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An engineered eukaryotic protein glycosylation pathway in Escherichia coli

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Abstract

We performed bottom-up engineering of a synthetic pathway in Escherichia coli for the production of eukaryotic trimannosyl chitobiose glycans and the transfer of these glycans to specific asparagine residues in target proteins. The glycan biosynthesis was enabled by four eukaryotic glycosyltransferases, including the yeast uridine diphosphate-N-acetylglucosamine transferases Alg13 and Alg14 and the mannosyltransferases Alg1 and Alg2. By including the bacterial oligosaccharyltransferase PglB from Campylobacter jejuni, we successfully transferred glycans to eukaryotic proteins.

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Figure 1: Engineering eukaryotic glycan biosynthesis in E. coli.
Figure 2: Characterization of LLOs produced by glycoengineered E. coli.
Figure 3: Transfer of eukaryotic glycans to target proteins in E. coli.

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Acknowledgements

We thank B. Imperiali (Massachusetts Institute of Technology) for plasmid pBAD(ALG2)-DEST49, G. O'Toole (Dartmouth College) for plasmid pMQ70, T. Mansell for helpful discussions regarding RNaseA glycosylation, C. Hong for helpful discussions regarding glycan synthesis and individuals from the Functional Genomic Center Zürich for input and instrument support. This work was supported by the National Science Foundation Career Award CBET-0449080 (to M.P.D.), the New York State Office of Science, Technology and Academic Research Distinguished Faculty Award (to M.P.D.), the National Institutes of Health Small Business Innovation Research grants R43 GM087766 and R43 GM086965 (to A.C.F.), the National Institutes of Health National Center for Research Resources grant 1 P41 RR018502-01 (to the Complex Carbohydrate Research Center) and a graduate fellowship from LASPAU and the Universidad Antonio Nariño (to J.D.V.-R.).

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Author notes

  1. Juan D Valderrama-Rincon and Adam C Fisher: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA

    Juan D Valderrama-Rincon & Matthew P DeLisa

  2. Glycobia, Inc., Ithaca, New York, USA

    Adam C Fisher, Judith H Merritt & Craig A Reading

  3. Department of Biology, Institute of Microbiology, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland

    Yao-Yun Fan & Markus Aebi

  4. Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA

    Krishan Chhiba

  5. Complex Carbohydrate Research Center, University of Georgia, Athens, Gerogia, USA

    Christian Heiss & Parastoo Azadi

Authors
  1. Juan D Valderrama-Rincon
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  2. Adam C Fisher
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  3. Judith H Merritt
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  4. Yao-Yun Fan
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  5. Craig A Reading
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  6. Krishan Chhiba
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  7. Christian Heiss
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  8. Parastoo Azadi
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  9. Markus Aebi
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  10. Matthew P DeLisa
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Contributions

J.D.V.-R. and A.C.F. designed research, performed research, analyzed data and wrote the paper. J.H.M. designed research and performed research. Y.-Y.F. performed MS analysis and analyzed data. C.A.R. and K.C. performed research. C.H. and P.A. performed NMR analysis and analyzed data. M.A. designed research and analyzed data. M.P.D. designed research, analyzed data and wrote the paper.

Corresponding author

Correspondence to Matthew P DeLisa.

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Competing interests

A.C.F., J.H.M. and C.A.R. are employees of Glycobia, Inc.

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Valderrama-Rincon, J., Fisher, A., Merritt, J. et al. An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat Chem Biol 8, 434–436 (2012). https://doi.org/10.1038/nchembio.921

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  • DOI: https://doi.org/10.1038/nchembio.921

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