Catabolite repression mutants of Saccharomyces cerevisiae show altered fermentative metabolism as well as cell cycle behavior in glucose-limited chemostat cultures

In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates (D) and respiro-fermentative metabolism at high D. We have studied the onset of ethanol production and cell cycle behavior in glucose-limited chemostat cultures of the wild type S. cerevisiae strain CEN.PK122 (WT) and isogenic mutants, snf1 (cat1) and snf4 (cat3) defective in proteins involved in catabolite derepression and the mutant in glucose repression mig1 (cat4). The triggering of fermentative metabolism was dependent upon catabolite repression properties of yeast and was coincident with a significant decrease of G1 length. WT cells of the strain CEN.PK122 displayed respiratory metabolism up to a D of 0.2 h^-1 and exhibited longer G1 lengths than the snf1 and snf4 mutants that started fermenting after a D of 0.1 and 0.15 h^{- 1}, respectively. The catabolite derepression mutant snf4 showed a significant decrease in the duration of G1 with respect to the WT. An increase of 300% to 400% in the expression of CDC28 (CDC28-lacZ) with a noticeable shortening in G1 to values lower than -150 min, was detected in the transformed wild type CEN.SC13-9B in glucose-limited chemostat cultures. The expression of CDC28-lacZ was analyzed in the wild type and isogenic mutant strains growing at maximal rate on glucose or in the presence of ethanol or glycerol. Two- to three-fold lower expression of the CDC28-lacZ fusion gene was detected in the snf1 or snf4 disruptants with respect to the WT and mig1 strains in the presence of all carbon sources. This effect was further shown to be growth rate-dependent exhibiting apparently, a threshold effect in the expression of the fusion gene with respect to the length of G1, similar to that shown in chemostat cultures. At the onset of fermentation, the control of the glycolytic flux was highly distributed between the uptake, hexokinase, and phosphofructokinase steps. Particularly interesting was the fact that the snf1 mutant exhibited the lowest fluxes of ethanol production, the highest of respiration and correspondingly, the branch to the tricarboxylic acid cycle was significantly rate-controling of glycolysis.