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

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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.

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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).

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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).

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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

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  1. Simon Ostergaard
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  2. Lisbeth Olsson
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  4. Jens Nielsen
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Correspondence to Jens Nielsen.

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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

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