Physiological Effects of GLT1 Modulation in Saccharomyces cerevisiae Strains Growing on Different Nitrogen Sources

Marco  Brambilla; Giusy Manuela  Adamo; Gianni  Frascotti; Danilo  Porro; Paola  Branduardi

doi:10.4014/jmb.1508.08002

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Research article

Bioprocess and Metabolic Engineering

Physiological Effects of GLT1 Modulation in Saccharomyces cerevisiae Strains Growing on Different Nitrogen Sources

Marco Brambilla ¹, Giusy Manuela Adamo ¹, Gianni Frascotti ¹, Danilo Porro ^{1, 2*} and Paola Branduardi ¹

¹Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2 - 20126 Milan, Italy, ¹SYSBIO – Centre of Systems Biology, Milano and Roma, Italy

Received: August 3, 2015; Accepted: October 28, 2015

J. Microbiol. Biotechnol. 2016; 26(2): 326-336

Published February 28, 2016 https://doi.org/10.4014/jmb.1508.08002

Abstract

Saccharomyces cerevisiae is one of the most employed cell factories for the production of bioproducts. Although monomeric hexose sugars constitute the preferential carbon source, this yeast can grow on a wide variety of nitrogen sources that are catabolized through central nitrogen metabolism (CNM). To evaluate the effects of internal perturbations on nitrogen utilization, we characterized strains deleted or overexpressed in GLT1, encoding for one of the key enzymes of the CNM node, the glutamate synthase. These strains, together with the parental strain as control, have been cultivated in minimal medium formulated with ammonium sulfate, glutamate, or glutamine as nitrogen source. Growth kinetics, together with the determination of protein content, viability, and reactive oxygen species (ROS) accumulation at the single cell level, revealed that GLT1 modulations do not significantly influence the cellular physiology, whereas the nitrogen source does. As important exceptions, GLT1 deletion negatively affected the scavenging activity of glutamate against ROS accumulation, when cells were treated with H2O2, whereas Glt1p overproduction led to lower viability in glutamine medium. Overall, this confirms the robustness of the CNM node against internal perturbations, but, at the same time, highlights its plasticity in respect to the environment. Considering that side-stream protein-rich waste materials are emerging as substrates to be used in an integrated biorefinery, these results underline the importance of preliminarily evaluating the best nitrogen source not only for media formulation, but also for the overall economics of the process.

Keywords

Central Nitrogen Metabolism, GLT1, Saccharomyces cerevisiae, Glutamate, Glutamine, Ammonium sulphate

References

Abdel-Rahman MA, Tashiro Y, Sonomoto K. 2013. Recent advances in lactic acid production by microbial fermentation processes. Biotechnol. Adv. 31: 877-902.
Bertram PG, Choi JH, Carvalho J, Chan TF, Ai W, Zheng XF. 2002. Convergence of TOR-nitrogen and Snf1-glucose signaling pathways onto Gln3. Mol. Cell. Biol. 22: 1246-1252.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Branduardi P, Porro D. 2012. Yeasts in Biotechnology, pp. 347-370. In Feldmann H (ed). Yeast: Molecular and Cell Biology. John Wiley & Sons Inc., Germany.
Branduardi P, Fossati T, Sauer M, Pagani R, Mattanovich D, Porro D. 2007. Biosynthesis of vitamin C by yeast leads to increased stress resistance. PLoS One 2: e1092.
Cao J, Barbosa JM, Singh NK, Locy RD. 2013. GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production. Yeast 30: 129-144.
Cogoni C, Valenzuela L, González-Halphen D, Olivera H, Macino G, Ballario P, González A. 1995. Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide. J. Bacteriol. 177: 792-798.
Coleman ST, Fang TK, Rovinsky SA, Turano FJ, MoyeRowley WS. 2001. Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae. J. Biol. Chem. 276: 244-250.
Crespo JL, Powers T, Fowler B, Hall MN. 2002. The TORcontrolled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proc. Natl. Acad. Sci. USA 99: 6784-6789.
DeLuna A, Avendano A, Riego L, Gonzalez A. 2001. NADPglutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles. J. Biol. Chem. 276: 43775-43783.
Edwards AN, Patterson-Fortin LM, Vakulskas CA, Mercante JW, Potrykus K, Vinella D, et al. 2011. Circuitry linking the Csr and stringent response global regulatory systems. Mol. Microbiol. 80: 1561-1580.
Gietz RD, Woods RA. 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350: 87-96.
Guillamón JM, van Riel NA, Giuseppin ML, Verrips CT. 2001. The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism. FEMS Yeast Res. 1: 169-175.
Hohenblum H, Borth N, Mattanovich D. 2003. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J. Biotechnol. 102: 281-290.
Holmes AR, Collings A, Farnden KJ, Shepherd MG. 1989. Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. J. Gen. Microbiol. 135: 1423-1430.
Hong KK, Nielsen J. 2012. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell. Mol. Life Sci. 69: 2671-2690.
Huo YX, Cho KM, Rivera JG, Monte E, Shen CR, Yan Y, Liao JC. 2011. Conversion of proteins into biofuels by engineering nitrogen flux. Nat. Biotechnol. 29: 346-351.
Kayikci Ö, Nielsen J. 2015. Glucose repression in Saccharomyces cerevisiae. FEMS Yeast Res. 15. DOI: 10.1093/femsyr/fov068.
Kim YM, Hong SJ, Billiar TR, Simmons RL. 1996. Counterprotective effect of erythrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates. Infect. Immun. 64: 3074-3080.
Kingdon HS, Hubbard JS, Stadtman ER. 1968. Regulation of glutamine synthetase. XI. The nature and implications of a lag phase in the Escherichia coli glutamine synthetase reaction. Biochemistry 7: 2136-2142.
Ljungdahl PO, Daignan-Fornier B. 2012. Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae. Genetics 190: 885-929.
Madeo F , Fröhlich E, Ligr M, Grey M, S igrist S J, Wolf DH, Fröhlich KU. 1999. Oxygen stress: a regulator of apoptosis in yeast. J. Cell Biol. 145: 757-767.
Magasanik B, Kaiser CA. 2002. Nitrogen regulation in Saccharomyces cerevisiae. Gene 290: 1-18.
Miller JS, Quarles JM. 1990. Flow cytometric identification of microorganisms by dual staining with FITC and PI. Cytometry 11: 667-675.
Moreira dos Santos M, Thygesen G, Kötter P, Olsson L, Nielsen J. 2003. Aerobic physiology of redox-engineered Saccharomyces cerevisiae strains modified in the ammonium assimilation for increased NADPH availability. FEMS Yeast Res. 4: 59-68.
Murthy GS, Johnston DB, Rausch KD, Tumbleson ME, Singh V. 2012. A simultaneous saccharification and fermentation model for dynamic growth environments. Bioprocess Biosyst. Eng. 35: 519-534.
Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J. 2000. Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab. Eng. 2: 69-77.
Penninckx MJ. 2002. An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res. 2: 295-305.
Perrone GG, Tan SX, Dawes IW. 2008. Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta 1783: 1354-1368.
Porro D, Gasser B, Fossati T, Maurer M, Branduardi P, Sauer M, Mattanovich D. 2011. Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems? Appl. Microbiol. Biotechnol. 89: 939-948.
Riego L, Avendaño A, DeLuna A, Rodríguez E, González A. 2002. GDH1 expression is regulated by GLN3, GCN4, and HAP4 under respiratory growth. Biochem. Biophys. Res. Commun. 293: 79-85.
Roca C, Nielsen J, Olsson L. 2003. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production. Appl. Environ. Microbiol. 69: 4732-4736.
Soberón M, González A. 1987. Physiological role of glutaminase activity in Saccharomyces cerevisiae. J. Gen. Microbiol. 133: 1-8.
Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, Yates III JR. 2008. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression Saccharomyces cerevisiae strains: the comparison of two quantitative methods. J. Proteome Res. 7: 266-275.
Valenzuela L, Ballario P, Aranda C, Filetici P, González A. 1998. Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae. J. Bacteriol. 180: 3533-3540.
Verduyn C , Postma E , Schef fers WA, V an D ijken J P. 1 992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8: 501-517.
Wach A, Brachat A, Pöhlmann R, Philippsen P. 1994. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-1808.
Wang J, Liu W, Ding W, Zhang G, Liu J. 2013. Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae. Biotechnol. Lett. 35: 1859-1864.
Zhao X, Zou H, Fu J, Chen J, Zhou J, Du G. 2013. Nitrogen regulation involved in the accumulation of urea in Saccharomyces cerevisiae. Yeast 30: 437-447.

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Article

Research article

J. Microbiol. Biotechnol. 2016; 26(2): 326-336

Published online February 28, 2016 https://doi.org/10.4014/jmb.1508.08002

Physiological Effects of GLT1 Modulation in Saccharomyces cerevisiae Strains Growing on Different Nitrogen Sources

Marco Brambilla ¹, Giusy Manuela Adamo ¹, Gianni Frascotti ¹, Danilo Porro ^{1, 2*} and Paola Branduardi ¹

¹Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2 - 20126 Milan, Italy, ¹SYSBIO – Centre of Systems Biology, Milano and Roma, Italy

Received: August 3, 2015; Accepted: October 28, 2015

Abstract

Saccharomyces cerevisiae is one of the most employed cell factories for the production of
bioproducts. Although monomeric hexose sugars constitute the preferential carbon source,
this yeast can grow on a wide variety of nitrogen sources that are catabolized through central
nitrogen metabolism (CNM). To evaluate the effects of internal perturbations on nitrogen
utilization, we characterized strains deleted or overexpressed in GLT1, encoding for one of the
key enzymes of the CNM node, the glutamate synthase. These strains, together with the
parental strain as control, have been cultivated in minimal medium formulated with
ammonium sulfate, glutamate, or glutamine as nitrogen source. Growth kinetics, together
with the determination of protein content, viability, and reactive oxygen species (ROS)
accumulation at the single cell level, revealed that GLT1 modulations do not significantly
influence the cellular physiology, whereas the nitrogen source does. As important exceptions,
GLT1 deletion negatively affected the scavenging activity of glutamate against ROS
accumulation, when cells were treated with H2O2, whereas Glt1p overproduction led to lower
viability in glutamine medium. Overall, this confirms the robustness of the CNM node against
internal perturbations, but, at the same time, highlights its plasticity in respect to the
environment. Considering that side-stream protein-rich waste materials are emerging as
substrates to be used in an integrated biorefinery, these results underline the importance of
preliminarily evaluating the best nitrogen source not only for media formulation, but also for
the overall economics of the process.

Keywords: Central Nitrogen Metabolism, GLT1, Saccharomyces cerevisiae, Glutamate, Glutamine, Ammonium sulphate

Abstract

References

Abdel-Rahman MA, Tashiro Y, Sonomoto K. 2013. Recent advances in lactic acid production by microbial fermentation processes. Biotechnol. Adv. 31: 877-902.
Bertram PG, Choi JH, Carvalho J, Chan TF, Ai W, Zheng XF. 2002. Convergence of TOR-nitrogen and Snf1-glucose signaling pathways onto Gln3. Mol. Cell. Biol. 22: 1246-1252.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Branduardi P, Porro D. 2012. Yeasts in Biotechnology, pp. 347-370. In Feldmann H (ed). Yeast: Molecular and Cell Biology. John Wiley & Sons Inc., Germany.
Branduardi P, Fossati T, Sauer M, Pagani R, Mattanovich D, Porro D. 2007. Biosynthesis of vitamin C by yeast leads to increased stress resistance. PLoS One 2: e1092.
Cao J, Barbosa JM, Singh NK, Locy RD. 2013. GABA shunt mediates thermotolerance in Saccharomyces cerevisiae by reducing reactive oxygen production. Yeast 30: 129-144.
Cogoni C, Valenzuela L, González-Halphen D, Olivera H, Macino G, Ballario P, González A. 1995. Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide. J. Bacteriol. 177: 792-798.
Coleman ST, Fang TK, Rovinsky SA, Turano FJ, MoyeRowley WS. 2001. Expression of a glutamate decarboxylase homologue is required for normal oxidative stress tolerance in Saccharomyces cerevisiae. J. Biol. Chem. 276: 244-250.
Crespo JL, Powers T, Fowler B, Hall MN. 2002. The TORcontrolled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proc. Natl. Acad. Sci. USA 99: 6784-6789.
DeLuna A, Avendano A, Riego L, Gonzalez A. 2001. NADPglutamate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, kinetic properties, and physiological roles. J. Biol. Chem. 276: 43775-43783.
Edwards AN, Patterson-Fortin LM, Vakulskas CA, Mercante JW, Potrykus K, Vinella D, et al. 2011. Circuitry linking the Csr and stringent response global regulatory systems. Mol. Microbiol. 80: 1561-1580.
Gietz RD, Woods RA. 2002. Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350: 87-96.
Guillamón JM, van Riel NA, Giuseppin ML, Verrips CT. 2001. The glutamate synthase (GOGAT) of Saccharomyces cerevisiae plays an important role in central nitrogen metabolism. FEMS Yeast Res. 1: 169-175.
Hohenblum H, Borth N, Mattanovich D. 2003. Assessing viability and cell-associated product of recombinant protein producing Pichia pastoris with flow cytometry. J. Biotechnol. 102: 281-290.
Holmes AR, Collings A, Farnden KJ, Shepherd MG. 1989. Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. J. Gen. Microbiol. 135: 1423-1430.
Hong KK, Nielsen J. 2012. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell. Mol. Life Sci. 69: 2671-2690.
Huo YX, Cho KM, Rivera JG, Monte E, Shen CR, Yan Y, Liao JC. 2011. Conversion of proteins into biofuels by engineering nitrogen flux. Nat. Biotechnol. 29: 346-351.
Kayikci Ö, Nielsen J. 2015. Glucose repression in Saccharomyces cerevisiae. FEMS Yeast Res. 15. DOI: 10.1093/femsyr/fov068.
Kim YM, Hong SJ, Billiar TR, Simmons RL. 1996. Counterprotective effect of erythrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates. Infect. Immun. 64: 3074-3080.
Kingdon HS, Hubbard JS, Stadtman ER. 1968. Regulation of glutamine synthetase. XI. The nature and implications of a lag phase in the Escherichia coli glutamine synthetase reaction. Biochemistry 7: 2136-2142.
Ljungdahl PO, Daignan-Fornier B. 2012. Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae. Genetics 190: 885-929.
Madeo F , Fröhlich E, Ligr M, Grey M, S igrist S J, Wolf DH, Fröhlich KU. 1999. Oxygen stress: a regulator of apoptosis in yeast. J. Cell Biol. 145: 757-767.
Magasanik B, Kaiser CA. 2002. Nitrogen regulation in Saccharomyces cerevisiae. Gene 290: 1-18.
Miller JS, Quarles JM. 1990. Flow cytometric identification of microorganisms by dual staining with FITC and PI. Cytometry 11: 667-675.
Moreira dos Santos M, Thygesen G, Kötter P, Olsson L, Nielsen J. 2003. Aerobic physiology of redox-engineered Saccharomyces cerevisiae strains modified in the ammonium assimilation for increased NADPH availability. FEMS Yeast Res. 4: 59-68.
Murthy GS, Johnston DB, Rausch KD, Tumbleson ME, Singh V. 2012. A simultaneous saccharification and fermentation model for dynamic growth environments. Bioprocess Biosyst. Eng. 35: 519-534.
Nissen TL, Kielland-Brandt MC, Nielsen J, Villadsen J. 2000. Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation. Metab. Eng. 2: 69-77.
Penninckx MJ. 2002. An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res. 2: 295-305.
Perrone GG, Tan SX, Dawes IW. 2008. Reactive oxygen species and yeast apoptosis. Biochim. Biophys. Acta 1783: 1354-1368.
Porro D, Gasser B, Fossati T, Maurer M, Branduardi P, Sauer M, Mattanovich D. 2011. Production of recombinant proteins and metabolites in yeasts: when are these systems better than bacterial production systems? Appl. Microbiol. Biotechnol. 89: 939-948.
Riego L, Avendaño A, DeLuna A, Rodríguez E, González A. 2002. GDH1 expression is regulated by GLN3, GCN4, and HAP4 under respiratory growth. Biochem. Biophys. Res. Commun. 293: 79-85.
Roca C, Nielsen J, Olsson L. 2003. Metabolic engineering of ammonium assimilation in xylose-fermenting Saccharomyces cerevisiae improves ethanol production. Appl. Environ. Microbiol. 69: 4732-4736.
Soberón M, González A. 1987. Physiological role of glutaminase activity in Saccharomyces cerevisiae. J. Gen. Microbiol. 133: 1-8.
Usaite R, Wohlschlegel J, Venable JD, Park SK, Nielsen J, Olsson L, Yates III JR. 2008. Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression Saccharomyces cerevisiae strains: the comparison of two quantitative methods. J. Proteome Res. 7: 266-275.
Valenzuela L, Ballario P, Aranda C, Filetici P, González A. 1998. Regulation of expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae. J. Bacteriol. 180: 3533-3540.
Verduyn C , Postma E , Schef fers WA, V an D ijken J P. 1 992. Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8: 501-517.
Wach A, Brachat A, Pöhlmann R, Philippsen P. 1994. New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-1808.
Wang J, Liu W, Ding W, Zhang G, Liu J. 2013. Increasing ethanol titer and yield in a gpd1Δ gpd2Δ strain by simultaneous overexpression of GLT1 and STL1 in Saccharomyces cerevisiae. Biotechnol. Lett. 35: 1859-1864.
Zhao X, Zou H, Fu J, Chen J, Zhou J, Du G. 2013. Nitrogen regulation involved in the accumulation of urea in Saccharomyces cerevisiae. Yeast 30: 437-447.