Technical Session 16: Yeast III Session
KEI ASADA, SAPPORO BREWERIES LTD., Yaizu, Shizuoka, Japan
Co-author(s): Ryouichi Fukuda and Akinori Ohta, University of Tokyo, Tokyo, Japan; Masahide Sato and Tatsuro Shigyo, Sapporo Breweries Ltd., Yaizu, Japan
ABSTRACT: The brewer’s yeast genome encodes a “Flo” flocculin family responsible for flocculation. Controlled floc formation or flocculation at the end of fermentation is of great importance in the brewing industry since it is a cost-effective and environmentally friendly technique for separating yeast cells from the final beer. Yeast flocculation is a very complex process that depends on the expression of specific flocculation genes such as FLO1, FLO5, FLO8, FLO11, and Lg-FLO1. Among these genes, Lg-FLO1 is the most effective gene for brewing beer because Lg-Flo1 protein recognizes not only mannose but also glucose and maltose, and it contributes to flocculation at the end of fermentation. For this reason, brewers want to understand the behavior of Lg-Flo1 protein during fermentation. In this study, we report the localization of Lg-Flo1 protein during the propagation of genetically modified bottom fermenting yeasts (Saccharomyces pastorianus W34/70). We used E. coli as a host for DNA cloning, and we subcloned the upper region of the Lg-FLO1 promoter, Lg-FLO1 promoter domain, and Lg-FLO1 N terminal domain. Subsequently, we ligated the upper region of the Lg-FLO1 promoter, drug resistance gene, Lg-FLO1 promoter domain, EGFP gene, and Lg-FLO1 N terminal domain in that order. We transformed this fragment into wild-type bottom fermenting yeast and screened the cells by drug resistance and obtained recombinant strain (EGFP-Lg-FLO1). We verified that this recombination occurred correctly in the specific location using the colony PCR method. Finally, we observed the localization of the protein with a fluorescence microscope. As a result, we found that the EGfp-Lg-Flo1 protein was localized in the cell wall.
Kei Asada received a master’s degree from the Graduate School of Biostudies, Kyoto University, Japan. He began employment with Sapporo Breweries Ltd. in 2008 as a microbiologist in the Frontier Laboratory of Value Creation. From April 2011 to March 2012, he studied the flocculation of bottom fermenting yeast as a researcher in the Department of Biotechnology, University of Tokyo.
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