Large-scale production of oligosaccharides using engineered bacteria

Endo, Tetsuo; Koizumi, Satoshi

doi:10.1016/s0959-440x(00)00127-5

Cited by 75 publications

(31 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…[8,18] A b1,3-Gal transferase was thus the only additional enzyme that was necessary for the production of the carbohydrate moiety of GM1 (Figure 1). Therefore, we subcloned the C. jejuni cgtB gene for b1,3-Gal transferase in the pACT3cgtA plasmid to yield the plasmid pACT3cgtAB, in which the two genes cgtA and cgtB were both under the control of the strong tac promoter, with cgtB inserted downstream of cgtA.…”

Section: Production Of IImentioning

confidence: 99%

“…[8] Sialyllactose (NeuAca-3Galb-4Glc), the carbohydrate moiety of GM3, and the common trisaccharide core of all gangliosides have been produced in high yield either through bacterial coupling [9] or by living bacteria. [10] In the latter process, sialyllactose was directly produced by growing cells of metabolically engineered Escher-ichia coli strains that overexpress the Neisseria meningitidis genes for a-2,3-sialyltransferase and CMP-NeuAc synthase (CMP cytidine monophosphate).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2003

View full text Add to dashboard Cite

Two metabolically engineered Escherichia coli strains have been constructed to produce the carbohydrate moieties of gangliosides GM2 (GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc; Gal = galactose, Glc = glucose, Ac = acetyl) and GM1 (Galbeta-3GalNAcbeta-4(NeuAcalpha-3)Galbeta-4Glc. The GM2 oligosaccharide-producing strain TA02 was devoid of both beta-galactosidase and sialic acid aldolase activities and overexpressed the genes for CMP-NeuAc synthase (CMP = cytidine monophosphate), alpha-2,3-sialyltransferase, UDP-GlcNAc (UDP = uridine diphosphate) C4 epimerase, and beta-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 beta-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.

show abstract

Section: Production Of IImentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2003

View full text Add to dashboard Cite

show abstract

“…However, alternative mechanisms are possible and a crystal structure is ultimately needed to reveal the structural basis for enzyme activity and specificity. A better understanding of substrate specificity of H. pylori FucTs may enable us to engineer new enzymes with improvedspecificityforeitherTypeIorTypeIIsubstrates.Suchenzymescould be used to synthesize biologically important carbohydrates that contain Lewis structures and might be potential pharmaceuticals in the prevention of bacterial or viral infection, in the neutralization of toxins, and in immunotherapy for cancer (50)(51)(52)(53)(54).…”

Section: Discussionmentioning

confidence: 99%

A Single Aromatic Amino Acid at the Carboxyl Terminus of Helicobacter pylori α1,3/4 Fucosyltransferase Determines Substrate Specificity

Lau

Palcic

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

Fucosyltransferases (FucT) from differentHelicobacter pylori strains display distinct Type I (Gal␤1,3GlcNAc) or Type II (Gal␤1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both ␣1,3 and ␣1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive ␣1,3 activity. Our domain swapping study ( Helicobacter pylori is associated with gastritis and peptic ulcer formation and is a risk factor for the development of gastric cancer and mucosa-associated lymphoid tissue lymphoma. One of the virulence factors of H. pylori is the lipopolysaccharide, which contains lipid A, core oligosaccharide and O-antigens. The O-antigens of H. pylori lipopolysaccharide contain fucosylated oligosaccharides, predominantly the Type II blood group antigens, Lewis X (Gal␤1,4(Fuc␣1,3)GlcNAc) and Lewis Y (Fuc␣1,2Gal␤1,4(Fuc␣1,3)GlcNAc) (1), but a small number of H. pylori strains also express the Type I blood group antigens, Lewis A (Gal␤1,3(Fuc␣1,4)GlcNAc), and Lewis B (Fuc␣1,2Gal␤1,3(Fuc␣1,4)-GlcNAc) (2).The role of Lewis antigens in H. pylori pathogenesis is still ambiguous. It has been suggested that Lewis antigens play a role in H. pylori adhesion to (3, 4) or internalization by (5) the gastric epithelial cells. Nevertheless, conflicting evidence argues that Lewis X and Lewis Y are not required for colonization of human gastric epithelium (6) or mouse stomach (7,8). Lewis antigens may also play an important role in the persistence of H. pylori infection by molecular mimicry, helping the bacteria to evade the host immune response (2, 9, 10). Environmental changes such as pH influence the expression of H. pylori O-antigens, particularly Lewis X and Lewis Y. This may aid in adaptation of the bacterium to its niche in the stomach (10).Fucosyltransferases (FucTs) 3 are enzymes responsible for the last steps in the synthesis of Lewis antigens in H. pylori (11,12). ␣1,2 and ␣1,3 or ␣1,3/4 FucTs have been identified and characterized in H. pylori (13-18). These FucTs catalyze the transfer of the L-fucose moiety from guanosine diphosphate ␤-L-fucose (GDP-Fuc) to the OH-2 of the galactose moiety and the OH-3 or the OH-3 and the OH-4 positions of the GlcNAc moiety in glycoconjugate acceptors, respectively. The H. pylori genome contains two homologous ␣1,3 or ␣1,3/4 FucT genes, futA and futB (19,20), but they do not always encode functional proteins. For instance, only the futA gene encodes an active FucT in H. pylori strains NCTC11639 and UA948 (13,17). Bacterial ␣1,3/4 FucTs are functionally equivalent to the mammalian ␣1,3/4 FucTs, which have been well characterized. Mammalian FucTs are Type II membrane proteins with a short N-terminal cytoplasmic tail, transmembrane domain, stem region, and C-terminal catalytic domain. H. pylori FucTs share weak homology with their mammalian counterparts in two small segments within the catalytic domain, called ␣1,3 FucT motifs (14, 21). H. pylori ␣1,3/4 FucTs lack the N...

show abstract

“…This technology was developed by Kyowa Hakko Kogyo Co. Ltd. in Japan [31][32][33][34][35]. The key in Kyowa Hakko's technology for the large-scale production of oligosaccharides is a C. ammoniagenes bacterial strain engineered to efficiently convert inexpensive orotic acid to UTP (Scheme 3).…”

Section: Scheme 2 Biosynthesis Of Oligosaccharide By Glycosyltransfementioning

confidence: 99%

Approaches to the Enzymatic Carbohydrate Synthesis

Kowal

Shao

Mei

et al. 2004

ACS Symposium Series

View full text Add to dashboard Cite

Polysaccharides are the least appreciated class of macromolecules though they constitute the largest part of the planet's bio-mass. This large bulk has perpetuated a compelling reason for their neglect: carbohydrates were thought to be biochemically uninteresting serving as structural and energy storage molecules. The second more insidious and perhaps contradictory reason for the relative slowness in carbohydrate research is that biochemically important carbohydrates are difficult to study. They seem haphazardly put together, without a template, creating enormous complexity that, at first, did not suggest specific functions. The past three decades have seen enormous strides in the understanding of the importance of carbohydrates feeding the quickly expanding and vibrant field of enzymatic carbohydrate synthesis. The clear goal of the research in this field is to supplant Nature as the supreme oligosaccharide maker to generate desired saccharides, be it natural or unnatural, in high yields, large quantities and at low cost.

show abstract

Large-scale production of oligosaccharides using engineered bacteria

Cited by 75 publications

References 49 publications

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

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

A Single Aromatic Amino Acid at the Carboxyl Terminus of Helicobacter pylori α1,3/4 Fucosyltransferase Determines Substrate Specificity

Approaches to the Enzymatic Carbohydrate Synthesis

Contact Info

Product

Resources

About