Eric Samain

GRAM-SCALE SYNTHESIS OF RECOMBINANT CHITOOLIGOSACCHARIDES IN ESCHERICHIA COLI

Cultivation of Escherichia coli harbouring heterologous genes of oligosaccharide synthesis is presented as a new method for preparing large quantities of high-value oligosaccharides. To test the feasibility of this method, we successfully produced in high yield (up to 2.5 g/L) penta-N-acetyl-chitopentaose (1) and its deacetylated derivative tetra-N-acetyl-chitopentaose (2) by cultivating at high density cells of E. coli expressing nodC or nodBC genes (nodC and nodB encode for chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively). These two products were easily purified by charcoal adsorption and ion-exchange chromatography. One important application of compound 2 could be its utilisation as a precursor for the preparation of synthetic nodulation factors by chemical acylation.

Transcriptional Responses toward Diffusible Signals from Symbiotic Microbes Reveal MtNFP- and MtDMI3-Dependent Reprogramming of Host Gene Expression by Arbuscular Mycorrhizal Fungal Lipochitooligosaccharides

The formation of root nodules and arbuscular mycorrhizal (AM) roots is controlled by a common signaling pathway including the calcium/calmodulin-dependent kinase Doesn’t Make Infection3 (DMI3). While nodule initiation by lipochitooligosaccharide (LCO) Nod factors is well characterized, diffusible AM fungal signals were only recently identified as sulfated and nonsulfated LCOs. Irrespective of different outcomes, the perception of symbiotic LCOs in Medicago truncatula is mediated by the LysM receptor kinase M. truncatula Nod factor perception (MtNFP). To shed light on transcriptional responses toward symbiotic LCOs and their dependence on MtNFP and Ca2+ signaling, we performed genome-wide expression studies of wild-type, Nod-factor-perception mutant1, and dmi3 mutant roots challenged with Myc- and Nod-LCOs. We show that Myc-LCOs lead to transient, quick responses in the wild type, whereas Nod-LCOs require prolonged incubation for maximal expression activation. While Nod-LCOs are most efficient for an induction of persistent transcriptional changes, sulfated Myc-LCOs are less active, and nonsulfated Myc-LCOs display the lowest capacity to activate and sustain expression. Although all symbiotic LCOs up-regulated a common set of genes, discrete subsets were induced by individual LCOs, suggesting common and specific functions for these in presymbiotic signaling. Surprisingly, even sulfated fungal Myc-LCOs and Sinorhizobium meliloti Nod-LCOs, having very similar structures, each elicited discrete subsets of genes, while a mixture of both Myc-LCOs activated responses deviating from those induced by single treatments. Focusing on the precontact phase, we identified signaling-related and transcription factor genes specifically up-regulated by Myc-LCOs. Comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNFP and largely requires MtDMI3.

Large-Scale In Vivo Synthesis of the Carbohydrate Moieties of Gangliosides GM1 and GM2 by Metabolically Engineered Escherichia coli

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcβ‐4(NeuAcα‐3)Galβ‐4Glc; Gal=galactose, Glc=glucose, Ac=acetyl) and GM1 (Galβ‐3GalNAcβ‐4(NeuAcα‐3)Galβ‐4Glc. The GM2 oligosaccharide‐producing strain TA02 was devoid of both β‐galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP‐NeuAc synthase (CMP=cytidine monophosphate), α‐2,3‐sialyltransferase, UDP‐GlcNAc (UDP=uridine diphosphate) C4 epimerase, and β‐1,4‐GalNAc transferase. When this strain was cultivated on glycerol, exogenously added lactose and sialic acid were shown to be actively internalized into the cytoplasm and converted into GM2 oligosaccharide. The in vivo synthesis of GM1 oligosaccharide was achieved by taking a similar approach but using strain TA05, which additionally overexpressed the gene for β‐1,3‐galactosyltransferase. In high‐cell‐density cultures, the production yields for the GM2 and GM1 oligosaccharides were 1.25 g L−1 and 0.89 g L−1, respectively.

In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori α-1,3 fucosyltransferase in engineered Escherichia coli

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc, lacto-N-neodifucohexaose Galβ1-4(Fucα1-3)Glc-NAcβ1-3Galβ1-4(Fucα1-3)Glc, and lacto-N-neodifucooctaose Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an α-1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon.To prevent fucosylation at the glucosyl residue, lactulose Galβ1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.

Production of O-acetylated and sulfated chitooligosaccharides by recombinant Escherichia coli strains harboring different combinations of nod genes

High cell density cultivation of recombinant Escherichia coli strains harboring the nodBC genes (encoding chitooligosaccharide synthase and chitooligosaccharide N-deacetylase, respectively) from Azorhizobium caulinodans has been previously described as a practical method for the preparation of gram-scale quantities of penta-N-acetyl-chitopentaose and tetra-N-acetylchitopentaose (Samain, E., Drouillard, S., Heyraud, A., Driguez, H., Geremia, R.A., 1997. Carbohydr. Res. 30, 235-242). We have now extended this method to the production of sulfated and O-acetylated derivatives of these two compounds by coexpressing nodC or nodBC with nodH and/or nodL that encode chitooligosaccharide sulfotransferase and chitooligosaccharide O-acetyltransferase, respectively. In addition, these substituted chitooligosaccharides were also obtained as tetramers by using nodC from Rhizobium meliloti instead of nodC from A. caulinodans. These compounds should be useful precursors for the preparation of Nod factor analogues by chemical modification.

Production of lewis x tetrasaccharides by metabolically engineered Escherichia coli

Two tetrasaccharides carrying the trisaccharidic Lewis x motif on a GlcNAc or a Gal residue were produced on the gram‐scale by high‐cell‐density cultures of metabolically engineered Escherichia coli strains that overexpressed the Helicobacter pylori futA gene for α‐3 fucosyltransferase and the Neisseria meningitidis lgtB gene for β‐4 galactosyltransferase. The first compound Galβ‐4(Fucα‐3)GlcNAcβ‐4GlcNAc was produced by glycosylation of chitinbiose, which was endogenously generated in the bacterial cytoplasm by the successive action of the rhizobial chitin‐synthase NodC and the Bacillus circulans chitinase A1, whose genes were additionally expressed in the E. coli strain. The second compound, Galβ‐4(Fucα‐3)GlcNAcβ‐3Gal, was produced from exogenously added Gal by a strain that was deficient in galactokinase activity and overexpressed the additional N. meningitidis lgtA gene for β‐3 N‐acetylglucosaminyltransferase.

Lipo-chitooligosaccharidic Symbiotic Signals Are Recognized by LysM Receptor-Like Kinase LYR3 in the Legume Medicago truncatula

While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.

Assessment of the Two Helicobacter pylori α‐1,3‐Fucosyltransferase Ortholog Genes for the Large‐Scale Synthesis of LewisX Human Milk Oligosaccharides by Metabolically Engineered Escherichia coli

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto‐ N‐neotetraose Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)Glc (LNnT). The major product obtained was lacto‐ N‐neofucopentaose‐V Gal(β1–4)GlcNAc(β1–3)Gal(β1–4)[Fuc(α1–3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N‐acetylglucosaminyl residues and thus carrying the LewisX group (LeX) was also produced. We report here a fermentation process for the large‐scale production of LeX oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better LeX activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli ( E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of LeX carrying oligosaccharides. Lacto‐ N‐neodifucohexaose‐II Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)[Fuc(α1–3)]Glc represented 70% of the total oligosaccharide amount of futA‐on‐driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futBled to equal amounts of both lacto‐ N‐neofucopentaose‐V and lacto‐ N‐neofucopentaose‐II Gal(β1–4)[Fuc(α1–3)]GlcNAc(β1–3)Gal(β1–4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3‐fucosyllactose Gal(β1–4)[Fuc(α1–3)]Glc was produced in futA‐on‐driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.

Differential recognition and hydrolysis of host carbohydrate-antigens by Streptococcus pneumoniae family 98 glycoside hydrolases

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-β-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-β-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.