Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae

Aar, P.C. van der; Lopes, T. S.; Klootwijk, J.; Ph, Groeneveld; Verseveld, Henk W. van; Stouthamer, A. H.

doi:10.1007/bf00173731

Cited by 11 publications

(8 citation statements)

References 45 publications

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“…There are two possible explanations for this question. First, previous reports have indicated that an increase in the glycolytic flux can lead to an increase in ethanol production very similar to what we have observed here (Van der Aar et al, 1990). However, it is unlikely that the action of VHb is to directly influence the enzymatic activities of the glycolytic pathway since we have shown that is it linked to respiration.…”

Section: Discussionsupporting

confidence: 89%

Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae

Chen

Hughes²,

Bailey

1994

Biotechnology Progress

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Vitreoscilla hemoglobin (VHb) has been expressed in Saccharomyces cerevisiae, and its influence on yeast aerobic metabolism has been investigated. New expression vectors were constructed to express VHb constitutively under the control of the ADH-1 promoter. The presence of VHb was shown by Western blot analysis. VHb has been shown to localize predominantly in the cytoplasm. Batch fermentation results indicated that the wild-type strain expressing VHb exhibited a shift in the carbon flux toward ethanol production, with no significant alteration in the specific growth rate. This effect was not observed if cells were grown under respiration inhibition, indicating that the metabolic effect of VHb is likely linked to respiration. Expression of VHb in the adh degrees strain MC65-2A, which produces ethanol only via a respiration-coupled pathway, revealed that ethanol production was decreased and cells reached a higher final cell density in a culture of the VHb-expressing strain. Growth enhancement due to expression of VHb was observed only during the final stage of culture growth when the acetaldehyde produced during the first growth phase was used as a substrate. This metabolic effect of intracellular VHb was seen more clearly in an acetaldehyde fed-batch fermentation in which VHb-expressing cells grew to at least 3-fold higher final cell density. These results suggest that the action of VHb is likely linked to electron transfer.

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Section: Discussionsupporting

confidence: 89%

Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae

Chen

Hughes²,

Bailey

1994

Biotechnology Progress

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“…In batch culture, the PGK1 promoter is maximally induced by the presence of glucose (Kingsman et al, 1990;Shuster, 1989;Romanos et al, 1992), whereas the ADH2 promoter is repressed during growth on glucose and derepressed during the transition to growth on ethanol (Price et al, 1990;Noronha et al, 1998;Shuster, 1989). As the produced xylanase had no known catalytic function in yeast metabolism, and was secreted efficiently by the recombinant strains, the quantified metabolic burden was strictly related to the presence and activity of the recombinant expression system (Van der Aar et al, 1990aAar et al, , 1990bVan Hoek et al, 1998). Stable maintenance of the recombinant plasmids in nonselective cultivation media has been ensured through the inclusion of the fur1 ura3 autoselective system (Loison et al, 1986) in the recombinant strains (La Grange et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium

Görgens

Zyl

Knoetze

et al. 2001

Biotech & Bioengineering

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Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.

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“…In other studies the workers associated nitrogen deficiency with a high sugar transporter turnover rate, which resulted in a loss of sugar uptake capacity in the cells (5,32). The cellular membrane is the primary region for controlling sugar uptake and the subsequent ethanol production (34,39).…”

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confidence: 99%

Biomass Content Governs Fermentation Rate in Nitrogen-Deficient Wine Musts

Varela

Pizarro

Agosín

2004

Appl Environ Microbiol

197

179

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Problematic fermentations are common in the wine industry. Assimilable nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce fermentation rates significantly. A lack of nitrogen diminishes a yeast's metabolic activity, as well as the biomass yield, although it has not been clear which of these two interdependent factors is more significant in sluggish fermentations. Under winemaking conditions with different initial nitrogen concentrations, metabolic flux analysis was used to isolate the effects. We quantified yeast physiology and identified key metabolic fluxes. We also performed cell concentration experiments to establish how biomass yield affects the fermentation rate. Intracellular analysis showed that trehalose accumulation, which is highly correlated with ethanol production, could be responsible for sustaining cell viability in nitrogen-poor musts independent of the initial assimilable nitrogen content. Other than the higher initial maintenance costs in sluggish fermentations, the main difference between normal and sluggish fermentations was that the metabolic flux distributions in nitrogen-deficient cultures revealed that the specific sugar uptake rate was substantially lower. The results of cell concentration experiments, however, showed that in spite of lower sugar uptake, adding biomass from sluggish cultures not only reduced the time to finish a problematic fermentation but also was less likely to affect the quality of the resulting wine as it did not alter the chemistry of the must.Sluggish and stuck fermentations are common in the wine industry. Factors that affect the yeast growth rate and that lead to problematic fermentations include limited nutrient contents, ethanol toxicity, fatty acid toxicity, temperature extremes (5), the ratio of nitrogen sources to carbon sources in the medium (21), and the initial quantity and quality of the amino acids (29). Sluggish cultures may prolong the process time from days to weeks. In stuck fermentations, high levels of residual sugar are left following the arrest of fermentation. Several difficulties occur at the cellular level; these include diminished sugar transport capacity, the inability of yeasts to produce compatible solutes in response to high osmolality (20,28), and cell membrane integrity issues in the presence of high concentrations of ethanol (8). As such mechanisms may act simultaneously in cells and to various extents, it is difficult to isolate and reproduce the mechanisms in stuck and sluggish fermentations.Quantitatively, nitrogen is the second most abundant nutrient in wine fermentations. It is essential for yeast metabolism and growth. Consequently, a lack of nitrogen triggers sluggish fermentations (1,8,13). In previous studies workers found differences in nitrogen-related curves (e.g., curves of biomass versus assimilable nitrogen) which indicated that the fermentation rate and biomass yield functions are distinct (9, 10; W. Agenbach, Proc. S. Afr. Soc. Enol. Vitic., p. 66-88, 1977). In other studies the ...

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Consequences of phosphoglycerate kinase overproduction for the growth and physiology of Saccharomyces cerevisiae

Cited by 11 publications

References 45 publications

Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae

Intracellular Expression of Vitreoscilla Hemoglobin Alters the Aerobic Metabolism of Saccharomyces cerevisiae

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium

Biomass Content Governs Fermentation Rate in Nitrogen-Deficient Wine Musts

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