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Ability of Saccharomyces cerevisiae MPY3 cells to flocculate during fermentation in wort was found to be triggered after growth limitation by oxygen shortage and to coincide with a sharp increase in cell surface hydrophobicity of the cells. Presence of oxygen in the pitching wort influenced final cell number, flocculence of the cells and cell surface hydrophobicity. Flocculation ability of cells grown in air-depleted pitching wort was hampered, concomitant with a decrease in final cell number and in final cell surface hydrophobicity. Addition of ergosterol and Tween 80 to air-depleted wort restored normal growth of the cells as well as flocculation ability and the increase in cell surface hydrophobicity. The same parameters increased in value after addition of ergosterol and Tween 80 to a fermentation with air-saturated pitching wort. Hydrophobicity of a non-flocculent mutant of S. cerevisiae strain MPY3, fermenting in air-saturated pitching wort, did not increase at cell division arrest. These results support the hypothesis that cell surface hydrophobicity is a major determinant for yeast cells to become flocculent, and suggest that shortage of sterols and unsaturated fatty acids precedes flocculence under brewing conditions.

In this report the effects of phosphoglycerate kinase (PGK) overproduction on the physiology and plasmid stability in baker's yeast Saccharomyces cerevisiae containing the PGK1 gene on an episomal plasmid are described. This examination reveals that there is a preferred intracellular level for this enzyme, amounting to 10-15% of the total soluble protein. Strains containing the plasmid and the host strain were grown in non-selective batch cultures and continuous culture, under different growth conditions. Plasmid-containing yeast strains stabilize the copy number of the episomal plasmid at a level at which the PGK concentration is about 12%. This stabilization is due to an equilibrium between normal plasmid loss and selective pressure because of advantages resulting from the increased amount of PGK under glucose-limited conditions. During respiro-fermentative growth, PGK-overproducing cells showed an increased respiration rate and decreased fermentative activity, compared to the host strain. The PGK1 gene can be applied as a direct positive selection marker to obtain a high episomal plasmid stability during growth on glucose. The results are consistent with previously reported data on the physiology and gene stability of PGK-overproducing yeast cells that contain multiple copies of the PGK1 gene integrated into the genome.

The physiological consequences of overproduction of the homologous glycolytic enzyme 3-phosphoglycerate kinase (PGK), integrated in 80 PGK1 gene copies in the genome of Saccharomyces cerevisiae are described. This multiple integration and the strong PGK overproduction (maximum 47% of the total soluble cell protein) do not affect the maximal specific growth rate, but cause 40% reduction of the molar growth yield, compared with that of the wild-type host. The extra energy that is needed for protein overproduction is mainly provided by extra fermentation (respirofermentative growth), but respiration is also elevated compared with the reference strains. The increase in the specific oxygen uptake rate indicates that the respiratory capacity of the yeasts is higher than that in the wild-type host, in which the limited capacity of respiration is generally supposed to be at its maximal level at the critical dilution rate, and is thus responsible for the switch to respirofermentative growth. In a medium PGK1 gene copy integrant (about 25 copies), overproduction of 10%-12% PGK has a stimulating effect on the growth yield and energy efficiency. In these cells the growth benefits of overproduction of the glycolytic enzyme are higher than the disadvantages of extra protein synthesis. The overproduction of PGK has also consequences for the glucose affinity of the yeasts: In the more overproducing strain the Ks is increased, compared to its reference strains. Elimination of strong overproducing cells from a glucose-limited chemostat culture is caused by two factors: (a) the excision of the PGK genes from the genome, which is of minor importance for wash-out, but the induction process for this overall decline of overproduction, and (b) the physiolOffprint requests to: P. C. van der Aar ogical selection process for less overproducing cells, caused by differences in affinity for glucose, most obvious at # ~, l/2#max. However in batch culture and in a chemostat at low specific growth rates, all the overproducing strains show high genetic stability and constantly provide high PGK quantities.

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