Advertisement

Screening Mutant Libraries in Saccharomyces cerevisiae

  • Thomas Bulter
  • Volker Sieber
  • Miguel Alcalde
Part of the Methods in Molecular Biology™ book series (MIMB, volume 230)

Abstract

Functional gene expression is a prerequisite for directed evolution with Escherichia coli (E. coli), the preferred host organism. However, bacterial expression of eukaryotic genes can be impossible, or produce proteins with substantially altered properties, because of differences between bacterial and native expression systems (1). Different codon usage, missing chaperones, and posttranslational modifications like disulfide bridges or glycosylation can all cause low expression levels, misfolding, and inclusion bodies (2). Some of these problems can be avoided if these genes are expressed in a eukaryotic host whose expression machinery is more similar to the native one. Considering transformation efficiency, stability of plasmid DNA, and growth rate, Saccharomyces cerevisiae (S. cerevisiae) (3,4) is best suited for directed evolution (5, 6, 7, 8), but the potential of this host has not been widely appreciated. Growing and manipulating S. cerevisiae is regarded as time consuming and more complicated than working with E. coli. The advantages of this host for directed evolution, such as homologous recombination that facilitates library construction (see  Chapter 3 in companion volume, “Directed Evolution Library Creation”) or secretion of mutant proteins, are frequently overlooked.

Keywords

Minimal Medium Mutant Library Laccase Gene Stability Plate Yeast Plasmid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Romanos, M. A., Scorer, C. A., and Clare, J. J. (1992) Foreign gene expression in yeast—a review. Yeast 8, 423–488.PubMedCrossRefGoogle Scholar
  2. 2.
    Georgiou, G. (1996) in Expression of proteins in bacteria. (eds. Cleland, J. L. and Craik, C. S.) Wiley-Liss, New York, NY.Google Scholar
  3. 3.
    Broach, J. R., Jones, E. W., and Pringle, J. R. (eds.) (1991) The Molecular Biology of the yeast Saccharomyces, Vol. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).Google Scholar
  4. 4.
    Sherman, F. (1991) Getting started with yeast. Meth. Enzymol. 194, 3–21.PubMedCrossRefGoogle Scholar
  5. 5.
    Cherry, J. R., Lamsa, M. H., Schneider, P., et al. (1999) Directed evolution of a fungal peroxidase. Nat. Biotechnol. 17, 379–384.PubMedCrossRefGoogle Scholar
  6. 6.
    Morawski, B., Lin, Z., Cirino, P., Joo, H., Bandara, G., and Arnold, F. H. (2000) Functional expression of horseradish peroxidase in Saccharomyces cerevisiae and Pichia pastoris. Protein Eng. 13, 377–384.PubMedCrossRefGoogle Scholar
  7. 7.
    Morawski, B., Quan, S., and Arnold, F. H. (2001) Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae. Biotechnol. Bioeng. 76, 99–107.PubMedCrossRefGoogle Scholar
  8. 8.
    Abecassis, V., Pompon, D., and Truan, G. (2000) High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2. Nucl. Acids Res. 28, E88.PubMedCrossRefGoogle Scholar
  9. 9.
    Schuster, J. R. (1991) Gene expression in yeast: protein secretion. Curr. Opin. Biotechnol. 2, 685–690.CrossRefGoogle Scholar
  10. 10.
    Otterbein, L., Record, E., Longhi, S., Asther, M., and Moukha, S. (2000) Molecular cloning of the cDNA encoding laccase from Pycnoporus cinnabarinus I-937 and expression in Pichia pastoris. Eur. J. Biochem. 267, 1619–1625.PubMedCrossRefGoogle Scholar
  11. 11.
    Gianfreda, L., Xu, F., and Bollag, J.-M. (1999) Laccases: a useful group of oxidoreductive enzymes. Bioremediation J. 3, 1–25.CrossRefGoogle Scholar
  12. 12.
    Kojima, Y., Tsukuda, Y., Kawai, Y., et al. (1990) Cloning, sequence analysis, and expression of ligninolytic phenoloxidase genes of the white-rot basidiomycete Coriolus hirsutus. J. Biol. Chem. 265, 15,224–15,230.PubMedGoogle Scholar
  13. 13.
    Cassland, P. and Jonsson, L. J. (1999) Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature. Appl. Microbiol. Biotechnol. 52, 393–400.PubMedCrossRefGoogle Scholar
  14. 14.
    Larsson, S., Cassland, P., and Jonsson, L. J. (2001) Development of a Saccharomyces cerevisiae strain with enhanced resistance to phenolic fermentation inhibitors in lignocellulose hydrolysates by heterologous expression of laccase. Appl. Environ. Microbiol. 67, 1163–1170.PubMedCrossRefGoogle Scholar
  15. 15.
    Yasuchi, K., Yukio, K., and Yukiko, T. (1990) DNA for expression and secretion. European patent application EP 0 388 166.Google Scholar
  16. 16.
    Jonsson, L. J., Saloheimo, M., and Penttila, M. (1997) Laccase from the white-rot fungus Trametes versicolor: cDNA cloning of Icc1 and expression in Pichia pastoris. Curr. Genet. 32, 425–430.Google Scholar
  17. 17.
    Majcherczyk, A., Johannes, C., and Huttermann, A. (1998) Oxidation of polycyclic aromatic hydrocarbons (PAH) by laccase of Trametes versicolor. Enzyme Microb. Technol. 22, 335–341.CrossRefGoogle Scholar
  18. 18.
    Bajpai, P. (1999) Application of enzymes in the pulp and paper industry. Biotechnol. Prog. 15, 147–157.PubMedCrossRefGoogle Scholar
  19. 19.
    Bourbonnais, R., Paice, M. G., Freiermuth, B., Bodie, E., and Borneman, S. (1997) Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl. Env. Microbiol. 63, 4627–4632.Google Scholar
  20. 20.
    Chen, T., Barton, S. C., Binyamin, G., et al. (2001) A miniature biofuel cell. J. Am. Chem. Soc. 123, 8630–8631.PubMedCrossRefGoogle Scholar
  21. 21.
    Jones, E. W. (1991) Tackling the protease problem in Saccharomyces cerevisiae. Meth. Enzymol. 194, 428–453.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2003

Authors and Affiliations

  • Thomas Bulter
    • 1
  • Volker Sieber
    • 2
  • Miguel Alcalde
    • 3
  1. 1.Department of Chemical EngineeringUniversity of California at Los AngelesLos Angeles
  2. 2.Degussa AGProjecthouse BiotechnologyFreisingGermany
  3. 3.Division of Chemistry and Chemical EngineeringCalifornia Institute of TechnologyPasadena

Personalised recommendations