O-10
Improving maltotriose fermentation by Saccharomyces cerevisiae
Presenter: Boris Stambuk, Departamento de Bioquimica, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900, Brazil
Co-Authors: Claudia Hollatz, Sergio Alves-Jr., Ricardo Herberts, and Luiz Miletti, Departamento de Bioquimica, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900, Brazil

Although maltotriose is the second most abundant sugar of brewer's wort, it has the lowest priority for uptake by yeast cells. In general, only when half of the wort glucose has been taken up by the yeast will the uptake of maltose and maltotriose commence, with a slower uptake rate for maltotriose than for maltose. This slower, and sometimes incomplete, utilization of maltotriose leads to one of the problems experienced by some breweries, namely a high content of fermentable sugars in the finished beer, and atypical beer flavor profiles. The rate of uptake and fermentation of maltotriose during wort fermentation is, therefore, one of the major determinants of fermentation efficiency and product quality. In order to gain insights into the molecular basis of such slow and delayed utilization of maltotriose by S. cerevisiae, we have analyzed the rates of active maltose and maltotriose transport and utilization by industrial yeast strains and strains with defined genotypes, deleted or not in specific transporters. Although there are conflicting reports on whether these two sugars share common transporters, maltases and/or regulatory mechanisms, our results clearly show that maltose fermentation is uncoupled from maltotriose fermentation by yeast cells. While all strains analyzed fermented maltose, distinct phenotypes for maltotriose utilization were found: some industrial and laboratory strains fermented maltotriose efficiently, and other strains utilized this sugar without fermenting it into ethanol. Although all maltotriose fermenting strains were MAL constitutive, transformation of several maltotriose non-fermenting strains with a constitutive MAL63(^c) gene did not improve ethanol production from maltotriose. Fermentation of this sugar was impaired when the AGT1 permease was deleted from the genome of yeast cells, while maltose fermentation was unaffected. This AGT1 permease has a lower affinity for maltotriose active transport (K(m) ~20 mM), when compared with the affinity of the maltose permeases (e.g., MAL61, MAL21) for maltose uptake (K(m) ~4 mM). Furthermore, our results indicate that maltotriose is a poor inducer of this permease in several yeast strains. The determinant role of this permease was confirmed by transforming an industrial strain that utilizes but doesn't ferment maltotriose with the AGT1 permease expressed from a strong and constitutive promoter: this modified yeast gained the ability to ferment maltotriose efficiently. Thus, our results demonstrate that the enhanced and constitutive expression of the low-affinity maltotriose permease encoded by the AGT1 gene is required for efficient maltotriose fermentation by S. cerevisiae cells.

Boris Stambuk received a B.Sc. degree in biological sciences from Universidade de São Paulo, completed a M.Sc. degree in microbiology at Escola Paulista de Medicina, and holds a Ph.D. degree in biochemistry from Universidade de São Paulo, Brazil. In 1994 he move to Universidade Federal de Santa Catarina in Florianopolis, Brazil, where became a lecturer and senior lecturer in biochemistry. Since 1997, Boris has held a research fellowship in applied microbiology from CNPq (Brazil) investigating yeast physiology, sugar fermentation, and biotechnology of industrial yeasts. In 2001, he also performed studies on pentose fermentation by yeasts as senior research associate at the National Renewable Energy Laboratory in Golden, CO.