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High-throughput screening methodology for the directed evolution of glycosyltransferases

Nature Methods volume 3, pages 609–614 (2006)Cite this article

Abstract

Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of new oligosaccharides holds great potential for the development of the field of glycobiology. However, engineering of GTs by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of a new fluorescence-based high-throughput screening (HTS) methodology for the directed evolution of sialyltransferases (STs). Using this methodology, we detected the formation of sialosides in intact Escherichia coli cells by selectively trapping the fluorescently labeled transfer products in the cell and analyzing and sorting the resulting cell population using a fluorescence-activated cell sorter (FACS). We screened a library of >106 ST mutants using this methodology and found a variant with up to 400-fold higher catalytic efficiency for transfer to a variety of fluorescently labeled acceptor sugars, including a thiosugar, yielding a metabolically stable product.

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Figure 1: Cell-based assay for ST activity.
Figure 2: Donor sugars and fluorescent acceptor sugars used in this study.
Figure 3: Cell-based ST assay for cells expressing wild-type CstII (WT) and containing the pUC18 plasmid (control).
Figure 4: Library selection through three iterative rounds of sorting by FACS.
Figure 5: Structure of the CstII F91Y mutant.

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References

  1. Crocker, P.R. & Feizi, T. Carbohydrate recognition systems: functional triads in cell-cell interactions. Curr. Opin. Struct. Biol. 6, 679–691 (1996).

    Article  CAS  Google Scholar 

  2. Rudd, P.M., Elliott, T., Cresswell, P., Wilson, I.A. & Dwek, R.A. Glycosylation and the immune system. Science 291, 2370–2376 (2001).

    Article  CAS  Google Scholar 

  3. Lowe, J.B. Glycan-dependent leukocyte adhesion and recruitment in inflammation. Curr. Opin. Cell Biol. 15, 531–538 (2003).

    Article  CAS  Google Scholar 

  4. Sacks, D. & Kamhawi, S. Molecular aspects of parasite-vector and vector-host interactions in leishmaniasis. Annu. Rev. Microbiol. 55, 453–483 (2001).

    Article  CAS  Google Scholar 

  5. Qasba, P.K., Ramakrishnan, B. & Boeggeman, E. Substrate-induced conformational changes in glycosyltransferases. Trends Biochem. Sci. 30, 53–62 (2005).

    Article  CAS  Google Scholar 

  6. Arnold, F.H., Wintrode, P.L., Miyazaki, K. & Gershenson, A. How enzymes adapt: lessons from directed evolution. Trends Biochem. Sci. 26, 100–106 (2001).

    Article  CAS  Google Scholar 

  7. Tao, H. & Cornish, V.W. Milestones in directed enzyme evolution. Curr. Opin. Chem. Biol. 6, 858–864 (2002).

    Article  CAS  Google Scholar 

  8. Dalby, P.A. Optimising enzyme function by directed evolution. Curr. Opin. Struct. Biol. 13, 500–505 (2003).

    Article  CAS  Google Scholar 

  9. Goddard, J.P. & Reymond, J.L. Enzyme assays for high-throughput screening. Curr. Opin. Biotechnol. 15, 314–322 (2004).

    Article  CAS  Google Scholar 

  10. Aharoni, A., Griffiths, A.D. & Tawfik, D.S. High-throughput screens and selections of enzyme-encoding genes. Curr. Opin. Chem. Biol. 9, 210–216 (2005).

    Article  CAS  Google Scholar 

  11. Becker, S., Schmoldt, H.U., Adams, T.M., Wilhelm, S. & Kolmar, H. Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts. Curr. Opin. Biotechnol. 15, 323–329 (2004).

    Article  CAS  Google Scholar 

  12. Griffiths, A.D. & Tawfik, D.S. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J. 22, 24–35 (2003).

    Article  CAS  Google Scholar 

  13. Harduin-Lepers, A. et al. The human sialyltransferase family. Biochimie 83, 727–737 (2001).

    Article  CAS  Google Scholar 

  14. Chiu, C.P. et al. Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. Nat. Struct. Mol. Biol. 11, 163–170 (2004).

    Article  CAS  Google Scholar 

  15. Antoine, T., Heyraud, A., Bosso, C. & Samain, E. Highly efficient biosynthesis of the oligosaccharide moiety of the GD3 ganglioside by using metabolically engineered Escherichia coli. Angew. Chem. Int. Edn. Engl. 44, 1350–1352 (2005).

    Article  CAS  Google Scholar 

  16. Aharoni, A., Amitai, G., Bernath, K., Magdassi, S. & Tawfik, D.S. High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. Chem. Biol. 12, 1281–1289 (2005).

    Article  CAS  Google Scholar 

  17. Gosselin, S., Alhussaini, M., Streiff, M.B., Takabayashi, K. & Palcic, M.M. A continuous spectrophotometric assay for glycosyltransferases. Anal. Biochem. 220, 92–97 (1994).

    Article  CAS  Google Scholar 

  18. Fernandez-Gacio, A., Uguen, M. & Fastrez, J. Phage display as a tool for the directed evolution of enzymes. Trends Biotechnol. 21, 408–414 (2003).

    Article  CAS  Google Scholar 

  19. Varadarajan, N., Gam, J., Olsen, M.J., Georgiou, G. & Iverson, B.L. Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proc. Natl. Acad. Sci. USA 102, 6855–6860 (2005).

    Article  CAS  Google Scholar 

  20. Mastrobattista, E. et al. Discovering novel evolutionary pathways to β-galactosidases using in vitro compartmentalization and fluorescence activated sorting of double emulsions. Chem. Biol. 12, 1291–1300 (2005).

    Article  CAS  Google Scholar 

  21. Kim, Y.W., Lee, S.S., Warren, R.A. & Withers, S.G. Directed evolution of a glycosynthase from Agrobacterium sp. increases its catalytic activity dramatically and expands its substrate repertoire. J. Biol. Chem. 279, 42787–42793 (2004).

    Article  CAS  Google Scholar 

  22. Lin, H., Tao, H. & Cornish, V.W. Directed evolution of a glycosynthase via chemical complementation. J. Am. Chem. Soc. 126, 15051–15059 (2004).

    Article  CAS  Google Scholar 

  23. Witczak, Z.J. & Culhane, J.M. Thiosugars: new perspectives regarding availability and potential biochemical and medicinal applications. Appl. Microbiol. Biotechnol. 69, 237–244 (2005).

    Article  CAS  Google Scholar 

  24. Rich, J.R., Szpacenko, A., Palcic, M.M. & Bundle, D.R. Glycosyltransferase-catalyzed synthesis of thiooligosaccharides. Angew. Chem. Int. Edn. Engl. 43, 613–615 (2004).

    Article  CAS  Google Scholar 

  25. Karwaski, M.F., Wakarchuk, W.W. & Gilbert, M. High-level expression of recombinant Neisseria CMP-sialic acid synthetase in Escherichia coli. Protein Expr. Purif. 25, 237–240 (2002).

    Article  CAS  Google Scholar 

  26. Vartanian, J.P., Henry, M. & Wain-Hobson, S. Simulating pseudogene evolution in vitro: determining the true number of mutations in a lineage. Proc. Natl. Acad. Sci. USA 98, 13172–13176 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to A. Johnson for his devoted assistance with the FACS. We thank E. Samain for his provision of JM107 Nan A− cells. A.A. is supported by a long term fellowship from the Human Frontiers Science Program (HFSP), K.T. by a Deutscher Akademischer Austausch Dienst (DAAD) fellowship, S.B. by a Swiss National Science Foundation Fellowship, C.P.C.C. by a Canadian Institutes for Health Research (CIHR) and Michael Smith Foundation for Health Research (MSFHR) fellowships, and L.L.L. by Natural Sciences and Engineering Research Council (NSERC) and MSFHR fellowships. We thank the Canadian Institutes for Health Research, the Howard Hughes Medical Institute (to N.C.J.S.) and the Natural Sciences and Engineering Research Council of Canada for financial support.

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Authors and Affiliations

  1. Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, V6T 1Z1, British Columbia, Canada

    Amir Aharoni, Karena Thieme, Sabrina Buchini, Luke L Lairson, Hongming Chen & Stephen G Withers

  2. Department of Biochemistry, Life Sciences Center, 2350 Health Sciences Mall, Vancouver, V6T 1Z3, British Columbia, Canada

    Cecilia P C Chiu & Natalie C J Strynadka

  3. Institute of Biological Sciences, National Research Council, Room 3157, 100 Sussex Drive, Ottawa, K1A OR6, Ontario, Canada

    Warren W Wakarchuk

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  1. Amir Aharoni
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Contributions

A.A. conceived the strategy and carried out the majority of the work. K.T., S.B., L.L.L. and H.C. carried out the synthesis. C.P.C.C. and N.C.J.S. carried out the crystallography. W.W.W. and S.G.W. helped in development of the strategy. A.A. and S.G.W. wrote the manuscript.

Corresponding author

Correspondence to Stephen G Withers.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Reaction scheme for 〈-2,3 sialyltransferase Cst-II. (PDF 74 kb)

Supplementary Fig. 2

FACS density plots. (PDF 426 kb)

Supplementary Fig. 3

Activity analysis by TLC of the best four clones isolated following three rounds of FACS enrichment of the Cst-II library relative to the wt Cst-II. The (PDF 256 kb)

Supplementary Fig. 4

Transfer activity analysis of CMP-Neu5Ac (1 mM) or CMP-KDN (5 mM) to bodipy-3SH-lactose or bodipy-lactose (0.5 mM) for different samples analysed by TLC. (PDF 145 kb)

Supplementary Table 1

Catalytic efficiency (kcat/KM) of wt Cst-II and F91Y mutant for the transfer of CMPNeu5Ac to different acceptors. (PDF 75 kb)

Supplementary Table 2

Data collection and refinement statistics. (PDF 92 kb)

Supplementary Methods (PDF 124 kb)

Supplementary Note (PDF 72 kb)

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Aharoni, A., Thieme, K., Chiu, C. et al. High-throughput screening methodology for the directed evolution of glycosyltransferases. Nat Methods 3, 609–614 (2006). https://doi.org/10.1038/nmeth899

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