Synthesis of Neuraminidase-Resistant Sialyllactose Mimetics from N-Acyl Mannosamines using Metabolically Engineered Escherichia coli.

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 % (200-700 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.

Biotechnological production of sialylated solid lipid microparticles as inhibitors of influenza A virus infection.

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.

Production of perdeuterated fucose from glyco-engineered bacteria.

l-Fucose and l-fucose-containing polysaccharides, glycoproteins or glycolipids play an important role in a variety of biological processes. l-Fucose-containing glycoconjugates have been implicated in many diseases including cancer and rheumatoid arthritis. Interest in fucose and its derivatives is growing in cancer research, glyco-immunology, and the study of host-pathogen interactions. l-Fucose can be extracted from bacterial and algal polysaccharides or produced (bio)synthetically. While deuterated glucose and galactose are available, and are of high interest for metabolic studies and biophysical studies, deuterated fucose is not easily available. Here, we describe the production of perdeuterated l-fucose, using glyco-engineered Escherichia coli in a bioreactor with the use of a deuterium oxide-based growth medium and a deuterated carbon source. The final yield was 0.2 g L-1 of deuterated sugar, which was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We anticipate that the perdeuterated fucose produced in this way will have numerous applications in structural biology where techniques such as NMR, solution neutron scattering and neutron crystallography are widely used. In the case of neutron macromolecular crystallography, the availability of perdeuterated fucose can be exploited in identifying the details of its interaction with protein receptors and notably the hydrogen bonding network around the carbohydrate binding site.

Determining the Site of Action of Strigolactones during Nodulation.

Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa.

Activation of symbiosis signaling by arbuscular mycorrhizal fungi in legumes and rice.

Establishment of arbuscular mycorrhizal interactions involves plant recognition of diffusible signals from the fungus, including lipochitooligosaccharides (LCOs) and chitooligosaccharides (COs). Nitrogen-fixing rhizobial bacteria that associate with leguminous plants also signal to their hosts via LCOs, the so-called Nod factors. Here, we have assessed the induction of symbiotic signaling by the arbuscular mycorrhizal (Myc) fungal-produced LCOs and COs in legumes and rice (Oryza sativa). We show that Myc-LCOs and tetra-acetyl chitotetraose (CO4) activate the common symbiosis signaling pathway, with resultant calcium oscillations in root epidermal cells of Medicago truncatula and Lotus japonicus. The nature of the calcium oscillations is similar for LCOs produced by rhizobial bacteria and by mycorrhizal fungi; however, Myc-LCOs activate distinct gene expression. Calcium oscillations were activated in rice atrichoblasts by CO4, but not the Myc-LCOs, whereas a mix of CO4 and Myc-LCOs activated calcium oscillations in rice trichoblasts. In contrast, stimulation of lateral root emergence occurred following treatment with Myc-LCOs, but not CO4, in M. truncatula, whereas both Myc-LCOs and CO4 were active in rice. Our work indicates that legumes and non-legumes differ in their perception of Myc-LCO and CO signals, suggesting that different plant species respond to different components in the mix of signals produced by arbuscular mycorrhizal fungi.

Efficient Conjugation of Oligosaccharides to Polymer Particles through Furan/Maleimide Diels-Alder Reaction: Application to the Capture of Carbohydrate-Binding Proteins.

Glycan-protein interactions play a crucial role in physiological and pathological events. Hence, improving the isolation of carbohydrate-binding proteins (i.e., lectins and anti-glycan antibodies) from complex media might not only lead to a better understanding of their function, but also provide solutions for public health issues, such as water contamination or the need for universal blood plasma. Here we report a rapid and efficient method for producing carbohydrate-based affinity adsorbents combining enzymatic synthesis and metal-free click chemistry. Both simple and complex glycans (maltose, blood group antigens A, B, and H) were readily modified by the addition of a furyl group at the reducing end without the need for protecting groups and were then efficiently conjugated to maleimide-activated Sepharose particles through Diels-Alder cycloaddition. These neoglycoconjugates showed high efficiency for the purification of lectins (concanavalin A and Ulex europaeus agglutinin), as well as for the capture of anti-A and anti-B blood group antibodies, opening new prospects for glycoproteomics and for the development of universal blood plasma.

Chemobacterial Synthesis of a Sialyl-Tn Cyclopeptide Vaccine Candidate.

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. 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.

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.

Chemoenzymatic Syntheses of Sialylated Oligosaccharides Containing C5-Modified Neuraminic Acids for Dual Inhibition of Hemagglutinins and Neuraminidases.

A fast chemoenzymatic synthesis of sialylated oligosaccharides containing C5-modified neuraminic acids is reported. Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of NeuNAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria. GM3 analogues were either obtained by chemoselective modification of biosynthetic N-acetyl-sialyllactoside (GM3 NAc) or by direct bacterial synthesis using C5-modified neuraminic acid precursors. The latter strategy proved to be very versatile as it led to an efficient synthesis of GM2 analogues. These glycomimetics were assessed against hemagglutinins and sialidases. In particular, the GM3 NPhAc displayed a binding affinity for Maackia amurensis agglutinin (MAA) similar to that of GM3 NAc, while being resistant to hydrolysis by Vibrio cholerae (VC) neuraminidase. A preliminary study with influenza viruses also confirmed a selective inhibition of N1 neuraminidase by GM3 NPhAc, suggesting potential developments for the detection of flu viruses and for fighting them.

Aniline-catalyzed reductive amination as a powerful method for the preparation of reducing end-"clickable" chitooligosaccharides.

Functionalized oligosaccharides are useful intermediates to prepare products for biological research or for the development of advanced functional materials. Here, we report the unprecedented use of aniline as an efficient organocatalyst reaction with "clickable" (azide or alkyne) amine for the transimination-mediated reductive amination of a chitooligosaccharide. Moreover, we demonstrate that alkyne-bearing aniline constitutes an excellent tool for the easy derivatization of chitosan oligosaccharides. Evidence for such improvement has been illustrated by the straightforward design of a FRET substrate to probe chitinase activity and of amphiphilic polycaprolactone-grafted-chitosan. This efficient methodology paves the way to the preparation of novel chitosan oligosaccharide-based advanced materials.

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 Ca(2+) 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.

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-beta-galactosidase activity on the LewisY antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-beta-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.

Glucuronylation in Escherichia coli for the bacterial synthesis of the carbohydrate moiety of nonsulfated HNK-1.

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.

Genetic engineering of Escherichia coli for the economical production of sialylated oligosaccharides.

We have previously described a microbiological process for the conversion of lactose into 3'sialyllactose and other ganglioside sugars by living Escherichia coli cells expressing the appropriate recombinant glycosyltransferase genes. In this system the activated sialic acid donor (CMP-Neu5Ac) was generated from exogenous sialic acid, which was transported into the cells by the permease NanT. Since sialic acid is an expensive compound, a more economical process has now been developed by genetically engineering E. coli K12 to be capable of generating CMP-Neu5Ac using its own internal metabolism. Mutant strains devoid of Neu5Ac aldolase and of ManNAc kinase were shown to efficiently produce 3'sialyllactose by coexpressing the alpha-2,3-sialyltransferase gene from Neisseria meningitidis with the neuC, neuB and neuACampylobacter jejuni genes encoding N-acetylglucosamine-6-phosphate-epimerase, sialic acid synthase and CMP-Neu5Ac synthetase, respectively. A sialyllactose concentration of 25 g l(-1) was obtained in long-term high cell density culture with a continuous lactose feed. This high concentration and low cost of fermentation medium should make possible to use sialylated oligosaccharides in new fields such as the food industry.

Synthesis of globopentaose using a novel beta1,3-galactosyltransferase activity of the Haemophilus influenzae beta1,3-N-acetylgalactosaminyltransferase LgtD.

We have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding beta1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87, 197-203]. Here, we found that LgtD has an additional beta1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galbeta-3GalNAcbeta-3Galalpha-4Galbeta-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both beta1,3-GalT and beta1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.

Unexpected regioselectivity of Humicola insolens Cel7B glycosynthase mutants.

Four Humicola insolens Cel7B glycoside hydrolase mutants have been evaluated for the coupling of lactosyl fluoride on O-allyl N(I)-acetyl-2(II)-azido-beta-chitobioside. Double mutants Cel7B E197A H209A and Cel7B E197A H209G preferentially catalyze the formation of a beta-(1-->4) linkage between the two disaccharides, while single mutant Cel7B E197A and triple mutant Cel7B E197A H209A A211T produce predominantly the beta-(1-->3)-linked tetrasaccharide. This result constitutes the first report of the modulation of the regioselectivity through site-directed mutagenesis for an endoglycosynthase.

Nod Factor Effects on Root Hair-Specific Transcriptome of Medicago truncatula: Focus on Plasma Membrane Transport Systems and Reactive Oxygen Species Networks.

Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli.

Large amounts of globotriose (Galalpha-4Galbeta-4Glc) are shown to be produced by the high cell density culture of an Escherichia coli strain over-expressing the Neisseria meningitidis lgtC gene for alpha-1,4-Gal transferase. The strain which was devoid of both alpha and beta galactosidase activity was fed with glycerol as the energy and carbon source and with lactose as precursor for globotriose synthesis. After complete exhaustion of lactose, globotriose could serve as an alternative acceptor for LgtC and the formation of a series of polygalactosylated compounds was observed. The system was extended to the synthesis of globotetraose (GalNAcbeta-3Galalpha-4Galbeta-4Glc) by overexpressing two additional genes: lgtD from Haemophilus influenzae Rd which encodes a beta-1,3-GalNAc transferase and wbpP from Pseudomonas aeruginosa which encodes a UDP-GalNAc C4 epimerase. Globotetraose could also be produced from exogenous globotriose which was shown to be actively taken up by the cells.

Biosynthesis of conjugatable saccharidic moieties of GM2 and GM3 gangliosides by engineered E. coli.

Oligosaccharidic moieties of GM(2) and GM(3) gangliosides bearing an allyl or a propargyl aglycon, are efficiently biosynthesized on the gram scale by growing metabolically engineered Escherichia coli cells in the presence of the corresponding lactoside acceptors and sialic acid.

Assessment of the two Helicobacter pylori alpha-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(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]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 (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) 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 Le(X) 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 Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]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 futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)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(beta1-4)[Fuc(alpha1-3)]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.