Remodeling bacterial polysaccharides by metabolic pathway engineering

Yi, Wen; Liu, Xianwei; Li, Yanhong; Li, Jianjun; Xia, Chengfeng; Zhou, Guangyan; Zhang, Wenpeng; Zhao, Wei; Chen, Xi; Wang, Peng George

doi:10.1073/pnas.0812432106

Cited by 108 publications

(115 citation statements)

References 19 publications

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“…Recently, Wang et al showed that the His 6 -tagged recombinant FKP from B. fragilis has relaxed specificity toward fucose analogs bearing unnatural substituents at the C-5 position (19). To quantitatively evaluate the specific activity of the recombinant FKP toward C-5 substituted fucose analogs, we determined the fucokinase activity of the recombinant FKP toward a panel of unnatural substrates, using a simple coupled enzymatic assay with a spectophotometric readout.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Chemoenzymatic synthesis of GDP- l -fucose and the Lewis X glycan derivatives

Wang¹,

Hu²,

Frantom³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

122

100

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Lewis X (Le x )-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Le x and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5-diphosphate-␤-L-fucose (GDPfucose), the universal fucosyl donor, the Le x trisaccharide, and their C-5 substituted derivatives can be synthesized on preparative scales, using a chemoenzymatic approach. This method exploits L-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme isolated from Bacteroides fragilis 9343, which converts L-fucose into GDP-fucose via a fucose-1-phosphate (Fuc-1-P) intermediate. Combining the activities of FKP and a Helicobacter pylori ␣1,3 fucosyltransferase, we prepared a library of Le x trisaccharide glycans bearing a wide variety of functional groups at the fucose C-5 position. These neoglycoconjugates will be invaluable tools for studying Le x -mediated biological processes.glycobiology ͉ catalysis ͉ enzyme

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Section: Resultsmentioning

confidence: 99%

“…Connecting these two domains is a 150-aa linker, whose function is currently unknown. Wang and co-workers demonstrated recently that a His 6 -tagged recombinant FKP, expressed in Escherichia coli, is a promiscuous enzyme with relaxed specificity toward fucose analogs bearing unnatural substituents at the C-5 position (19).…”

mentioning

confidence: 99%

Chemoenzymatic synthesis of GDP- l -fucose and the Lewis X glycan derivatives

Wang¹,

Hu²,

Frantom³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

122

100

View full text Add to dashboard Cite

show abstract

“…Furthermore, this result may also demonstrate the promiscuity of the glycosyltransferases which are responsible for the synthesis of the oligosaccharides that are eventually displayed on the cell surface. In conjunction with the incorporation of modified fucose derivatives into the LPS of E. coli O86:B7, we further demonstrate proof of principle that other unique monosaccharides can be engineered into the cell surface oligosaccharides in bacteria [27]. Unlike our previous work where we could control the exact location of the modification to the LPS, because of the possible pathways that 2-ketoGlc may take, exact identification of which residue is being modified in the core oligosaccharides is challenging.…”

Section: Resultsmentioning

confidence: 94%

“…Previous work by our lab has used E. coli O86 as a model system for the pin point modification of the fucosylated LPS by using metabolic pathway engineering, and in this work we sought to expand our ability to modify this model strain with 2-ketoGlc [27]. Structurally 2-ketoGlc is very similar to GlcNAc, with the exception of the 2 amide, and thus may be incorporated as GlcNAc in the LPS.…”

Section: Labeling Lpsmentioning

confidence: 99%

C2-Carbon Isostere of N-acetylglucosamine as Substrate for Bacterial Polysaccharide Remodeling

Pettit¹,

Cai²,

Han³

et al. 2011

Am. J. Biomed. Sci.

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Introduction of unique structural modifications into a living cell presents opportunities to investigate and manipulate specific biological pathways and functions. Cell surface oligosaccharides represent one area where structural modifications can easily be made which allow for the investigation and manipulation of such processes. Bacterial cell surface oligosaccharides, while both structurally diverse and biological relevant, have not been extensively probed as a source for introducing chemically modified structures. In this study, we demonstrate the incorporation of an unnatural ketone containing monosaccharide, which heterogeneously is incorporated into the cell surface oligosaccharides of E. coli. Through bioorthogonal chemistry we selectively label the cell surface oligosaccharides containing the ketone modified monosaccharide with various probes, which are used to image and identify the cell surface oligosaccharides. In summary, this study presents a facile and effective method for the in vivo incorporation of a ketone containing monosaccharide which can be exploited for studying the bacterium.

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“…Significantly, exogenous salvage pathways can serve as convenient routes to introduce nonnatural sugar analogs into glycans. For example, an artificial fucose salvage pathway was recently exploited to install fucose analogs into cell-surface polysaccharides of E. coli in which de novo biosynthesis of the donor nucleotide-sugar GDPfucose had been disrupted (31). We conducted several experiments to determine if the GlcNAc salvage pathway in gna1Δ yeast could assimilate unnatural GlcNAc analogs into secreted glycoproteins.…”

Section: Resultsmentioning

confidence: 99%

Targeted metabolic labeling of yeast N-glycans with unnatural sugars

Breidenbach¹,

Gallagher

King

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridinediphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Δ strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.click chemistry | GlcNAc | metabolic engineering | N-glycosylation | GNA1

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Remodeling bacterial polysaccharides by metabolic pathway engineering

Cited by 108 publications

References 19 publications

Chemoenzymatic synthesis of GDP- l -fucose and the Lewis X glycan derivatives

Chemoenzymatic synthesis of GDP- l -fucose and the Lewis X glycan derivatives

C2-Carbon Isostere of N-acetylglucosamine as Substrate for Bacterial Polysaccharide Remodeling

Targeted metabolic labeling of yeast N-glycans with unnatural sugars

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