Research Article Microaerobic glycerol formation in Saccharomyces cerevisiae Roeland Costenoble 1{ , Hadi Valadi 2{ , Lena Gustafsson 2 , Claes Niklasson 1 and Carl Johan Franze Ân 1 * 1 Department of Chemical Reaction Engineering, Chalmers University of Technology, SE-412 96 Goteborg, Sweden 2 Department of Molecular Biotechnology, Chalmers University of Technology, SE-412 96 Goteborg, Sweden * Correspondence to: C. J. Franze Ân, Department of Chemical Reaction Engineering, Chalmers University of Technology, SE-412 96 Go Èteborg, Sweden. E-mail: franzen@cre.chalmers.se {These authors have contributed equally. Received: 15 March 2000 Accepted: 20 June 2000 Abstract The yeast Saccharomyces cerevisiae produces large amounts of glycerol as an osmoregulator during hyperosmotic stress and as a redox sink at low oxygen availability. NAD + -dependent glycerol-3-phosphate dehydrogenase in S. cerevisiae is present in two isoforms, coded for by two different genes, GPD1 and GPD2. Mutants for either one or both of these genes were investigated under carefully controlled static and dynamic conditions in continuous cultures at low oxygen transfer rates. Our results show that S. cerevisiae controls the production of glycerol in response to hypoxic conditions by regulating the expression of several genes. At high demand for NADH reoxidation, a strong induction was seen not only of the GPD2 gene, but also of GPP1, encoding one of the molecular forms of glycerol-3-phosphatase. Induction of the GPP1 gene appears to play a decisive role at elevated growth rates. At low demand for NADH reoxidation via glycerol formation, the GPD1, GPD2, GPP1, and GPP2 genes were all expressed at basal levels. The dynamics of the gene induction and the glycerol formation at low demand for NADH reoxidation point to an important role of the Gpd1p; deletion of the GPD1 gene strongly altered the expression patterns of the GPD2 and GPP1 genes under such conditions. Furthermore, our results indicate that GCY1 and DAK1, tentatively encoding glycerol dehydrogenase and dihydroxyacetone kinase, respectively, may be involved in the redox regulation of S. cerevisiae. Copyright # 2000 John Wiley & Sons, Ltd. Keywords: yeast; redox regulation; glycerol metabolism; dihydroxyacetone pathway; glycerol-3-phosphate dehydrogenase; glycerol-3-phosphatase; oxygen limitation Introduction Glycerol metabolism has proved to be of central importance in yeast metabolism, especially in media with low osmotic potential (Albertyn et al., 1994; Ansell et al., 1997; Blomberg and Adler, 1992; Nevoigt and Stahl, 1997) and under anaerobic conditions (Ansell et al., 1997; Bjo È rkqvist et al., 1997; Nordstro È m, 1966; Van Dijken and Scheffers, 1986). Under anaerobic conditions, glycerol is formed in order to reoxidize the NADH formed in anabolism and in the synthesis of organic acids (Albers et al., 1996; 1998; Holzer et al., 1963; Nordstro Èm, 1966; Oura, 1977; Van Dijken and Scheffers, 1986). Depending on the cultivation conditions, 4±10% of the carbon source may be converted to glycerol (Radler and Schutz, 1982). Glycerol may also be formed under aerobic condi- tions, but normally at much lower yields than under anaerobic conditions. An exception to this occurs in the presence of a low osmotic potential, in which case the glycerol acts as an osmoregulator and substantial amounts may be produced (Blomberg and Adler, 1992). The reactions surrounding the dihydroxyacetone phosphate node are illustrated in Figure 1. Glycerol is formed by the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate, and the subse- quent dephosphorylation of the glycerol-3-phos- phate. The ®rst reaction is catalysed by NAD + - dependent glycerol-3-phosphate dehydrogenase. Two genes encoding different molecular forms of this enzyme have been identi®ed. One isoform is induced by osmotic stress (Andre  et al., 1991) and is encoded by the GPD1 gene (Larsson et al., 1993). A second gene, called GPD2 and highly homologous Yeast Yeast 2000; 16: 1483±1495. Copyright # 2000 John Wiley & Sons, Ltd.