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Redox engineering by ectopic expression of glutamate dehydrogenase genes links NADPH availability and NADH oxidation with cold growth in Saccharomyces cerevisiae.

Ballester-Tomás L, Randez-Gil F, Pérez-Torrado R, Prieto JA - Microb. Cell Fact. (2015)

Bottom Line: Indeed, neither the Trp(-) character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes.However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells.We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker's yeast cells display higher fermentative capacity than those cultivated at 30°C.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain. lballester@iata.csic.es.

ABSTRACT

Background: Cold stress reduces microbial growth and metabolism being relevant in industrial processes like wine making and brewing. Knowledge on the cold transcriptional response of Saccharomyces cerevisiae suggests the need of a proper redox balance. Nevertheless, there are no direct evidence of the links between NAD(P) levels and cold growth and how engineering of enzymatic reactions requiring NAD(P) may be used to modify the performance of industrial strains at low temperature.

Results: Recombinant strains of S. cerevisiae modified for increased NADPH- and NADH-dependent Gdh1 and Gdh2 activity were tested for growth at low temperature. A high-copy number of the GDH2-encoded glutamate dehydrogenase gene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused by cofactor preferences. Indeed, neither the Trp(-) character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes. However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells. We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker's yeast cells display higher fermentative capacity than those cultivated at 30°C. Thus, increasing NADH oxidation by overexpression of GDH2 would help to avoid perturbations in the redox metabolism induced by a higher fermentative/oxidative balance at low temperature. Finally, it is shown that overexpression of GDH2 increases notably the cold growth in the wine yeast strain QA23 in both standard growth medium and synthetic grape must.

Conclusions: Redox constraints limit the growth of S. cerevisiae at temperatures below the optimal. An adequate supply of NAD(P) precursors as well as a proper level of reducing equivalents in the form of NADPH are required for cold growth. However, a major limitation is the increased need of oxidation of NADH to NAD(+) at low temperature. In this scenario, our results identify the ammonium assimilation pathway as a target for the genetic improvement of cold growth in industrial strains.

No MeSH data available.


Related in: MedlinePlus

Overexpression of GDH2 improves the cold performance of the QA23 ho wine yeast strain. a Ura− derivatives of the wine strain QA23 ho were transformed with YEplac195-based plasmids (URA3) containing GDH1 or GDH2 and assayed for growth at the indicated temperatures on SCD-Ura agar medium. Transformants carrying the empty plasmid were also tested (Control). In all cases, cells were pre-grown and treated as described in the Figure 2. b The same strains were tested for growth at 12°C in synthetic grape must (left graph) or liquid SCD-Ura (right graph) medium. Growth of yeast cultures was monitored by measuring the cell suspension’s optical density at 600 nm (OD600) for the indicated period. A representative experiment is shown.
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Fig6: Overexpression of GDH2 improves the cold performance of the QA23 ho wine yeast strain. a Ura− derivatives of the wine strain QA23 ho were transformed with YEplac195-based plasmids (URA3) containing GDH1 or GDH2 and assayed for growth at the indicated temperatures on SCD-Ura agar medium. Transformants carrying the empty plasmid were also tested (Control). In all cases, cells were pre-grown and treated as described in the Figure 2. b The same strains were tested for growth at 12°C in synthetic grape must (left graph) or liquid SCD-Ura (right graph) medium. Growth of yeast cultures was monitored by measuring the cell suspension’s optical density at 600 nm (OD600) for the indicated period. A representative experiment is shown.

Mentions: High growth at low temperature has become one of the most important oenological criteria used to select industrial wine yeast strains. Uncovering genes and metabolic pathways that determine the adaptation of industrial yeast strains to cold is therefore highly relevant to targeting the genetic improvement of these microorganisms. Therefore, we investigated whether the ectopic expression of GDH1 and GDH2 in a wine yeast strain may have a similar impact on cold growth as that observed in lab yeast strains. Industrial wine strains differ genetically and physiologically from S. cerevisiae lab strains [43] and thus changes in the copy number of a particular gene might result in distinct responses. To address this, an Ura− auxotrophic derivative (MJHL201, ura3) of the QA23 ho wine strain [44] was transformed with the URA3-based plasmids YEpGDH1 and YEpGDH2 and tested for growth at low temperature in SCD-Ura plates. Enhanced growth was observed for YEpGDH2 transformants exposed to a range of low temperatures from 8 to 15°C (Figure 6a). Overexpression of GDH1 impaired again cold-growth although its effects appeared less pronounced in the prototroph wine yeast (Figure 6a) than those found in laboratory strains (Figure 2). Similar results were observed in SCD-Ura liquid cultures at 12°C (Figure 6b, right graph). In this point, we investigated the cold response of the wine yeast transformants in a synthetic grape must. As it is shown, the overexpression of GDH1 had no noticeable effects on cold growth (Figure 6b, left graph), a result that stress the importance of checking physiological responses in wine yeasts under these conditions. However, ectopic expression of GDH2 stimulated again the growth at low temperature of the QA23 strain (Figure 6b, left graph), confirming thus the data for laboratory strains of S. cerevisiae and the potential of this tool to manipulate the redox balance and the performance of industrial strains at low temperature.Figure 6


Redox engineering by ectopic expression of glutamate dehydrogenase genes links NADPH availability and NADH oxidation with cold growth in Saccharomyces cerevisiae.

Ballester-Tomás L, Randez-Gil F, Pérez-Torrado R, Prieto JA - Microb. Cell Fact. (2015)

Overexpression of GDH2 improves the cold performance of the QA23 ho wine yeast strain. a Ura− derivatives of the wine strain QA23 ho were transformed with YEplac195-based plasmids (URA3) containing GDH1 or GDH2 and assayed for growth at the indicated temperatures on SCD-Ura agar medium. Transformants carrying the empty plasmid were also tested (Control). In all cases, cells were pre-grown and treated as described in the Figure 2. b The same strains were tested for growth at 12°C in synthetic grape must (left graph) or liquid SCD-Ura (right graph) medium. Growth of yeast cultures was monitored by measuring the cell suspension’s optical density at 600 nm (OD600) for the indicated period. A representative experiment is shown.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4496827&req=5

Fig6: Overexpression of GDH2 improves the cold performance of the QA23 ho wine yeast strain. a Ura− derivatives of the wine strain QA23 ho were transformed with YEplac195-based plasmids (URA3) containing GDH1 or GDH2 and assayed for growth at the indicated temperatures on SCD-Ura agar medium. Transformants carrying the empty plasmid were also tested (Control). In all cases, cells were pre-grown and treated as described in the Figure 2. b The same strains were tested for growth at 12°C in synthetic grape must (left graph) or liquid SCD-Ura (right graph) medium. Growth of yeast cultures was monitored by measuring the cell suspension’s optical density at 600 nm (OD600) for the indicated period. A representative experiment is shown.
Mentions: High growth at low temperature has become one of the most important oenological criteria used to select industrial wine yeast strains. Uncovering genes and metabolic pathways that determine the adaptation of industrial yeast strains to cold is therefore highly relevant to targeting the genetic improvement of these microorganisms. Therefore, we investigated whether the ectopic expression of GDH1 and GDH2 in a wine yeast strain may have a similar impact on cold growth as that observed in lab yeast strains. Industrial wine strains differ genetically and physiologically from S. cerevisiae lab strains [43] and thus changes in the copy number of a particular gene might result in distinct responses. To address this, an Ura− auxotrophic derivative (MJHL201, ura3) of the QA23 ho wine strain [44] was transformed with the URA3-based plasmids YEpGDH1 and YEpGDH2 and tested for growth at low temperature in SCD-Ura plates. Enhanced growth was observed for YEpGDH2 transformants exposed to a range of low temperatures from 8 to 15°C (Figure 6a). Overexpression of GDH1 impaired again cold-growth although its effects appeared less pronounced in the prototroph wine yeast (Figure 6a) than those found in laboratory strains (Figure 2). Similar results were observed in SCD-Ura liquid cultures at 12°C (Figure 6b, right graph). In this point, we investigated the cold response of the wine yeast transformants in a synthetic grape must. As it is shown, the overexpression of GDH1 had no noticeable effects on cold growth (Figure 6b, left graph), a result that stress the importance of checking physiological responses in wine yeasts under these conditions. However, ectopic expression of GDH2 stimulated again the growth at low temperature of the QA23 strain (Figure 6b, left graph), confirming thus the data for laboratory strains of S. cerevisiae and the potential of this tool to manipulate the redox balance and the performance of industrial strains at low temperature.Figure 6

Bottom Line: Indeed, neither the Trp(-) character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes.However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells.We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker's yeast cells display higher fermentative capacity than those cultivated at 30°C.

View Article: PubMed Central - PubMed

Affiliation: Department of Biotechnology, Instituto de Agroquímica y Tecnología de los Alimentos (CSIC), Avda. Agustín Escardino 7, 46980, Paterna, Valencia, Spain. lballester@iata.csic.es.

ABSTRACT

Background: Cold stress reduces microbial growth and metabolism being relevant in industrial processes like wine making and brewing. Knowledge on the cold transcriptional response of Saccharomyces cerevisiae suggests the need of a proper redox balance. Nevertheless, there are no direct evidence of the links between NAD(P) levels and cold growth and how engineering of enzymatic reactions requiring NAD(P) may be used to modify the performance of industrial strains at low temperature.

Results: Recombinant strains of S. cerevisiae modified for increased NADPH- and NADH-dependent Gdh1 and Gdh2 activity were tested for growth at low temperature. A high-copy number of the GDH2-encoded glutamate dehydrogenase gene stimulated growth at 15°C, while overexpression of GDH1 had detrimental effects, a difference likely caused by cofactor preferences. Indeed, neither the Trp(-) character of the tested strains, which could affect the synthesis of NAD(P), nor changes in oxidative stress susceptibility by overexpression of GDH1 and GDH2 account for the observed phenotypes. However, increased or reduced NADPH availability by knock-out or overexpression of GRE3, the NADPH-dependent aldose reductase gene, eliminated or exacerbated the cold-growth defect observed in YEpGDH1 cells. We also demonstrated that decreased capacity of glycerol production impairs growth at 15 but not at 30°C and that 15°C-grown baker's yeast cells display higher fermentative capacity than those cultivated at 30°C. Thus, increasing NADH oxidation by overexpression of GDH2 would help to avoid perturbations in the redox metabolism induced by a higher fermentative/oxidative balance at low temperature. Finally, it is shown that overexpression of GDH2 increases notably the cold growth in the wine yeast strain QA23 in both standard growth medium and synthetic grape must.

Conclusions: Redox constraints limit the growth of S. cerevisiae at temperatures below the optimal. An adequate supply of NAD(P) precursors as well as a proper level of reducing equivalents in the form of NADPH are required for cold growth. However, a major limitation is the increased need of oxidation of NADH to NAD(+) at low temperature. In this scenario, our results identify the ammonium assimilation pathway as a target for the genetic improvement of cold growth in industrial strains.

No MeSH data available.


Related in: MedlinePlus