O-8
Application of shotgun DNA microarray technology to gene expression analysis in lager yeast
Presenter: Naoyuki Kobayashi, Sapporo Breweries Ltd., Yaizu, Shizuoka, Japan
Co-Authors: M. Sato, S. Fukuhara, S. Yokoi, and M. Takashio, Sapporo Breweries Ltd., Shizuoka, Japan; and T. Yokoi, Y. Kaku, and T. Saito, Life Science Group, Hitachi Ltd., Saitama, Japan

For organisms with fully sequenced genomes, DNA microarrays are a powerful technology for measuring the mRNA expression response of almost every gene. This technology has been applied to the gene expression analyses of several species, including brewing yeast, during the past few years. Lager yeast (bottom-fermenting yeast) Saccharomyces pastorianus is known to be a polyploid consisting of the chromosomes from Saccharomyces cerevisiae and Saccharomyces bayanus. All the ORFs obtained from the complete genomes of S. pastorianus enable us to accomplish a transcriptomic analysis. However, this strategy requires us to undertake expensive and time-consuming genome sequencing projects to study the function of just a particular set of genes. We applied a shotgun DNA microarray technology to the gene-expression analysis in lager yeast for the first time, bypassing the need for previous knowledge of the genome sequence. The technology consists of the following steps: (i) A random genomic library from a lager yeast strain Weihenstephan 34/70, widely used for beer production, was constructed. (ii) About 20,000 clones from the genomic library were spotted onto a glass chip. (iii) The gene expression analysis was carried out with total RNA extracted from lager yeast strains for a series of test brewings. (iv) Relevant spots on the shotgun DNA microarray showing large differences in the gene expression after competitive hybridization were sequenced. Moreover, to study the relationship between the results obtained from the gene expression analysis and the important flavor produced by these yeast strains during fermentation, more than 80 flavor-active compounds in the fermented and finished beers were analyzed by gas chromatography. Several flavor-active compounds that highly correlated with the sensory evaluation of the finished beer were selected. The relevant genes responsible for producing these compounds were determined from the gene-expression analysis using the shotgun DNA microarray. This approach allowed us to select the important genes in the lager yeast strains responsible for the important flavors in finished beer. In the present paper, we describe the integration of transcriptome (gene-expression analysis) and metabolome (flavor-active compounds) which involve the important flavor produced by the lager yeast strains during fermentation, indicating that our strategy using shotgun DNA microarray technology was a very useful technique to accomplish gene-expression studies with lager yeast strains that have a complicated genetic background.

Naoyuki Kobayashi graduated from the Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo. In 1991, He began to work as a research scientist in the Brewing Research Laboratories, Sapporo Breweries, Ltd. In March 1998, he received a Ph.D. in analytical chemistry from The University of Tokyo. From 2002 to 2004, he studied as a research fellow at the Laboratory for Protein Analytics in Max-Planck-Institute of Biochemistry, Martinsried, Germany. Since June 2004, he has worked as lead biochemist in system solution, Frontier Laboratories of Value Creation, Sapporo Breweries, Ltd. In January 2005, he started to serve the Brewery Convention of Japan (BCOJ) on its Program Committee as a vice-chairman.