Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase

Abstract

In Saccharomyces cerevisiae, the glutamate dehydrogenase (GDH) enzymes play a pivotal role in glutamate biosynthesis and nitrogen assimilation. It has been proposed that, in GDH-deficient yeast, either the proline utilization (PUT) or the glutamine synthetase-glutamate synthase (GS/GOGAT) pathway serves as the alternative pathway for glutamate production and nitrogen assimilation to the exclusion of the other. Using a gdh-null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway-specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). The GDH-null mutant was viable on all nitrogen sources tested, confirming that alternate pathways for nitrogen assimilation exist in the gdh-null strain. Enzyme assays point to GS/GOGAT as the primary alternative pathway on the preferred nitrogen sources ammonia and glutamine, whereas growth on proline required both the PUT and GS/GOGAT pathways. In contrast, growth on glucose-urea media elicited a decrease in GOGAT activity along with an increase in activity of the PUT pathway specific enzyme Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). Together, these results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH-dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serves as the sole compensatory pathway.

Glutamate dehydrogenases in the oleaginous yeast Yarrowia lipolytica

“…But, when ammonia nitrogen is replaced with glutamate nitrogen (glucose/glutamate or glutamate/glutamate), ylGdh1p activity is significantly lowered (Figure 4b), suggesting that glutamate is a negative regulator of (Freese et al, 2011;Riego et al, 2002). Despite this regulatory difference, such as S. cerevisiae lacking scGDH1 (Miller, S. M. & Magasanik, 1990;Sieg & Trotter, 2014), the loss of ylGDH1 causes impaired growth on glucose/ammonia synthetic medium (Figure 5a), supporting a role in nitrogen assimilation.…”

“…The only partial effect on glucose/ammonia growth also suggests that, like S. cerevisiae , Y. lipolytica possesses other pathways for the glutamate synthesis and/or nitrogen assimilation, perhaps via proline or glutamine metabolism (Sieg & Trotter, ). The observation that Y. lipolytica is capable of growth in medium containing glutamate (Figure c), aspartate, or proline as sole carbon and nitrogen sources (Freese et al, ) is an indication of alternative modes of nitrogen assimilation in this organism.…”

“…GDHs have been well studied in the yeast Saccharomyces cerevisiae, which possesses three GDH-encoding genes ( Figure 1). Two NADP-dependent GDHs (NADP-GDHs) are encoded by scGDH1 and scGDH3 and catalyse the synthesis of glutamate (Avendano, Deluna, Olivera, Valenzuela, & Gonzalez, 1997;DeLuna, Avendano, Riego, & Gonzalez, 2001;Sieg & Trotter, 2014). The NAD-dependent GDH (NAD-GDH) encoded by scGDH2 degrades glutamate, producing α-ketoglutarate (Coschigano, Miller, & Magasanik, 1991;Miller & Magasanik, 1990).…”

The pleiotropic effects of the glutamate dehydrogenase (GDH) pathway in Saccharomyces cerevisiae

“…GS–GOGAT pathway in yeast has a marginal contribution in glutamate synthesis both in fermentation and respiratory conditions, while the GDH pathway has the prominent role [ 5 , 6 ]. Yeast strains lacking the GDH route, present a proline utilization pathway (PUT) that can contribute together with the GS–GOGAT in glutamate biosynthesis and nitrogen assimilation [ 7 ]. The relative contribution of PUT is being dictated by the nitrogen sources [ 7 ].…”

“…Yeast strains lacking the GDH route, present a proline utilization pathway (PUT) that can contribute together with the GS–GOGAT in glutamate biosynthesis and nitrogen assimilation [ 7 ]. The relative contribution of PUT is being dictated by the nitrogen sources [ 7 ]. To our knowledge, S. cerevisiae is the only organism having three pathways for glutamate synthesis (Fig.…”