Engineering alcohol tolerance in yeast

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Science  03 Oct 2014:
Vol. 346, Issue 6205, pp. 71-75
DOI: 10.1126/science.1257859

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  1. Fig. 1 Elevated extracellular potassium and pH enhance ethanol tolerance and production under high-glucose and high–cell-density conditions.

    (A) Ethanol titers (squares) and per-cell rates of production (triangles) from fermentations in unmodified YSC medium (dashed lines) or YSC supplemented with 40 mM KCl and 10 mM KOH (solid lines). Specific productivities are calculated from the mean viable population [thick lines from (B)] during each 24-hour period. DCW, dry cell weight. (B) Cell densities (DCW; thin squares) and the underlying viable populations (thick triangles) from the fermentations in (A). Data are mean ± SD from three biological replicates. (C) Net fermentation viability, expressed as time integrals of the viable cell population, as a function of potassium added to YSC in the form of KCl (pH 3.6), or 5 mM KOH + KCl (pH 5.8). (D) Time integral values from (C) regressed against their final ethanol titers.

  2. Fig. 2 Elevated potassium and pH are sufficient to enhance tolerance independently of strain genetics, sugar substrate, and alcohol species.

    (A) Ethanol titers from glucose fermentation (top) of one laboratory (S288C) and three industrial (PE-2, Ethanol Red, and Kyokai 7) yeast strains, or from xylose fermentation (bottom) of an engineered xylose strain, in unmodified YSC or YSC supplemented with 40 mM KCl and 10 mM KOH. (B) Titers from S288C cultured in 20% YP medium or medium supplemented with potassium, at pH 6 and 3.7. (C) Population fractions of S288C after transfer from overnight growth in unmodified YSC (dashed lines) or medium supplemented with 48 mM KCl and 2 mM KOH (solid lines) into media containing the indicated concentrations of ethanol. (D and E) Same as (C), but with step increases of isopropanol or isobutanol, respectively. All data are mean ± SD from three biological replicates.

  3. Fig. 3 Genetic augmentation of the plasma membrane potassium (TRK1) and proton (PMA1) pumps increases ethanol production to levels exceeding those of industrial strains.

    Shown are ethanol titers from a wild-type laboratory strain (S288C) transformed with empty overexpression plasmid, S288C transformed with a plasmid overexpressing PMA1, S288C containing hyperactivated TRK1 (via deletions of PPZ1 and PPZ2) and transformed with empty overexpression plasmid, the TRK1 hyperactivated strain transformed with a plasmid overexpressing PMA1, and bioethanol production strains from Brazil (PE-2) and the United States (Ethanol Red), all cultured in unmodified YSC lacking uracil. Data are mean ± SD from three biological replicates.

  4. Fig. 4 Potential biophysical mechanism depicting how elevated potassium and pH counteract rising alcohol toxicity.

    In the absence of stress (top row), the opposing K+ and H+ pumps maximally maintain steep gradients of K+ and H+ across the plasma membrane. Rising alcohol levels perturb these gradients by permeabilizing the membrane and increasing ion leakage (middle row, left). Elevated potassium and pH, however, bolster the gradients by slowing rates of ion leakage (due to reduced concentration differences) and allowing transporters to pump against a less-precipitous differential (middle row, right). Therefore, the threshold alcohol concentration that collapses these gradients is raised, allowing cells to maintain viability at higher toxicity levels (bottom row). Red corresponds to K+ and blue to H+, the size of the triangles corresponds to concentration gradient steepness, and the thickness of the arrows corresponds to the magnitude of ion flux.

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