Prevention of HXT1 endocytosis by ROD1 deletion in Saccharomyces cerevisiae for co-fermentation of glucose and xylose

Abstract

For economical production of bioethanol, it is important to develop the strains consuming a mixture of sugars from biomass efficiently. Saccharomyces cerevisiae, the traditional strain producing bioethanol, has many advantages such as high ethanol production ability and high tolerance to ethanol. However, there are several barriers in using lignocellulosic biomass. First, cofactors for xylose metabolic enzymes are imbalanced between xylose reductase (NADPH) and xylitol dehydrogenase (NADH). Second, glucose and xylose, the major sugars of lignocellulosic biomass, share common hexose trasnporters. Finally, there are regulatory systems in S. cerevisiae such as catabolite repression and catabolite inactivation. Above all, the catabolite inactivation was studied in this thesis. It is already known that ROD1, an arrestin-like protein, mediates endocytosis of catabolite inactivation by many researchers. It was reported that the knock-out of the ROD1 gene of S. cerevisiae prevented catabolite inactivation, hence elevated both sugar consumption and final ethanol concentration in glucose and xylose co-fermentation. However, it is still not understood how prevention of catabolite inactivation by ROD1 deletion increased sugar consumption and what transporters are connected. In addition, research efforts on catabolite inactivation have been made in the media containing glucose only. Therefore, it needs to be considered whether the endocytosis occurs in xylose containing media or not, because the ethanol fermentation is conducted in a mixture of glucose and xylose. It was explored what transporters are related with ROD1 by tagging several hexose transporters with EGFP and by using fluorescence microscopy and confocal laser scanning microscopy. Consequently, it was discovered that the HXT1 protein is a target transporter of ROD1-mediated endocytosis. Also, the internalization of hexose transporters happened in the media containing not only glucose but xylose. Furthermore, it was also confirmed whether the prevention of HXT1 endocytosis targeted by ROD1 deletion affects sugar consumption. As a result, the glucose consumption rate of the ROD1 deleted strain was 14.1% higher than that of the wild-type strain in complex medium. Moreover, in minimal medium, the glucose consumption rate of the ROD1 deleted strain increased by 73.7% compared with the wild-type strain.