Physiological and morphological effects of genetic alterations leading to a reduced synthesis of UDP-glucose in Saccharomyces cerevisiae

Authors

  • Jean Marc Daran,

    1. Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, Laboratoire associé INRA, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex 04, France
    Search for more papers by this author
    • 1

      Manchester Biotechnology Centre, UMIST, P.O. Box 88, Manchester, UK.

  • Walter Bell,

    1. Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, Laboratoire associé INRA, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex 04, France
    Search for more papers by this author
  • Jean François

    Corresponding author
    1. Centre de Bioingenierie Gilbert Durand, UMR-CNRS 5504, Laboratoire associé INRA, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex 04, France
    Search for more papers by this author

Corresponding author. Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, Complexe Scientifique de Rangueil, 31077 Toulouse Cedex 04, France. Tel.: +33 (5) 61559492; Fax: +33 (5) 61559400; E-mail: fran-jm@insatlse.insa-tlse.fr

Abstract

Yeast cells lacking UDP-Glc pyrophosphorylase (UGPase) encoded by UGP1 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 GAL10/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 β-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.

Ancillary