YEAST VOL. zyxwvutsrqpo 11: zyxwvutsr 4 0 7 4 8 (1995) Regulation of Carbon Metabolism in Chemostat Cultures of zyxwv Saccharomyces cerevisiae Grown on Mixtures of Glucose and Ethanol PATRICIA DE JONG-GUBBELS, PETER VANROLLEGHEMI, SEF HEIJNENI, JOHANNES P. VAN DIJKEN AND JACK T. PRONK* Department of Microbiology and Enzymology and ?Department of Bioprocess Engineering, Kluyver Laboratory of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands Received 2 September 1994; accepted 6 December 1994 Growth efficiency and regulation of key enzyme activities were studied in carbon- and energy-limited chemostat cultures of Saccharomyces cerevisiae grown on mixtures of glucose and ethanol at a fixed dilution rate. Biomass yields on substrate carbon and oxygen could be adequately described as the net result of growth on the single substrates. Activities of isocitrate lyase and malate synthase were not detected in cell-free extracts of glucose-limited cultures. However, both enzymes were present when the ethanol fraction in the reservoir medium exceeded the theoretical minimum above which the glyoxylate cycle is required for anabolic reactions. Fructose-1,6- bisphosphatase activity was only detectable at high ethanol fractions in the feed, when activity of this enzyme was required for synthesis of hexose phosphates. zyxwvu Phospho-enol-pyruvate-carboxykinase activity was not detectable in extracts from glucose-grown cultures and increased with the ethanol fraction in the feed. It is concluded that, during carbon-limited growth of zyxwvutsrq S. cerevisiae on mixtures of glucose and ethanol, biosynthetic intermediates with three or more carbon atoms are preferentially synthesized from glucose. Synthesis of the key enzymes of gluconeogenesis and the glyoxylate cycle is adapted to the cells’ requirement for these intermediates. The gluconeogenic enzymes and their physiological antagonists (pyruvate kinase, pyruvate carboxylase and phosphofructokinase) were expressed simul- taneously at high ethanol fractions in the feed. If futile cycling is prevented under these conditions, this is not primarily achieved by tight control of enzyme synthesis. KEY WORDS zyxwvutsrqponm - chemostat; mixed substrates; gluconeogenesis; glyoxylate cycle; Saccharomyces cerevisiae INTRODUCTION Growth of Saccharomyces cerevisiae in batch cul- tures on glucose follows a characteristic pattern. Glucose is initially mainly fermented to ethanol, which, in a separate second growth phase, serves as a carbon and energy source (Fiechter et al., 1981). Adaptation to growth on ethanol involves the induction of a number of key enzyme activities, including those of the glyoxylate cycle (isocitrate lyase and malate synthase) and gluconeogenesis (phospho-enol-pyruvate (PEP)-carboxykinase and fructose- 1,6-bisphosphatase (FBPase); Haarasilta and Oura, 1975; situations I and VI in Figure 1). In S cerevisiae, activity of the key enzymes of the glyoxylate cycle and gluconeogenesis is tightly *Corresponding author CCC 0749-503X/95/050407-12 ( 1995 by John Wiley & Sons Ltd regulated (Wills, 1990). In particular, the regu- lation of FBPase has been extensively studied. Activity of this enzyme is subject to a multilayered regulation, which suggests that different mech- anisms have evolved to avoid the operation of a futile cycle caused by the simultaneous activity of FBPase and the glycolytic enzyme phospho- fructokinase (Navas et al., 1993). In zyx S. cerevisiae, synthesis of FBPase is strongly repressed by glucose (Gancedo et al., 1967). Furthermore, the enzyme is rapidly inactivated when glucose is added to ethanol-grown cultures (Lenz and Holzer, 1980). Catabolite inactivation by excess glucose has also been demonstrated for PEP- carboxykinase (Gancedo and Schwerzmann. 1976) and isocitrate lyase (Lopez-Boado et al., 1987). In addition to regulation of gene expression and