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.
