Variation inα-acetolactate production within the hybrid lager yeast groupSaccharomyces pastorianusand affirmation of the central role of theILV6gene

Abstract

A screen of 14 S. pastorianus lager-brewing strains showed as much as a nine-fold difference in wort total diacetyl concentration at equivalent stages of fermentation of 15°Plato brewer's wort. Two strains (A153 and W34), with relatively low and high diacetyl production, respectively, but which did not otherwise differ in fermentation performance, growth or flavour production, were selected for further investigation. Transcriptional analysis of key genes involved in valine biosynthesis showed differences between the two strains that were consistent with the differences in wort diacetyl concentration. In particular, the ILV6 gene, encoding a regulatory subunit of acetohydroxy acid synthase, showed early transcription (only 6 h after inoculation) and up to five-fold greater expression in W34 compared to A153. This earlier transcription was observed for both orthologues of ILV6 in the S. pastorianus hybrid (S. cerevisiae × S. eubayanus), although the S. cerevisiae form of ILV6 in W34 also showed a consistently higher transcript level throughout fermentation relative to the same gene in A153. Overexpression of either form of ILV6 (by placing it under the control of the PGK1 promoter) resulted in an identical two-fold increase in wort total diacetyl concentration relative to a control. The results confirm the role of the Ilv6 subunit in controlling α-acetolactate/diacetyl concentration and indicate no functional divergence between the two forms of Ilv6. The greater contribution of the S. cerevisiae ILV6 to acetolactate production in natural brewing yeast hybrids appears rather to be due to higher levels of transcription relative to the S. eubayanus form. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: Saccharomyces pastorianus; lager beer; diacetyl; ILV6; acetohydroxyacid synthase IntroductionDiacetyl or 2,3-butanedione is a vicinal diketone (VDK) formed during beer fermentation as well as other yeast fermentations (Inoue, 2008). It is typically perceived as a butter-or butterscotch-like flavour and has a low flavour threshold (20-100 ppb) (Krogerus and Gibson 2013a). In lager beer, which is characterized by a clean flavour profile, diacetyl is generally considered to be an off-flavour and considerable effort is taken to ensure that concentrations are lower than threshold levels before sale. Diacetyl is formed as an indirect by-product of valine biosynthesis in yeast, and the amount formed during fermentation varies greatly, depending on wort valine content and other parameters, such as the yeast strain used, the wort quality (e.g. free amino nitrogen content, initial oxygen content and pH) and the fermentation conditions (e.g. temperature, pressure and yeast pitching rate) (Garcia et al., 1994;Kobayashi et al., 2005;Pugh et al., 1997;Verbelen et al., 2008Verbelen et al., , 2009. The concentration of diacetyl in beer typically peaks during the first half of the fermentation and thereafter is lowered toward the end of the fermentation. Freshly fermented 'green' beer undergoes a time-and resource-consum...

Evolutionary Engineering in Chemostat Cultures for Improved Maltotriose Fermentation Kinetics in Saccharomyces pastorianus Lager Brewing Yeast

“…The catalytic subunit is encoded by ILV2 and the regulatory subunit encoded by ILV6 a gene located on CHRIII. Although the ILV6 copy number has been associated with variation in diacetyl production in S. pastorianus , a reduction in copy number was associated with lower diacetyl (Duong et al, 2011 ) while overexpression of either of the ILV6 alleles led to increased diacetyl formation (Gibson et al, 2015 ). These results would disagree with the results of this study which would associate a doubling of SeILV6 copy number with a reduction of diacetyl.…”

Novel brewing yeast hybrids: creation and application

“…Proteome and transcriptome studies have, however, shown that significant differences in subgenome activity can occur in lager yeast (Caesar et al 2007; Horinouchi et al 2010; Minato et al 2009; Yoshida et al 2007), suggesting that gene regulation in interspecies hybrids is not seamlessly integrated across subgenomes. Gibson et al (2015) showed that differences in expression of the two different alleles of the regulatory gene ILV6 in S. pastorianus could influence strain phenotypes (in this case, the production of α-acetolactate). Otherwise, there was no functional difference between the gene products as determined by their over-expression with the same promoter.…”

Aroma formation by immobilized yeast cells in fermentation processes

“…These acids are intermediates in the synthesis of valine and isoleucine, respectively. The amounts and profile of α ‐acetohydroxyacids produced during fermentation are influenced by yeast strain, medium composition, and fermentation conditions (Dufour et al, ; Brányik et al, ; Gibson et al, ). It is also suggested that VDK formation is linked to amino acid metabolism.…”