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 Δmep strain was dependent on the extracellular NH₃ concentrations. Metabolomic analysis revealed a significantly higher intracellular NH_X concentration (3.3-fold) in the Δmep 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 Δmep strain than in the reference strain, which suggests that the lower biomass yield of the Δmep strain was related to higher turnover rates of biomass components.

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Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the in vivo intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we were able to measure the intracellular ammonium concentration in N-limited aerobic chemostat cultivations using three different N sources (ammonium, urea, and glutamate) at the same growth rate (0.05 h-1). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH4 +/literIC, is required to supply the reactions in the central N metabolism, independent of the N source. Based on the experimental results and different assumptions, the vacuolar and cytosolic ammonium concentrations were estimated. Furthermore, we identified a futile cycle caused by NH3 leakage into the extracellular space, which can cost up to 30% of the ATP production of the cell under N-limited conditions, and a futile redox cycle between Gdh1 and Gdh2 reactions. Finally, using shotgun proteomics with protein expression determined relative to a labeled reference, differences between the various environmental conditions were identified and correlated with previously identified N compound-sensing mechanisms.@en

Ammonium (NH4+) is the most common N-source for yeast fermentations, and N-limitation is frequently applied to reduce growth and increase product yields. While there is significant molecular knowledge on NH4 + transport and assimilation, there have been few attempts to measure the in vivo concentration of this metabolite. In this article, we present a sensitive and accurate analytical method to quantify the in vivo intracellular ammonium concentration in Saccharomyces cerevisiae based on standard rapid sampling and metabolomics techniques. The method validation experiments required the development of a proper sample processing protocol to minimize ammonium production/consumption during biomass extraction by assessing the impact of amino acid degradation—an element that is often overlooked. The resulting cold chloroform metabolite extraction method, together with quantification using ultra high performance liquid chromatography-isotope dilution mass spectrometry (UHPLC-IDMS), was not only more sensitive than most of the existing methods but also more accurate than methods that use electrodes, enzymatic reactions, or boiling water or boiling ethanol biomass extraction because it minimized ammonium consumption/production during sampling processing and interference from other metabolites in the quantification of intracellular ammonium. Finally, our validation experiments showed that other metabolites such as pyruvate or 2-oxoglutarate (KG) need to be extracted with cold chloroform to avoid measurements being biased by the degradation of other metabolites (e.g., amino acids).@en

The field of metabolic engineering has delivered new microbial cell factories and processes for the production of different compounds including biofuels, (di)carboxylic acids, alcohols, and amino acids. Most of these processes are aerobic, with few exceptions (e.g., alcoholic fermentation), and attention is focused on assembling a high-flux product pathway with a production limit usually set by the oxygen transfer rate. By contrast, anaerobic product synthesis offers significant benefits compared to aerobic systems: higher yields, less heat generation, reduced biomass production, and lower mechanical energy input, which can significantly reduce production costs. Using simple thermodynamic calculations, we demonstrate that many products can theoretically be produced under anaerobic conditions using several conventional and non-conventional substrates.

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