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 UGPI are not viable. Two strategies were developed to drastically reduce the intracellular concentration of UDP-Glc in order to study the consequences of this metabolic engineering on physiology and morphology. Firstly, UGP1 was placed under the strongly regulatable THI4 promoter. This resulted in a 95% reduction of UGPase activity in the presence of thiamine. The phenotypic effects of this reduction were slightly stronger than those of glucose on the GALI0/CYC1-UGP1 gene fusion [Daran et al. (1995) Eur. J. Biochem. 230, 520-530]. A further reduction of flux towards UDP-Glc was achieved by deletion of the two phosphoglucomutase genes in the ugp1 conditional strain. The growth of this new mutant strain was hardly affected, while it was extremely sensitive to cell wall interfering drugs. Surprisingly, UDP-Glc levels were reduced only by 5-fold, causing a proportional decrease in both glycogen and beta-glucans. Taken altogether, these results indicate that a few percent of enzymatic activities leading to the formation of UDP-Glc appears sufficient to provide the UDP-Glc demands required for cell viability, and that the loss of function of UGP1 is lethal mainly because of the inability of yeast cells to properly form the cell wall.
Daran
J M
JM
Centre de Bioingenierie Gilberi Durand, UMR-CNRS 5504, Laboratoire associé INRA, Toulouse, France.
Bell
W
W
François
J
J
D002473:Cell Wall; D009154:Mutation; D010733:Phosphoglucomutase / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013831:Thiamine / Q000494:pharmacology; D014199:Trehalose / Q000096:biosynthesis; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000096:biosynthesis*
D011993:Recombinant Fusion Proteins; D014199:Trehalose; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D010733:Phosphoglucomutase; D014532:Uridine Diphosphate Glucose; D013831:Thiamine