PHYSIOLOGICAL AND MORPHOLOGICAL EFFECTS OF GENETIC ALTERATIONS LEADING TO A REDUCED SYNTHESIS OF UDP-GLUCOSE IN SACCHAROMYCES-CEREVISIAE

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGP1 are not viable. Two strategies were developed to drastically reduce t he intracellular concentration of UDP-Glc in order to study the conseq uences of this metabolic engineering on physiology and morphology. Fir stly, UGP1 was placed under the strongly regulatable TH14 promoter. Th is resulted in a 95% reduction of UGPase activity in the presence of t hiamine. The phenotypic effects of this reduction were slightly strong er than those of glucose on the GAL10/CYC1-UGP1 gene fusion [Daran et al. (1995) fur. J. Biochem. 230, 520-530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutas e genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell w all interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and beta-g lucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears suff icient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the ina bility of yeast cells to properly form the cell wall.