Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network

Nature Biotechnology volume 18, pages 1283–1286 (2000)Cite this article

Abstract

Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pathway, and ultimately, increasing metabolic flux through the pathway of interest. By manipulating the GAL gene regulatory network of Saccharomyces cerevisiae, which is a tightly regulated system, we produced prototroph mutant strains, which increased the flux through the galactose utilization pathway by eliminating three known negative regulators of the GAL system: Gal6, Gal80, and Mig1. This led to a 41% increase in flux through the galactose utilization pathway compared with the wild-type strain. This is of significant interest within the field of biotechnology since galactose is present in many industrial media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The galactose utilization pathway.
Figure 2: Various control mechanisms of the GAL system in S. cerevisiae .
Figure 3: Correlation between the maximum specific ethanol production rate and the maximum specific galactose uptake rate for the wild-type strain (WT), SO3 (Δgal80 Δmig1), SO7 (pGAL4, 2μ), SO15 (Δgal6), SO16 (Δgal6 Δgal80 Δ mig1), and SO37 (Δgal6 Δgal80 Δmig1 & pGAL4, 2μ).

Similar content being viewed by others

References

  1. Bailey, J.E. Lessons from metabolic engineering for functional genomics and drug discovery . Nat. Biotechnol. 17, 616– 618 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Cameron, D.C. & Tong, I.T. Cellular and metabolic engineering: an overview. Appl. Biochem. Biotechnol. 38, 105–140 (1993).

    Article  CAS  PubMed  Google Scholar 

  3. Ostergaard, S., Olsson, L. & Nielsen, J. Metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 64, 34–50 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Schaaff, I., Heinisch, J. & Zimmermann, F.K. Overproduction of glycolytic enzymes in yeast. Yeast 5, 285–290 ( 1989).

    Article  CAS  PubMed  Google Scholar 

  5. Acerenza, L. & Kacser, H. Enzyme kinetics and metabolic control. Biochem. J. 269, 697– 707 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Niederberger, P., Prasad, R., Miozzari, G. & Kacser, H. A strategy for increasing an in vivo flux by genetic manipulations. Biochem. J. 287, 473–479 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Small, R. & Kacser, H. Responses of metabolic systems to large changes in enzyme activities and effectors. J. Biochem. 213, 613–624 ( 1993).

    CAS  Google Scholar 

  8. Johnston, M. & Carlson, M. In The molecular and cellular biology of the yeast Saccharomyces: Gene expression. (eds Jones, E.W., Pringel, J.R. & Broach, J.) 193– 281 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1992).

    Google Scholar 

  9. Zheng, W., Xu, H.E. & Johnston, S.A. The cysteine–peptidase bleomycin hydrolase is a member of the galactose regulon in yeast. J. Biol. Chem. 272, 30350–30355 (1997).

    Article  CAS  PubMed  Google Scholar 

  10. Wiselogel, A., Tyson, S. & Johnson, D. In Handbook on bioethanol: production and utilization. (ed. Wyman, C.E.) 105–118 (Taylor & Francis, Washington, DC; 1996).

    Google Scholar 

  11. Ostergaard, S., Roca, C., Rønnow, B., Nielsen, J. & Olsson, L. Physiological characterisation in aerobic batch cultivations of Saccharomyces cerevisiae strains harboring the MEL1 gene. Biotechnol. Bioeng. 68, 252–259 ( 2000).

    Article  CAS  PubMed  Google Scholar 

  12. Igarashi, M., Segawa, T., Nogi, Y., Suzuki, Y. & Fukasawa, T. Autogenous regulation of the Saccharomyces cerevisiae regulatory gene GAL80. Mol. Gen. Genet. 207, 273–279 (1987).

    Article  CAS  PubMed  Google Scholar 

  13. Adam, A.C., Rubio-Texeira, M. & Polaina, J. Induced expression of bacterial β-glucosidase activity in Saccharomyces. Yeast 11, 395– 406 (1995).

    Article  CAS  PubMed  Google Scholar 

  14. Martegani, E., Brambilla, L., Porro, D., Ranzi, B.M. & Alberghina, L. Alteration of cell population structure due to cell lysis in Saccharomyces cerevisiae cells overexpressing the GAL4 gene. Yeast 9, 575–582 (1993).

    Article  CAS  PubMed  Google Scholar 

  15. Gill, G. & Ptashne, M. Negative effect of the transcriptional activator GAL4. Nature 334, 721–724 (1988).

    Article  CAS  PubMed  Google Scholar 

  16. Güldener, U., Heck, S., Fiedler, T., Beinhauer, J. & Hegemann, J.H. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res. 24, 2519–2524 (1996).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Wach, A., Brachat, A., Pöhlmann, R. & Philippsen, P. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast. 10, 1793–1808 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. & Cullin, C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329–3330 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hoffman, C.S. & Winston, F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267– 272 (1987).

    Article  CAS  PubMed  Google Scholar 

  20. Sambrook, J., Fritsch, E.F. & Maniatis, T.M. Molecular cloning: a laboratory manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989 ).

    Google Scholar 

  21. Johnston, M. & Dover, J. Mutational analysis of the gal4-encoded transcriptional regulatory protein of Saccharomyces cerevisiae . Genetics 102, 63– 74 (1988).

    Google Scholar 

  22. Gietz, D., St. Jean, A., Woods, R.A. & Schiestl, R.H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425 ( 1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rose, M.D., Winston, F. & Hieter, P. Methods in yeast genetics: a laboratory course manual . (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1990).

    Google Scholar 

  24. Leuther, K.K. & Johnston, S.A. Nondissociation of GAL4 and GAL80 in vivo after galactose induction. Science 256, 1333–1335 (1992).

    Article  CAS  PubMed  Google Scholar 

  25. Wu, Y., Reece, R.J. & Ptashne, M. Quantitation of putative activator–target affinities predicts transcriptional activating potentials. EMBO J. 15, 3951–3963 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Suzuki-Fujimoto, T. et al. Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p. Mol. Cell. Biol. 16, 2504–2508 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Yano, K. & Fukasawa, T. Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 94, 1721–1726 ( 1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Keleher, C.A., Redd, M.J., Schultz, J., Carlson, M. & Johnson, A.D. Ssn6-Tup1 is a general repressor of transcription in yeast. Cell 68, 709–719 (1992).

    Article  CAS  PubMed  Google Scholar 

  29. Nehlin, J.O., Carlberg, M. & Ronne, H. Control of yeast GAL genes by MIG1 repressor: a transcriptional cascade in the glucose response. EMBO J. 10, 3373–3377 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Treitel, M.A. & Carlson, M. Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein. Proc. Natl. Acad. Sci. USA 92, 3132–3136 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The research on galactose metabolism and glucose repression at the Center for Process Biotechnology has been financially supported by the Danish Programme for Food Technology II (project 2409) as well as by the European Commission Framework IV “Cell Factory” (contract BIO-CT95-0107). Peter Kötter, Goethe Universität Frankfurt, is acknowledged for providing the Δmig1Δgal80 mutant strains (SO3 and SO4).

Author information

Authors and Affiliations

  1. Department of Biotechnology, Center for Process Biotechnology, Building 223

    Simon Ostergaard, Lisbeth Olsson & Jens Nielsen

  2. Technical University of Denmark, Lyngby, DK-2800, Denmark

    Simon Ostergaard, Lisbeth Olsson & Jens Nielsen

  3. Department of Genetics, Washington University School of Medicine, St. Louis, 63110, MO

    Mark Johnston

Authors
  1. Simon Ostergaard
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lisbeth Olsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mark Johnston
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jens Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jens Nielsen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ostergaard, S., Olsson, L., Johnston, M. et al. Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network. Nat Biotechnol 18, 1283–1286 (2000). https://doi.org/10.1038/82400

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/82400

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing