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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Haukeli A, Lie S (1978) Conversion of alpha-acetolatate and removal of diacetyl a kinatic study. J Inst Brew 84:85–89
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):638–647
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):1206–1215
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):989–994
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):175–183
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):32–37
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):360–370
Dunn B, Sherlock G (2008) Reconstruction of the genome origins and evolution of the hybrid lager yeast Saccharomyces pastorianus. Genome Res 18(10):1610–1623
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):20–26
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):2519–2524
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed., vol. I. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Burke D, Dawson DS, Stearns T (2000) Methods in yeast genetics: A Cold Spring Harbor laboratory course manual. CSHL, Press, USA
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):322–328
Daniel Gietz R, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96
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):18–24
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):1039–1051
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):1539–1545
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):2013–2018
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):233–237
Liu ZR, Zhang GY, Liu SG (2004) Constructing an amylolytic brewing yeast Saccharomyces pastorianus suitable for accelerated brewing. J Biosci Bioeng 98(6):414–419
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer-Verlag Berlin Heidelberg
About this paper
Cite this paper
Lu, J., Chen, Y., Wu, D., Xiao, D. (2014). Application of the Cre-loxP Recombination System for Two ILV2 Alleles Disruption in an Industrial Brewer’s Yeast Strain. In: Zhang, TC., Ouyang, P., Kaplan, S., Skarnes, B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 249. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37916-1_3
Download citation
DOI: https://doi.org/10.1007/978-3-642-37916-1_3
Published:
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-37915-4
Online ISBN: 978-3-642-37916-1
eBook Packages: EngineeringEngineering (R0)