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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

Schematic representation of some yeast NADH- and NADPH-dependent reactions and pathways cited in the text. Adh1-3 alcohol dehydrogenase, Gpd1 glycerol-3-phosphate dehydrogenase, Gre3 aldose reductase, Nde1,2 external NADH dehydrogenase, Ndi1 internal NADH dehydrogenase, AcCoA acetyl coenzyme A, EtOH ethanol, DHAP dihydroxy acetone phosphate, G3P glycerol 3-phosphate, MG methylglyoxal. Note that the glutamate dehydrogenase isoenzyme Gdh2 usually catalyzes the catabolic reaction from l-glutamate to α-ketoglutarate. Nevertheless, when overexpressed [21] or when NH4+ is plentiful [22], contributes to the glutamate production using NADH as cofactor [19]. For other details see the text.
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Fig1: Schematic representation of some yeast NADH- and NADPH-dependent reactions and pathways cited in the text. Adh1-3 alcohol dehydrogenase, Gpd1 glycerol-3-phosphate dehydrogenase, Gre3 aldose reductase, Nde1,2 external NADH dehydrogenase, Ndi1 internal NADH dehydrogenase, AcCoA acetyl coenzyme A, EtOH ethanol, DHAP dihydroxy acetone phosphate, G3P glycerol 3-phosphate, MG methylglyoxal. Note that the glutamate dehydrogenase isoenzyme Gdh2 usually catalyzes the catabolic reaction from l-glutamate to α-ketoglutarate. Nevertheless, when overexpressed [21] or when NH4+ is plentiful [22], contributes to the glutamate production using NADH as cofactor [19]. For other details see the text.

Mentions: A major source of NADH mitochondrial is the synthesis of α-ketoglutarate from pyruvate [18] (Figure 1) that precedes the cytosolic NADPH-dependent production of glutamate by the activity of the glutamate dehydrogenase (GDH) isoenzymes Gdh1 and Gdh3 [19]. GDH1 is highly expressed in actively growing cells while the Ghdh3-encoding gene shows a stationary-phase specific expression [20]. A third enzyme, Gdh2, which usually catalyzes the catabolic reaction, contributes when overexpressed [21] or when NH4+ is plentiful [22], to the glutamate production using NADH as cofactor [19] (Figure 1). Recently, a study of the yeast proteome variation following a cold-shock allowed us identifying Gdh1 among the proteins showing increased abundance at low temperature (unpublished results). By using a systems biology approach, Paget et al. [6] have also identified GDH2 as a candidate cold growth-favoring gene. Altogether, these results suggest that regulation of the GDH activity could represent a mechanism of adaptation to low temperature, although no experimental evidences of this function have been provided.Figure 1


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)

Schematic representation of some yeast NADH- and NADPH-dependent reactions and pathways cited in the text. Adh1-3 alcohol dehydrogenase, Gpd1 glycerol-3-phosphate dehydrogenase, Gre3 aldose reductase, Nde1,2 external NADH dehydrogenase, Ndi1 internal NADH dehydrogenase, AcCoA acetyl coenzyme A, EtOH ethanol, DHAP dihydroxy acetone phosphate, G3P glycerol 3-phosphate, MG methylglyoxal. Note that the glutamate dehydrogenase isoenzyme Gdh2 usually catalyzes the catabolic reaction from l-glutamate to α-ketoglutarate. Nevertheless, when overexpressed [21] or when NH4+ is plentiful [22], contributes to the glutamate production using NADH as cofactor [19]. For other details see the text.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Schematic representation of some yeast NADH- and NADPH-dependent reactions and pathways cited in the text. Adh1-3 alcohol dehydrogenase, Gpd1 glycerol-3-phosphate dehydrogenase, Gre3 aldose reductase, Nde1,2 external NADH dehydrogenase, Ndi1 internal NADH dehydrogenase, AcCoA acetyl coenzyme A, EtOH ethanol, DHAP dihydroxy acetone phosphate, G3P glycerol 3-phosphate, MG methylglyoxal. Note that the glutamate dehydrogenase isoenzyme Gdh2 usually catalyzes the catabolic reaction from l-glutamate to α-ketoglutarate. Nevertheless, when overexpressed [21] or when NH4+ is plentiful [22], contributes to the glutamate production using NADH as cofactor [19]. For other details see the text.
Mentions: A major source of NADH mitochondrial is the synthesis of α-ketoglutarate from pyruvate [18] (Figure 1) that precedes the cytosolic NADPH-dependent production of glutamate by the activity of the glutamate dehydrogenase (GDH) isoenzymes Gdh1 and Gdh3 [19]. GDH1 is highly expressed in actively growing cells while the Ghdh3-encoding gene shows a stationary-phase specific expression [20]. A third enzyme, Gdh2, which usually catalyzes the catabolic reaction, contributes when overexpressed [21] or when NH4+ is plentiful [22], to the glutamate production using NADH as cofactor [19] (Figure 1). Recently, a study of the yeast proteome variation following a cold-shock allowed us identifying Gdh1 among the proteins showing increased abundance at low temperature (unpublished results). By using a systems biology approach, Paget et al. [6] have also identified GDH2 as a candidate cold growth-favoring gene. Altogether, these results suggest that regulation of the GDH activity could represent a mechanism of adaptation to low temperature, although no experimental evidences of this function have been provided.Figure 1

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