. More than 160 genes were found differentially expressed in the knock-out mutant. Parts of these genes, important for biofilm formation and motility regulation, are known to belong also to the AmrZ transcriptional regulator regulon. Here, we show that ErsA binds in vitro and positively regulates amrZ mRNA at post-transcriptional level in vivo suggesting an interesting contribution of the ErsA-amrZ mRNA interaction in biofilm development at several regulatory levels.

Domestication and saponins contents in a gradient of management intensity of agaves: Agave cupreata, A. inaequidens and A. hookeri in central Mexico

Saponins occur in numerous plants, including agaves, determining benefic and harmful properties to humans; their presence may favor using plants as soap and other products, but also they may cause caustic effects producing contact dermatitis. In domestication, favorable and unfavorable properties of saponins may cause an increase or decrease of their content, respectively. This study quantified and identified saponins among wild and managed populations of three agave species: A. cupreata Trel. et Berger, A. inaequidens Koch with wild and cultivated populations used for mescal production, and A. hookeri Jacobi, existing exclusively cultivated, used for production of the fermented beverage pulque. We studied 272 plants from 19 populations, quantifying contents of crude saponins through spectrometry. In 12 populations, the saponins types were identified by High Performance Liquid Chromatography–Mass-Spectrography-Time-of-Flight HPLC-MS-TOF. The highest crude saponins content was recorded in A. hookeri (26.09 mg/g), followed by A. cupreata (19.85 and 15.17 mg/g in wild and cultivated populations, respectively). For A. inaequidens, we recorded 14.21, 12.95, and 10.48 mg/g in wild, silvicultural managed and cultivated populations, respectively. We identified 18 saponins types, A. inaequidens showing all of them. A hecogenin glycoside (HG1) is found in high amounts in A. hookeri but in low quantities in A. inaequidens and A. cupreata. A. inaequidens had the greatest diversity of saponins. The contents of crude saponins in A. inaequidens and A. cupreata decrease with management intensity, but contrarily to what we expected, it was the highest in A. hookeri. We hypothesize that such high amount could be due to some saponins, probably HG1, may be precursors of sugars.

Glycobiotechnology of the Insect Cell-Baculovirus Expression System Technology.

The insect cell-baculovirus expression system technology (BEST) has a prominent role in producing recombinant proteins to be used as research and diagnostic reagents and vaccines. The glycosylation profile of proteins produced by the BEST is composed predominantly of terminal mannose glycans, and, in Trichoplusia ni cell lines, core α3 fucosylation, a profile different to that in mammals. Insects contain all the enzymatic activities needed for complex N- and O-glycosylation and sialylation, although few reports of complex glycosylation and sialylation by the BEST exist. The insect cell line and culture conditions determine the glycosylation profile of proteins produced by the BEST. The promoter used, dissolved oxygen tension, presence of sugar precursors, bovine serum or hemolymph, temperature, and the time of harvest all influence glycosylation, although more research is needed. The lack of activity of glycosylation enzymes possibly results from the transcription regulation and stress imposed by baculovirus infection. To solve this limitation, the glycosylation pathway of insect cells has been engineered to produce complex sialylated glycans and to eliminate α3 fucosylation, either by generating transgenic cell lines or by using baculovirus vectors. These strategies have been successful. Complex glycosylation, sialylation, and inhibition of α3 fucosylation have been achieved, although the majority of glycans still have terminal mannose residues. The implication of insect glycosylation in the proteins produced by the BEST is discussed. Graphical Abstract.

Nucleotide sugar precursor feeding strategy to enhance sialylation of albumin-erythropoietin in CHO cell cultures

Abstract Chinese hamster ovary (CHO) cells have been widely used for the production of glycoproteins due to various advantages such as high productivity, ease of genetic manipulation, adaptability to serum-free media, and human-like glycosylation. Glycosylation, which occurs sequentially using nucleotide sugars as donor substrates, can affect the therapeutic function of glycoproteins. Terminal sialic acid residues of N-glycans play an important role in determining in vivo half-life of glycoproteins. Therefore, various strategies have been developed to enhance sialylation of glycoproteins. In this study, a nucleotide sugar precursor feeding strategy was optimized to increase sialic acid content of albumin-erythropoietin (Alb-EPO). Glucosamine, galactose, and N-acetylmannosamine were added to CHO cell cultures in lag (day 0) or exponential phase (day 3). Central composite design and response surface methodology were used to evaluate the effects of nucleotide sugar precursors on cell growth, titer, and sialic acid content. For the exponential feeding strategy, culture performance was not altered by addition of optimal precursor concentrations, whereas sialic acid contents increased 1.43-fold compared to the control. Consequently, our nucleotide sugar precursor feeding strategy can provide an efficient culture process for production of highly sialylated glycans on Alb-EPO.

Economic viability of energy beets (Beta vulgaris) as advanced biofuel feedstocks

Low returns and volatility in agricultural prices have led farmers to pursue new cropping opportunities, including bioenergy and bioproduct industrial sugar precursors, such as energy beets. This article assesses the economic viability of energy beet production using integrated agronomic and economic geographic information systems based techniques to estimate yields and break-even prices in North Dakota. The results show that beet yields vary throughout the state, averaging 34.4t/ha, and are highest in the East, in the Red River Valley, where soil quality is high, moisture is plentiful and heat is sufficient. Production is also viable in the central and northern regions of the state, where soil quality and moisture are less favorable than in the Red River Valley. A smaller scale ethanol plant (75 million liters) would be able to source sufficient materials for production in many locations throughout the state for prices around $30/t.

Chemical Synthesis of Marine-Derived Sulfoglycolipids, a New Class of Molecular Adjuvants.

Vaccines play a primary role in the protection of human health by preventing infectious and chronic diseases. Recently we have reported 1,2-O-distearoyl-3-O-β-d-sulfoquinovosylglycerol (β-SQDG18), here named Sulfavant A (1), which shows promising properties as a new molecular adjuvant in in vitro and in vivo tests. In the present manuscript, we provide full details about a synthetic strategy for the preparation of 1, including a discussion of chemical determinants of the activity and the major technical hurdles we faced during the study. Synthesis of Sulfavant A (1) is achieved by a versatile procedure based on a trichloroacetimidate methodology and peracetate sugar precursors. The final design opens possibilities for the preparation of a series of interesting analogs for further pharmacological optimization and development, including derivatives containing different saturated and polyunsaturated fatty acids (e.g., 17 and 22).

Recent Advances in the Chemical Synthesis of C-Glycosides.

Advances in the chemical synthesis of C-pyranosides/furanosides are summarized, covering the literature from 2000 to 2016. The majority of the methods take advantage of the construction of the glycosidic C-C bond. These C-glycosylation methods are categorized herein in terms of the glycosyl donor precursors, which are commonly used in O-glycoside synthesis and are easily accessible to nonspecialists. They include glycosyl halides, glycals, sugar acetates, sugar lactols, sugar lactones, 1,2-anhydro sugars, thioglycosides/sulfoxides/sulfones, selenoglycosides/telluroglycosides, methyl glycosides, and glycosyl imidates/phosphates. Mechanistically, C-glycosylation reactions can involve glycosyl electrophilic/cationic species, anionic species, radical species, or transition-metal complexes, which are discussed as subcategories under each type of sugar precursor. Moreover, intramolecular rearrangements, such as the Claisen rearrangement, Ramberg-Bäcklund rearrangement, and 1,2-Wittig rearrangement, which usually involve concerted pathways, constitute another category of C-glycosylations. An alternative to the C-glycosylations is the formation of pyranoside/furanoside rings after construction of the predetermined glycosidic C-C bonds, which might involve cyclization of acyclic precursors or D-A cycloadditions. Throughout, the stereoselectivity in the formation of the resultant C-glycosidic linkages is highlighted.

Intracellular CHO Cell Metabolite Profiling Reveals Steady-State Dependent Metabolic Fingerprints in Perfusion Culture.

Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017.

New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors.

Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·gcdw−1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.

Cryptococcus neoformans UGT1 encodes a UDP-Galactose/UDP-GalNAc transporter.

Cryptococcus neoformans, an opportunistic fungal pathogen, produces a glycan capsule to evade the immune system during infection. This definitive virulence factor is composed mainly of complex polysaccharides, which are made in the secretory pathway by reactions that utilize activated nucleotide sugar precursors. Although the pathways that synthesize these precursors are known, the identity and the regulation of the nucleotide sugar transporters (NSTs) responsible for importing them into luminal organelles remain elusive. The UDP-galactose transporter, Ugt1, was initially identified by homology to known UGTs and glycan composition analysis of ugt1Δ mutants. However, sequence is an unreliable predictor of NST substrate specificity, cells may express multiple NSTs with overlapping specificities, and NSTs may transport multiple substrates. Determining NST activity thus requires biochemical demonstration of function. We showed that Ugt1 transports both UDP-galactose and UDP-N-acetylgalactosamine in vitro. Deletion of UGT1 resulted in growth and mating defects along with altered capsule and cellular morphology. The mutant was also phagocytosed more readily by macrophages than wild-type cells and cleared more quickly in vivo and in vitro, suggesting a mechanism for the lack of virulence observed in mouse models of infection.

Genetic alteration of UDP-rhamnose metabolism in Botrytis cinerea leads to the accumulation of UDP-KDG that adversely affects development and pathogenicity.

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs.

Biochemical and Structural Characterization of a Five-domain GH115 α-Glucuronidase from the Marine Bacterium Saccharophagus degradans 2-40T

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40(T), SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C(+) domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C(+) in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes.

Rhamnose synthase activity is required for pathogenicity of the vascular wilt fungus Verticillium dahliae.

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so-called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy-thymidine diphosphate (dTDP)-rhamnose, a precursor of L-rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal-host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)-rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.

Synthesis of glucopyranos-6′-yl purine and pyrimidine isonucleosides as potential cholinesterase inhibitors. Access to pyrimidine-linked pseudodisaccharides through Mitsunobu reaction

Abstract The synthesis of new isonucleosides comprising purine and pyrimidine-derived systems linked to methyl glucopyranosidyl units at C-6 and evaluation of their cholinesterase inhibitory profiles is reported. Their access was based on the Mitsunobu coupling of partially acetylated and benzylated methyl glucopyranosides with purine and pyrimidine derivatives. While the reactions with purines and theobromine proceeded with complete regioselectivity, affording exclusively N9- or N1-linked 6′-isonucleosides, respectively, the use of pyrimidine nucleobases led to N1 and/or N3-glucopyranosid-6′-yl pyrimidines and/or to N1,N3/2-O,4-O-pyrimidine-linked pseudodisaccharides through bis-coupling, depending on the substitution pattern of the sugar precursor and on the nature of the nucleobase. From this series of compounds, four were shown to be effective and selective inhibitors of acetylcholinesterase with inhibition constants in the micromolar concentration range. A tri-O-acetylated N1-glucopyranosid-6′-yl theobromine and a benzylated N1,N3-bis-glucopyranosid-6-yl thymine were the most active molecules with K i values of 4 μM. A tri-O-benzylated glucopyranosid-6′-yl uracil displayed good and selective inhibition of butyrylcholinesterase (K i=8.4±1.0 μM), similar to that exhibited by the standard galantamine. Molecular docking simulations, performed with the two most effective acetylcholinesterase inhibitors, showed interactions with key amino acid residues located at the enzyme’s active site gorge, which explain the competitive component of their inhibitory activities.

Regulation of O-antigen biosynthesis in Yersinia enterocolitica.

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane (OM) of Gram-negative bacteria. The LPS molecule consists of three regions: (i) the lipid A, a polar glycolipid in which a disaccharide backbone is replaced with six or seven fatty acid; (ii) the core, an oligosaccharide often rich in negatively charged groups and, (iii) the O-antigen, a polysaccharide that protrudes into the surroundings. It is widely known that the lipid A part is responsible for most of the biological effects of LPS; however, there is increasing body of evidence showing that O-antigen modulates the biological activity of lipid A and also plays a critical role in the initial interaction of bacterium with the host. The biosynthesis of heteropolymeric O-antigens whose O-units are composed of different sugar residues starts in the cytoplasm by the activation of sugar 1-phosphates in a reaction with one of the nucleoside triphosphates, followed by different biosynthetic pathways to give rise to cytoplasmically located individual nucleoside diphosphate (NDP)-activated sugar precursors. The assembly of the O-units takes place on the cytoplasmic face of the inner membrane and involves the transfer of sugar residues from the NDP-sugar precursors onto a lipid carrier molecule, undecaprenylphosphate (Und-P), by specific glycosyltransferases. The completed O-units still assembled on the Und-P are translocated by the O-unit flippase, Wzx, across the inner membrane to the periplasmic face. There the O-units are polymerized by the O-antigen polymerase, Wzy, into an O-antigen that is ligated to the lipid Acore structure by the O-antigen ligase encoded by the waaL gene of the core

Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation

Selective exo-enzymatic labeling (or SEEL) uses recombinant glycosyltransferases and nucleotide-sugar analogues to allow efficient labeling of cell surface glycans. SEEL can circumvent many of the possible issues associated with metabolic labeling, including low incorporation of sugar precursors, and allows for sugars to be added selectively to different types of glycans by virtue of the inherent specificity of the glycosyltransferases. Here we compare the labeling of sialoglycoproteins in undifferentiated and differentiated human erythroleukemia cells (HEL) using SEEL using the sialyltransferases ST6Gal1 and ST3Gal1, which label N- and O-glycans, respectively. Our results show that the profile of glycoproteins detected varies between undifferentiated HEL cells and those differentiated to megakaryocytes, with a shift to more N-linked sialoglycoproteins in the differentiated cells. The efficiency of SEEL for both sialyltransferases in HEL cells was greatly increased with prior neuraminidase treatment highlighting the necessity for the presence of available acceptors with this labeling method. Following metabolic labeling or SEEL, tagged glycoproteins were enriched by immunoprecipitation and identified using mass spectrometry. The proteomic findings demonstrated that the detection of many glycoproteins is markedly improved by SEEL labeling, and that unique glycoproteins can be identified using either ST6Gal1 or ST3Gal1. Furthermore, this analysis enabled the identification of increased surface expression of several sialylated cell adhesion molecules, including the known megakaryocytic markers integrinβ3 and CD44, upon differentiation of HEL cells to adherent megakaryocytes.

Synthesis and in vitro antitumour activity of tiazofurin analogues with nitrogen functionalities at the C-2′ position

Synthesis of three tiazofurin (1) isosteres with nitrogen functionalities at the C-2′ position (N3, NH2 and NH3+Cl−) has been achieved, in multistep sequences, starting from monoacetone d-glucose. A number of potential bioisosteres of 1 bearing acylamido functions at the C-2′ position have also been synthesized from the same sugar precursor. In vitro cytotoxicities of target molecules against a number of human tumour cell lines were recorded and compared with those observed for lead molecule 1. Some of the synthesized compounds showed potent in vitro antitumour activity, such as 2′-azido derivative 2, which is the most potent of all molecules under evaluation (IC50 0.004 μM against MCF-7 cells). Flow cytometry data suggest that cytotoxic effects of these compounds in the culture of K562 cells might be mediated by apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these cells. Results of Western blot analysis indicate that the synthesized tiazofurin analogues induce apoptosis in a caspase-dependent way.

An improved model framework linking the extracellular environment to antibody glycosylation

Background Glycoproteins make up the bulk of biologically-derived medicines, and are taking up an ever increasing share of the prescription pharmaceuticals market. As opposed to small molecule drugs, glycoproteins are large complex molecules with heterogeneity arising from a multitude of glycan moieties. Glycans are complex post-translation modifications, which result from a number of enzymatic reactions in the ER and Golgi collectively known as glycosylation and play an important role in pharmacokinetics such as drug safety, efficacy and half-life. It is known that the availability of the nucleotide sugar donors (NSDs), which are the co-substrates to the enzymatic glycosylation reactions of the Golgi, can be affected by a number of process conditions such as culture mode, temperature, dissolved oxygen and nutrient availability, as well as the addition of precursor molecules to the culture medium [1]. Consequently, feeding strategies of nucleotide and nucleotide sugar precursors have been explored to exert control over the glycoform [2,3]. In this work, a mathematical model platform is presented to quantify the impact of nutrient availability and feeding strategies on the glycosylation process with the aim to enable the design of feeding strategies to optimise the product glycoform.

Engineering a branch of the UDP-precursor biosynthesis pathway enhances the production of capsular polysaccharide in Escherichia coli O5:K4:H4.

Escherichia coli K4 produces a capsule with a chemical structure that resembles chondroitin, a molecule with established chondro protective properties. The endogenous genes pgm and galU are involved in the biosynthesis of UDP-glucose which is a critical intermediate in carbohydrate metabolism and biochemical precursor of UDP-glucuronic acid. Together with UDP-N-acetylgalactosamine, UDP-glucuronic acid is used as sugar donor for capsule biosynthesis. The aim of the study was to evaluate how a change in the pathways leading to UDP-glucuronic acid biosynthesis affected capsular polysaccharide production. One additional copy of pgm and galU was introduced in E. coli K4 and in the previously described recombinant strain EcK4r3. A microbioreactor was used to analyse strain performance with parallel batch experiments, demonstrating increased polysaccharide concentrations and providing data that are comparable to those obtained in larger fermenters. Further experiments on a glutamine enriched medium showed an additional 45% increase of capsule production, maybe indicating the need to balance both branches leading to polymer biosynthesis in order to maximize yields. In the effort towards the establishment of a feasible bio-chondroitin production process this study provides information on how the availability of sugar precursors impacts polysaccharide biosynthesis in E. coli K4, a complex unexplored aspect of a multifaceted process.