RESUMEN
We designed metabolically engineered non-pathogenic strains of Escherichia coli to produce unsulfated chondroitin with and without chondroitin lyase to produce the chondroitin polymer or its related oligosaccharides. Chondroitin was synthesized using chondroitin synthase KfoC and chondroitin was degraded using Pl35, a chondroitin lyase from Pedobacter heparinus. Pl35 behaved as a true endo-enzyme generating a large panel of oligosaccharides ranging from trimers to 18-mers instead of the di- and tetramers obtained with most chondroitin lyases. Two series of oligosaccharides were characterized, sharing an unsaturated uronic acid (4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid, â³UA) residue at their non-reducing end. The major "even-numbered" series was characterized by a terminal reducing N-acetylgalactosaminyl residue. The minor "odd-numbered" series oligosaccharides carried a terminal reducing glucuronic acid residue instead. Cultures were conducted in fed-batch conditions, and led to the production of up to 10 g L-1 chondroitin or chondroitin oligosaccharides. All products were purified and fully characterized using NMR and mass spectrometry analyses. This is the first report of the microbial production of large chondro-oligosaccharides.
RESUMEN
Herein, we describe the efficient gram-scale synthesis of α2,3- and α2,6-sialyllactose oligosaccharides as well as mimetics from N-acyl mannosamines and lactose in metabolically engineered bacterial cells grown at high cell density. We designed new Escherichia coli strains co-expressing sialic acid synthase and N-acylneuraminate cytidylyltransferase from Campylobacter jejuni together with the α2,3-sialyltransferase from Neisseria meningitidis or the α2,6-sialyltransferase from Photobacterium sp. JT-ISH-224. Using their mannose transporter, these new strains actively internalized N-acetylmannosamine (ManNAc) and its N-propanoyl (N-Prop), N-butanoyl (N-But) and N-phenylacetyl (N-PhAc) analogs and converted them into the corresponding sialylated oligosaccharides, with overall yields between 10 % and 39 % (200-700â mg.L-1 of culture). The three α2,6-sialyllactose analogs showed similar binding affinity for Sambucus nigra SNA-I lectin as for the natural oligosaccharide. They also proved to be stable competitive inhibitors of Vibrio cholerae neuraminidase. These N-acyl sialosides therefore hold promise for the development of anti-adhesion therapy against influenza viral infections.
Asunto(s)
Lactosa , Neuraminidasa , Neuraminidasa/metabolismo , Escherichia coli/metabolismo , Sialiltransferasas/metabolismo , Oligosacáridos/químicaRESUMEN
Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is, therefore, desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Herein, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N-acetyl-ß-D-glucosaminyl-(1â4)-1,6-anhydro-N-acetyl-ß-D-muramic acid (GlcNAc-anhMurNAc), a common precursor of Gram-negative and Gram-positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram-positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the inâ vivo production of Gram-negative muropeptides containing meso-diaminopimelic acid, a rarely available amino acid.
Asunto(s)
Escherichia coli , Peptidoglicano , Escherichia coli/metabolismo , Peptidoglicano/metabolismo , Bacterias/metabolismo , Pared Celular/metabolismo , HexosaminidasasRESUMEN
Influenza viruses bind to their target through a multivalent interaction of their hemagglutinins (HAs) with sialosides at the host cell surface. To fight the virus, one therapeutic approach consists in developing sialylated multivalent structures that can saturate the virus HAs and prevent the binding to host cells. We describe herein the biotechnological production of sialylated solid lipid microparticles (SSLMs) in 3 steps: (i) a microbiological step leading to the large-scale production of sialylated maltodextrins by metabolic engineering of an Escherichia coli strain, (ii) a new in vitro glycosylation process using the amylomaltase MalQ, based on the transglycosylation of the terminal sialoside ligand of the sialylated maltodextrin onto a long-chain alkyl glucoside, and (iii) the formulation of the final SSLMs presenting a multivalent sialic acid. We also describe the morphology and structure of the SSLMs and demonstrate their very promising properties as influenza virus inhibitors using hemagglutination inhibition and microneutralization assays on the human A/H1N1 pdm09 virus.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Virus de la Influenza A/metabolismo , Gripe Humana/tratamiento farmacológico , Hemaglutininas Virales , Lípidos , Glicoproteínas Hemaglutininas del Virus de la InfluenzaRESUMEN
Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.
Asunto(s)
Carbohidrato Epimerasas/química , Ácido Glucurónico/química , Heparina/química , Oligosacáridos/química , Sitios de Unión , Carbohidrato Epimerasas/genética , Catálisis , Cristalografía por Rayos X , Células HEK293 , Humanos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
A conjugatable form of the tumour-associated carbohydrate antigen sialyl-Tn (Neu5Ac-α-2,6-GalNAc) was efficiently produced in Escherichia coli. Metabolically engineered E.â coli strains overexpressing the 6-sialyltransferase gene of Photobacterium sp. and CMP-Neu5Ac synthetase genes of Neisseria meningitidis were cultivated at high density in the presence of GalNAc-α-propargyl as the exogenous acceptor. The target disaccharides, which were produced on the scale of several hundreds of milligrams, were then conjugated by using copper(I)-catalysed azide-alkyne cycloaddition click chemistry to a fully synthetic and immunogenic scaffold with the aim to create a candidate anticancer vaccine. Four sialyl-Tn epitopes were introduced on the upper face of an azido-functionalised multivalent cyclopeptide scaffold, the lower face of which was previously modified by an immunogenic polypeptide, PADRE. The ability of the resulting glycoconjugate to interact with oncofoetal sialyl-Tn monoclonal antibodies was confirmed in ELISA assays.
Asunto(s)
Antígenos de Carbohidratos Asociados a Tumores/metabolismo , Escherichia coli/metabolismo , Vacunas Sintéticas/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos de Carbohidratos Asociados a Tumores/química , Antígenos de Carbohidratos Asociados a Tumores/genética , Antígenos de Carbohidratos Asociados a Tumores/inmunología , Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/metabolismo , Cromatografía en Capa Delgada , Química Clic , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Ingeniería Metabólica , Neisseria/enzimología , Péptidos Cíclicos/genética , Péptidos Cíclicos/inmunología , Péptidos Cíclicos/metabolismo , Photobacterium/enzimología , Sialiltransferasas/genética , Sialiltransferasas/metabolismo , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Bacterial polysialyltransferases (PSTs) are processive enzymes involved in the synthesis of polysialic capsular polysaccharides. They can also synthesize polysialic acid in vitro from disialylated and trisialylated lactoside acceptors, which are the carbohydrate moieties of GD3 and GT3 gangliosides, respectively. Here, we engineered a non-pathogenic Escherichia coli strain that overexpresses recombinant sialyltransferases and sialic acid synthesis genes and can convert an exogenous lactoside into polysialyl lactosides. Several PSTs were assayed for their ability to synthesize polysialyl lactosides in the recombinant strains. Fed-batch cultures produced α-2,8 polysialic acid or alternate α-2,8-2,9 polysialic acid in quantities reaching several grams per liter. Bacterial culture in the presence of propargyl-ß-lactoside as the exogenous acceptor led to the production of conjugatable polysaccharides by means of copper-assisted click chemistry.
Asunto(s)
Glicósidos/biosíntesis , Ácidos Siálicos/biosíntesis , Sialiltransferasas/genética , Escherichia coli K12/genética , Gangliósidos , Regulación Enzimológica de la Expresión Génica/genética , Glicósidos/genética , Glicosilación , Lactosilceramidos , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/genética , Proteínas Recombinantes/genética , Ácidos Siálicos/genéticaRESUMEN
The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding ß-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2-10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.