Multiple pathways for glutamate biosynthesis in Saccharomyces cerevisiae and Kluyveromyces lactis: genetic and physiological characterization

DELUNA, A.; AVENDAÑO, A.; RIEGO, L.; ROMERO, M.; GONZALEZ, A.

Rimini, Italy.

Congreso; XIX International Conference on Yeast Genetics and Molecular Biology; 1999

Federation of European Microbiological Societies

Two pathways for ammonium assimilation and glutamate biosynthesis have been described in yeast. The first one is mediated by NADP-dependent glutamate dehydrogenase (GDH). An alternative pathway comprises a cycle between glutamine synthetase and glutamate synthase (GOGAT). We have characterized these pathways in Saccharomyces cerevisiae which has duplicated the GDH pathway, posessing two GDH isozymes- and our results suggest that each of the three genes involved in glutamate biosynthesis plays a particular physiological role. We have characterized mutants impaired in GDH and GOGAT enzymes, encoded by GDH1, GDH3, and GLT1, respectively. Only the triple mutant is a glutamate auxotroph. When cells were grown on glucose, only the Δgdh1 mutant showed a decreased growth rate phenotype compared with a wild type strain; however, both Δgdh1 or Δgdh3 strains had a slower duplication time when grown on ethanol. In order to study the expression of GDH1, GDH3 and GLT1 under different growth conditions, the promoter region of these genes were fused in frame with the Lac-Z gene. Our results indicate that each gene is differentially expresse. When cells were grown on minimal medium suplemented with glucose, GDH1 expression was 25-fold higher than that of GDH3, in contrast to growth on ethanol where both genes were highly expressed. The expression of each gene as studied in different nitrogen sources. GLT1 and GDH3, but not GDH1, were negatively modulated by-glutamate. Both GDH isozymes were purified in order to study their kinetical and biochemical properties. GDH1 showed a higher affinity for α-ketoglutarate and GDH3 was more sensitive to both inorganic phosphate inhibition and thermal inactivation. Both isozymes are strongly inhibited by citrate and to a lesser extent by malate. On the other hand, GDH1 product was susceptible to proteolysis during carbon starvation while that of GDH3 was not. The petite negative yeast Kfuyveromyces lactis represents a different situation, as single mutants in either GDH or GOGAT gene present no significant phenotype. In this case, the double mutant is a glutamate auxotroph, suggesting there are only two enzymes involved in glutamate biosynthesis in this phylogenetic related organism.