Background: Microbial production of nitrogen containing compounds requires a high uptake flux and assimilation of the N-source (commonly ammonium), which is generally coupled with ATP consumption and negatively influences the product yield. In the industrial workhorse Saccharomyces cerevisiae, ammonium (NH₄⁺) uptake is facilitated by ammonium permeases (Mep1, Mep2 and Mep3), which transport the NH₄⁺ ion, resulting in ATP expenditure to maintain the intracellular charge balance and pH by proton export using the plasma membrane-bound H⁺-ATPase.

Results: To decrease the ATP costs for nitrogen assimilation, the Mep genes were removed, resulting in a strain unable to uptake the NH₄⁺ ion. Subsequent analysis revealed that growth of this ∆mep strain was dependent on the extracellular NH₃ concentrations. Metabolomic analysis revealed a significantly higher intracellular NH_X concentration (3.3-fold) in the ∆mep strain than in the reference strain. Further proteomic analysis revealed significant up-regulation of vacuolar proteases and genes involved in various stress responses.

Conclusions: Our results suggest that the uncharged species, NH₃, is able to diffuse into the cell. The measured intracellular/extracellular NH_X ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NH_x compartmentalization was considered. On the other hand, proteomic analysis indicated a more pronounced N-starvation stress response in the ∆mep strain than in the reference strain, which suggests that the lower biomass yield of the ∆mep strain was related to higher turnover rates of biomass components.

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Journal Article

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Cueto-Rojas HF, Milne N, van Helmond W, Pieterse MM, van Maris AJA, Daran JM, Wahl SA

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