12. Self-cloning brewing yeast—New dimension of beer fermentation?

Susann Fischer (1), Thomas Becker (1); (1) Chair of Brewing and Beverage Technology, Technische Universität München, Freising, Germany

Technical Session 3: Yeast Biotechnology
Sunday, August 14  •  9:45–11:30 a.m.
Plaza Building, Concourse Level, Governor’s Square 15

Yeast is the primary microorganism used for fermented beverages such as beer. However, existing individual strains will not completely fulfill future demands for an efficient and high-quality fermentation. In this case, several research groups are focusing on the evaluation of wild Saccharomyces strains or non-Saccharomyces strains to enhance fermentation ability under industrial conditions. Another interesting possibility for targeted optimization of brewing yeast could be the self-cloning procedure. Self-cloning does not result in a GMO, due to the usage of homologous nucleic acid sequences. However, to avoid metabolic burden through constitutive gene expression and translation, induced gene expression of relevant genes is necessary for industrial fermentation. Induced gene expression with common inducible expression systems or equivalent inductors such as galactose and copper are prohibited for the production of foods and beverages. Therefore, temperature- and ethanol-induced gene expression under brewing conditions is the main focus of our research group. The industrial yeast S. cerevisiae is adapted to different stressors such as a high concentration of ethanol, osmotic pressure, limitation in nutrition and temperature shifts. This temperature shift leads to a modification of specific genetic regulation. The evaluation of 10 different homologue promoters of the lager yeast TUM 34/70 showed strong expression under temperature shifts from 12°C to 4°C and weak induction under varied ethanol concentrations at 12°C of 3 promoters. Further, the steady-state expression of these stress-relevant promoters is nearly constant during fermentation. Besides promoter screening, the ability of the evaluated promoters to enhance the content of volatile metabolites during the transition from fermentation to maturation under brewing conditions are the main aspects of this study. Therefore, self-cloning brewing yeast was created by assembling a homologue gene cassette containing the evaluated promoter, the target genes ATF1 or GPD1 and the homologous selection marker SMR1. The gene ATF1 encodes for the alcohol-acetyltransferase that is responsible for the formation of several volatile acetate esters such as ethyl acetate and isoamyl acetate during fermentation. GPD1 encodes for glycerol-3-phosphate dehydrogenase and is the key enzyme in glycerol synthesis. The gene cassette was inserted into the URA3 locus of the lager yeast TUM 34/70. Fermentations were carried out by use of synthetic wort (12°P) at 12°C in 2 L EBC tubes. In addition to biomass, extract decrease and ethanol production volatile metabolites and glycerol were analyzed. Temperature shift from 12°C to 4°C was carried out after 144 hr of fermentation and the increase of volatile metabolites and glycerol was observed during 72 hr.

After graduation from an apprenticeship as a winemaker, Susann Fischer started the study of enology (Dipl.-Ing. (FH)) at the University of Applied Science Wiesbaden-Geisenheim (Germany). In 2012 she graduated from Justus-Liebig University Gießen (Germany) with an M.S. degree in wine technology, with a focus on the genetic background of volatile metabolite synthesis of the non-Saccharomyces yeast Hanseniaspora uvarum. Susann is currently working toward a Ph.D. degree at the Technische Universität München (Germany), with a research focus on temperature-induced gene expression systems for self-cloning brewing yeast.