Application of the Cre-loxP Recombination System for Two ILV2 Alleles Disruption in an Industrial Brewer’s Yeast Strain

Conference paper
First Online:
Part of the Lecture Notes in Electrical Engineering book series (LNEE, volume 249)

Abstract

Diacetyl has long been considered an unpleasant off-flavor component in beer. A recombinant industrial brewer’s yeast strain in which two alleles of α-acetohydroxyacid synthase (AHAS) gene (ILV2) were disrupted using the Cre-loxP recombination system was constructed to produce the lower content of diacetyl. The results showed that the diacetyl production of recombinant yeast strain S-CSL5 is always lower than that of the parental strain S-6 at all stages of beer fermentation. The total process time (from the beginning of fermentation to the diacetyl reduction is finished) of beer fermented by the recombinant strain S-CSL5 could therefore be reduced to 12 days, in contrast to 15 days required for the parental strain. The AHAS activity of S-CSL5 was lowered by 58 % compared with that of the parental strain. In addition, the real-time PCR results revealed a low expression level of ILV2 as a potential molecular determinant for low diacetyl formation.

Keywords

Diacetyl Cre-loxP recombination system ILV2 Industrial brewer’s yeast 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

The current study was financially supported by the National Natural Science Foundation of China (No. 31271916), the Cheung Kong Scholars and Innovative Research Team Program in University of Ministry of Education, China (Grant No. IRT1166).

References

  1. 1.
    Haukeli A, Lie S (1978) Conversion of alpha-acetolatate and removal of diacetyl a kinatic study. J Inst Brew 84:85–89CrossRefGoogle Scholar
  2. 2.
    Duong CT, Strack L, Futschik M et al (2011) Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers’ yeast. Metab Eng 13(6):638–647CrossRefGoogle Scholar
  3. 3.
    Saison D, De Schutter DP, Uyttenhove B et al (2009) Contribution of staling compounds to the aged flavour of lager beer by studying their flavour thresholds. Food Chem 114(4):1206–1215CrossRefGoogle Scholar
  4. 4.
    Wang ZY, He XP, Liu N et al (2008) Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production. Int J Food Sci Tech 43(6):989–994CrossRefGoogle Scholar
  5. 5.
    Gjermansen C, Nilsson-Tillgren T, Petersen JGL et al (1988) Towards diacetyl-less brewers’ yeast. Influence of ilv2 and ilv5 mutations. J Basic Microbiol 28(3):175–183CrossRefGoogle Scholar
  6. 6.
    Liu Z, Zhang G, Li J et al (2008) Integrative expression of glucoamylase gene in a brewer’s yeast Saccharomyces pastorianus strain. Food Technol 46(1):32–37Google Scholar
  7. 7.
    Bond U, Neal C, Donnelly D et al (2004) Aneuploidy and copy number breakpoints in the genome of lager yeasts mapped by microarray hybridisation. Curr Genet 45(6):360–370CrossRefGoogle Scholar
  8. 8.
    Dunn B, Sherlock G (2008) Reconstruction of the genome origins and evolution of the hybrid lager yeast Saccharomyces pastorianus. Genome Res 18(10):1610–1623CrossRefGoogle Scholar
  9. 9.
    Ribeiro O, Gombert AK, Teixeira JA (2007) Application of the Cre-loxP system for multiple gene disruption in the yeast Kluyveromyces marxianus. J Biotechnol 131(1):20–26CrossRefGoogle Scholar
  10. 10.
    Güldener U, Heck S, Fiedler T et al (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24(13):2519–2524CrossRefGoogle Scholar
  11. 11.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed., vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  12. 12.
    Burke D, Dawson DS, Stearns T (2000) Methods in yeast genetics: A Cold Spring Harbor laboratory course manual. CSHL, Press, USAGoogle Scholar
  13. 13.
    Hao J, Dong J, Speers RA et al (2008) Construction of a single PEP 4 allele deletion in Saccharomyces carlsbergensis and a preliminary evaluation of its brewing performance. J Inst Brew 114(4):322–328CrossRefGoogle Scholar
  14. 14.
    Daniel Gietz R, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96Google Scholar
  15. 15.
    Zhang Y, Wang ZY, He XP et al (2008) New industrial brewing yeast strains with ILV2 disruption and LSD1 expression. Int J Food Microbiol 123(1−2):18–24CrossRefGoogle Scholar
  16. 16.
    Saerens SMG, Verbelen P, Vanbeneden N et al (2008) Monitoring the influence of high-gravity brewing and fermentation temperature on flavour formation by analysis of gene expression levels in brewing yeast. Appl Microbiol Biotechnol 80(6):1039–1051CrossRefGoogle Scholar
  17. 17.
    Wang JJ, Xiu PH, He XP et al (2010) Construction of amylolytic industrial brewing yeast strain with high glutathione content for manufacturing beer with improved anti-staling capability and flavor. J Microbiol Biotechnol 20(11):1539–1545CrossRefGoogle Scholar
  18. 18.
    Wang D, Wang Z, Liu N et al (2008) Genetic modification of industrial yeast strains to obtain controllable NewFlo flocculation property and lower diacetyl production. Biotechnol Lett 30(11):2013–2018CrossRefGoogle Scholar
  19. 19.
    Liu ZR, Zhang GY, Li J et al (2007) Stable expression of glucoamylase gene in industrial strain of Saccharomyces pastorianus with less diacetyl produced. Ann Microbiol 57(2):233–237CrossRefGoogle Scholar
  20. 20.
    Liu ZR, Zhang GY, Liu SG (2004) Constructing an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing. J Biosci Bioeng 98(6):414–419Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China

Personalised recommendations