Skip to main content
SpringerLink
Log in

Metabolic Flux and Nodes Control Analysis of Brewer’s Yeasts Under Different Fermentation Temperature During Beer Brewing

  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

The aim of this work was to further investigate the glycolysis performance of lager and ale brewer’s yeasts under different fermentation temperature using a combined analysis of metabolic flux, glycolytic enzyme activities, and flux control. The results indicated that the fluxes through glycolytic pathway decreased with the change of the fermentation temperature from 15 °C to 10 °C, which resulted in the prolonged fermentation times. The maximum activities (V max) of hexokinase (HK), phosphofructokinase (PFK), and pyruvate kinase (PK) at key nodes of glycolytic pathway decreased with decreasing fermentation temperature, which was estimated to have different control extent (22–84 %) on the glycolytic fluxes in exponential or flocculent phase. Moreover, the decrease of V max of PFK or PK displayed the crucial role in down-regulation of flux in flocculent phase. In addition, the metabolic state of ale strain was more sensitive to the variation of temperature than that of lager strain. The results of the metabolic flux and nodes control analysis in brewer’s yeasts under different fermentation temperature may provide an alternative approach to regulate glycolytic flux by changing V max and improve the production efficiency and beer quality.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Vidgren, V., Multanen, J., Ruohonen, L., & Londesborough, J. (2010). The temperature dependence of maltose transport in ale and lager strains of brewer’s yeast. FEMS Yeast Research, 10, 402–411.

    Article  CAS  Google Scholar 

  2. Lodolo, E. J., Kock, J. L. F., Axcell, B. C., & Brooks, M. (2008). The yeast Saccharomyces cerevisiae—the main character in beer brewing. FEMS Yeast Research, 8, 1018–1036.

    Article  CAS  Google Scholar 

  3. Kourkoutas, Y., Bekatorou, A., Banat, I. M., Marchant, R., & Koutinas, A. A. (2004). Immobilization technologies and support materials suitable in alcohol beverages production: a review. Food Microbiology, 21, 377–397.

    Article  CAS  Google Scholar 

  4. Piddocke, M. P., Kreisz, S., Heldt-Hansen, H. P., Nielsen, K. F., & Olsson, L. (2009). Physiological characterization of brewer’s yeast in high-gravity beer fermentations with glucose or maltose syrups as adjuncts. Applied Microbiology and Biotechnology, 84, 453–464.

    Article  CAS  Google Scholar 

  5. Verduyn, C., Postma, E., & Scheffers, W. (1990). Physiology of Saccharomyces cerevisiae in anaerobic glucose-limited chemostat cultures. Journal of the Genetics and Microbiology, 136, 395–403.

    Article  CAS  Google Scholar 

  6. Saerens, S. M. G., Verbelen, P. J., Vanbeneden, N., Thevelein, J. M., & Delvaux, F. R. (2008). Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast. Applied Microbiology and Biotechnology, 80, 1039–1051.

    Article  CAS  Google Scholar 

  7. Chowdhury, I., Watier, D., Leguerinel, I., & Hornez, J. P. (1997). Effect of Pectinatus cerevisiiphilus on Saccharomyces cerevisiae concerning its growth and alcohol production in wort medium. Food Microbiology, 14, 265–272.

    Article  CAS  Google Scholar 

  8. Landaud, S., Latrille, E., & Corrieu, G. (2001). Top pressure and temperature control the fusel alcohol/ester ratio through yeast growth in beer fermentation. Journal of the Institute of Brewing, 107, 107–117.

    Article  CAS  Google Scholar 

  9. Gibson, B. R., Lawrence, S. J., Leclaire, J. P. R., Powell, C. D., & Smart, K. A. (2007). Yeast responses to stresses associated with industrial brewery handling. FEMS Microbiology Reviews, 31, 535–569.

    Article  CAS  Google Scholar 

  10. Chen, Z., Liu, H. J., Zhang, J. A., & Liu, D. H. (2009). Cell physiology and metabolic flux response of Klebsiella pneumoniae to aerobic conditions. Process Biochemistry, 44, 862–868.

    Article  CAS  Google Scholar 

  11. Arense, P., Bernal, V., Iborra, J. L., & Cánovas, M. (2010). Metabolic adaptation of Escherichia coli to long-term exposure to salt stress. Process Biochemistry, 45, 1459–1467.

    Article  CAS  Google Scholar 

  12. Reuter, R., Naumann, M., Bär, J., Haferburg, D., & Kopperschläger, G. (2000). Purification, molecular and kinetic characterization of phosphofructokinase-I from the yeast Schizosaccharomyces pombe: evidence for an unusual subunit composition. Yeast, 16, 1273–1285.

    Article  CAS  Google Scholar 

  13. Susan-Resiga, D., & Nowak, T. (2003). The proton transfer step catalyzed by yeast pyruvate kinase. Journal of Biological Chemistry, 278, 12660–12671.

    Article  CAS  Google Scholar 

  14. Yu, M. A., Wei, Y. M., Zhao, L., Jiang, L., Zhu, X. B., & Qi, W. (2007). Bioconversion of ethyl 4-chloro-3-oxobutanoate by permeabilized fresh brewer’s yeast cells in the presence of allyl bromide. Journal of Industry Microbiology and Biotechnology, 34, 151–156.

    Article  CAS  Google Scholar 

  15. Bernal, V., Carinhas, N., Yokomizo, Y. Y., Carrondo, M. J. T., & Alves, P. M. (2009). Cell density effect in the bauculovirus-insect cells system: a quantitative analysis of energetic metabolism. Biotechnology and Bioengineering, 104, 162–178.

    Article  CAS  Google Scholar 

  16. Zhang, Q., Teng, H., Sun, Y., Xiu, Z., & Zeng, A. (2008). Metabolic flux and robustness analysis of glycerol metabolism in Klebsiella pneumoniae. Bioprocess and Biosystems Engineering, 31, 127–135.

    Article  CAS  Google Scholar 

  17. ven Eunen, K., Dool, P., Canelas, A. B., Kiewiet, J., Bouwman, J., van Gulik, W. M., Westerhoff, H. V., & Bakker, B. M. (2010). Time-dependent regulation of yeast glycolysis upon nitrogen starvation depends on cell history. IET Systems Biology, 4, 157–168.

    Article  Google Scholar 

  18. Liu, H. J., Li, Q., Liu, D. H., & Zhong, J. J. (2006). Impact of hyperosmotic condition on cell physiology and metabolic flux distribution of Candida krusei. Biochemistry Engineering Journal, 28, 92–98.

    Article  Google Scholar 

  19. Rossell, S., van der Weijden, C. C., Lindenbergh, A., Tuijl, A., Francke, C., Bakker, B. M., & Westerhoff, H. V. (2006). Unraveling the complexity of flux regulation: a new method demonstrated for nutrient starvation in Saccharomyces cerevisiae. PNAS, 103, 2166–2171.

    Article  CAS  Google Scholar 

  20. Shen, Y., Zhao, X. Q., Ge, X. M., & Bai, F. W. (2009). Metabolic flux and cell cycle analysis indicating new mechanism underlying process oscillation in continuous ethanol fermentation with Saccharomyces cerevisiae under VHG conditions. Biotechnology Advances, 27, 1118–1123.

    Article  CAS  Google Scholar 

  21. Çaklr, T., Arga, K. Y., Altmtas, M. M., & Ülgen, K. Ö. (2004). Flux analysis of recombinant Saccharomyces cerevisiae YPB-G utilizing starch for optimal ethanol production. Process Biochemistry, 39, 2097–2108.

    Article  Google Scholar 

  22. Gibson, B. R., Boulton, C. A., Box, W. G., Graham, N. S., Lawrence, S. J., Linforth, R. S. T., & Smart, K. A. (2008). Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation. Yeast, 25, 549–562.

    Article  CAS  Google Scholar 

  23. Li, J., Jiang, M., Chen, K. Q., Ye, Q., Shang, L. A., Wei, P., Ying, H. J., & Chang, H. N. (2010). Effect of redox potential regulation on succinic acid production by Actinobacillus succinogenes. Bioprocess and Biosystems Engineering, 33, 911–920.

    Article  CAS  Google Scholar 

  24. Liu, L., Li, Y., Shi, Z., Du, G., & Chen, J. (2006). Enhancement of pyruvate productivity in Torulopsis glabrata: increase of NAD+ availability. Journal of Biotechnology, 126, 173–185.

    Article  CAS  Google Scholar 

  25. Li, J., Yang, Y., Chu, J., Huang, M., Li, L., Zhang, X., Wang, Y., Zhuang, Y., & Zhang, S. (2010). Quantitative metabolic flux analysis revealed uneconomical utilization of ATP and NADPH in Acremonium chysogenum fed with soybean oil. Bioprocess and Biosystems Engineering, 33, 1119–1129.

    Article  CAS  Google Scholar 

  26. Andersen, H. W., Solem, C., Hammer, K., & Jensen, P. R. (2001). Twofold reduction of phosphofructokinase activity in Lactococcus lactis results in strong decreases in growth rate and in glycolytic flux. Journal of Bacteriology, 183, 3458–3467.

    Article  CAS  Google Scholar 

  27. Walsh, R. M., & Martin, P. A. (1977). Growth of Saccharomyces cerevisiae and Saccharomyces uvarum in a temperature gradient. Journal of the Institute of Brewing, 83, 169–172.

    Google Scholar 

  28. Tai, S. L., Daran-Lapujade, P., Luttik, M. A., Walsh, M. C., Diderich, J. A., Krijger, G. C., van Gulik, W. M., Pronk, J. T., & Daran, J. M. (2007). Control of the glycolytic flux in Saccharomyces cerevisiae grown at low temperature. Journal of Biological Chemistry, 282, 10243–10251.

    Article  CAS  Google Scholar 

  29. Verbelen, P. J., Dekoninck, T. M. L., Saerens, S. M. G., van Mulders, S. E., Thevelein, J. M., & Delvaux, F. R. (2009). Impact of pitching rate on yeast fermentation performance and beer flavour. Applied Microbiology and Biotechnology, 82, 155–167.

    Article  CAS  Google Scholar 

  30. Daran-Lapujade, P., Rossell, S., van Gulik, W. M., Luttik, M. A. H., de Groot, M. J. L., Slijper, M., Heck, A. J. R., Daran, J. M., de Winde, J. H., Westerhoff, H. V., Pronk, J. T., & Bakker, B. M. (2007). The fluxes through glycolytic enzymes in Saccharomyces cerevisiae are predominantly regulated at posttranscriptional levels. PNAS, 104, 15753–15758.

    Article  CAS  Google Scholar 

  31. Rautio, J. J., Huuskonen, A., Vuokko, H., Vidgren, V., & Londesborough, J. (2007). Monitoring yeast physiology during very high gravity wort fermentations by frequent analysis of gene expression. Yeast, 24, 741–760.

    Article  CAS  Google Scholar 

  32. Guimarães, P. M. R., & Londesborough, J. (2008). The adenylate energy charge and specific fermentation rate of brewer’s yeasts fermenting high- and very high-gravity worts. Yeast, 25, 47–58.

    Article  Google Scholar 

  33. van den Brink, J., Canelas, A. B., van Gulik, W. M., Pronk, J. T., Heijnen, J. J., de Winde, J. H., & Daran-Lapujade, P. (2008). Dynamics of glycolytic regulation during adaptation of Saccharomyces cerevisiae to fermentative metabolism. Applied and Environmental Microbiology, 78, 5710–5723.

    Article  Google Scholar 

  34. Ramos, A., Neves, A. R., Ventura, R., Maycock, C., Lopez, P., & Santos, H. (2004). Effect of pyruvate kinase overproduction on glucose metabolism of Lactococcus lactis. Microbiol-SGM, 150, 1103–1111.

    Article  CAS  Google Scholar 

  35. Breitenbach-Schmitt, I., Schmitt, H. D., Heinisch, J., & Zimmermann, F. K. (1984). Genetic and physiological evidence for the existence of a second glycolytic pathway in yeast parallel to the phosphofructokinase-aldolase reaction sequence. Molecular Genetics and Genomics, 195, 536–540.

    CAS  Google Scholar 

  36. Pearce, A. K., Crimmins, K., Groussac, E., Hewlins, M. J., Dickinson, J. R., Francois, J., Booth, I. R., & Brown, A. J. (2001). Pyruvate kinase (Pyk1) levels influence both the rate and direction of carbon flux in yeast under fermentative conditions. Microbiology, 147, 391–401.

    CAS  Google Scholar 

  37. Hauf, J., Zimmermann, F. K., & Muller, S. (2000). Simultaneous genomic overexpression of seven glycolytic enzymes in the yeast Saccharomyces cerevisiae. Enzyme and Microbial Technology, 26, 688–498.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors gratefully acknowledge the Key Technology R&D Program of Guangdong Province (Nos. 2011A020102001 and 2010A010500002), the National Natural Science Foundation of China (No. 31000810) and the Fundamental Research Funds for the Central Universities (No. 2012ZM0069) for their financial support.

Author information

Authors and Affiliations

  1. College of Light Industry and Food Sciences, South China University of Technology, Guangzhou, 510640, People’s Republic of China

    Zhimin Yu, Haifeng Zhao, Mouming Zhao & Hongjie Lei

  2. Guangzhou Zhujiang Brewery Co., Ltd, Guangzhou, 510308, People’s Republic of China

    Huiping Li

Authors
  1. Zhimin Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Haifeng Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mouming Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hongjie Lei
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Huiping Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Haifeng Zhao.

Appendices

Appendix 1: List of Metabolic Reactions

J1: GLC + MAL + MALT → 1/6 GLCin

J2: GLC + 1/6 ATP → G6P + 1/6 ADP

J3: G6P → F6P

J4: F6P + 1/6 ATP → F16P + 1/6 ADP

J5: F16P → 1/2 DHAP + 1/2 GAP + 1/6 H+

J6: DHAP → GAP

J7: DHAP + 1/3 NADH + 1/3 H+ → GOH3P + 1/3 NAD +

J8: GAP + 1/3 NAD+ + 1/3 ADP + 1/3 Pi → G3P + 1/3 ATP + 1/3 NADH + 1/3 H+

J9: G3P → PEP + 1/3 H2O

J10: PEP + 1/3 ADP + 1/3 H+ → PYR + 1/3 ATP

J11 − J12: G6P + 1/3 NADP+ + 1/2 H2O → 5/6 RU5P + 1/3 NADPH + 1/3 H+ + 1/6 CO2

RU5P → 2/5 E4P + 3/5 F6P

RU5P + 2/5 E4P → 6/9 F6P + 3/9 G3P

J13: 3/4 PYR + 1/4 CO2 + 1/4 H2O + 1/4 ATP → OAA + 1/4 ADP + 1/4 Pi + 1/4 H+

J14: PYR + 1/3 H+ → 2/3 ACET + 1/3 CO2

J15: ACET + 1/2 NADP+ +1/2 H2O → ACA + 1/2 NADPH + 1/2 H+

J16: ACA + 1/2 CoA + ATP → ACCOA + ADP + Pi

J17: 1/2 ACCOA + 1/2 OAA + 1/6 H2O → CIT + 1/3 CoA + 1/6 H+

J18: CIT + 1/3 NADP → 5/6 α-KTA + 1/6 CO2 + 1/3 NADPH

J19: OAA + 1/2 NADH + 1/2 H+ → SUC + 1/2 NAD+ + 1/2 H2O

J20: CIT → CITout

J21: PYR → PYRout

J22: ACET + 1/2 NADH + 1/2 H+ → ETHOH + 1/2 NAD+

J23: ACA → ACAout

J24: 2.52 G6P + 0.61 RU5P + 0.60 GOH3P + 0.007 G3P + 0.53 PEP + 1.76 PYR + 0.88

ACCOA + 1.16 α-KTA + 0.83 OAA → BIO

Appendix 2: Vector of Considered Metabolites

ACA: acetate; ACA out : extracellular acetate; ACCOA: acetyl-coenzyme A; ACET: acetaldehyde; ADP: adenosine-5′-diphosphate; ATP: adenosine-5′-triphosphate; BIO: biomass; CIT: citrate; CIT out : extracellular citrate; CO 2 : carbon dioxide; CoA: coenzyme A; DHAP: dihydroxyacetone-phosphate; E4P: erythrose-4-phosphate; ETHOH: ethanol; F16P: fructrose-1, 6-diphosphate; F6P: fructrose-6-phosphate; GAP: glyceraldehydes-3-phosphate; GLC: glucose; GLC in : intracellular glucose; G3P: 3-phosphoric acid glyceraldehyed; G6P: glucose-6-phosphate; H 2 O: water; H +: ion; α-KTA: α-ketoglutarate; MAL: maltose; MALT: maltotriose; NAD: nicotinamide adenine dinucleotide; NADH: nicotinamide adenine dinucleotide; NADP: nicotinamide adenine dinucleotide phosphate; NADPH: nicotinamide adenine dinucleotide phosphate; OAA: oxaloacetate; PEP: phosphoenolpyruvate; Pi: phosphate ion; PYR: pyruvate; PYR out : extracellular pyruvate; RU5P: ribulose-5-phosphate; SUC: succinate.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Yu, Z., Zhao, H., Zhao, M. et al. Metabolic Flux and Nodes Control Analysis of Brewer’s Yeasts Under Different Fermentation Temperature During Beer Brewing. Appl Biochem Biotechnol 168, 1938–1952 (2012). https://doi.org/10.1007/s12010-012-9909-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12010-012-9909-z

Keywords

Search

Navigation