Knockout Of ECM25 Gene In Brewing Yeast And Its Effects On Beer Flavor Stability

Flavor stability is a key attribute of beer and its staling during storage after packaging has long been a concern to brewers. Industrial brewing yeast is soul of beer production and it is one of the decisive factors on beer flavor and flavor stability. Beer is a complex system, several types of reactions have been thought to be responsible for the development of the various aged characters during storage of beer. However, industrial brewing yeast can synthesize and excrete GSH during fermentation. It can react with free radical, control the content of pre-staling compounds and flavor staling compounds, improve beer flavor stability during storage. Ecm25 gene was located in chromosome X of S. cerevisiae, knockout of ECM25 gene in S. cerevisiae could influence the transport pathway, and then could change GSH homeostasis and increase the extracellular GSH content.A primary aim of this research was to knockout of ECM25 gene in industrial brewing yeast by PCR-mediated gene disruption. We obtained a deficient transformant G-03/a based on homologous recombination with KanMX gene, and the transformant was verified to be genetically stable. The PCR analysis showed that ECM25 gene in the G-03/a was deleted. A second aim was to investigate the properties of the transformant during flask and EBC fermentation including its fermentation routine parameters, fermentation major staling parameters, the intracellular and extracellular GSH content, etc.Under aerobic conditions of 11℃and 28℃, compared with the host strain G-03, the excretive glutathione concentration of G-03/a increased by 21.4% (P<0.01) and 14.7% (P<0.01), respectively. Strains G-03 and G-03/a were inoculated in flasks and cultivated continuously for 3 generations. Compared with the host strain G-03, the glutathione concentration in the main fermentation broth and final beer of strain G-03/a increased by 32.1% (P<0.05) and 13.8% (P<0.05), the stability index (SI) increased by 7.7% (P<0.05) and 5.3% (P<0.05), respectively, and the flavor resistance staling value (RSV value) in final beer increased by 45.0% (P<0.01). During EBC fermentation, the glutathione concentration in the main fermentation broth of strain G-03/a increased by 34.0% (P<0.01), compared with the host strain G-03. Furthermore, no significant difference in routine fermentation parameters was found (P>0.05). Flavor test of beer demonstrated that the beer flavor was generally unchanged. The strain G-03/a is proved to be an excellent anti-staling brewing yeast to improve beer flavor stability.In this research, the deletion mutant, G03/a, was constructed by replacing the ECM25 gene with the KanMX gene. The KanMX gene must be knocked out because it is a foreign gene to the host strain, and then the transformant can be used in the brewery.