Cueto-Rojas H F, Maleki Seifar R, Ten Pierick A, van Helmond W, Pieterse M M, Heijnen J J, Wahl S A
Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, Delft, The Netherlands.
Appl Environ Microbiol. 2016 Dec 1;82(23):6831-6845. doi: 10.1128/AEM.01547-16. Epub 2016 Sep 16.
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 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). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH/liter, 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 NH 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. In our work, we studied central N metabolism using quantitative approaches. First, intracellular ammonium was measured under different N sources. The results suggest that cells maintain a constant NH concentration (around 3 mmol NH/liter), independent of the applied nitrogen source. We hypothesize that this amount of intracellular ammonium is required to obtain sufficient thermodynamic driving force. Furthermore, our calculations based on thermodynamic analysis of the transport mechanisms of ammonium suggest that ammonium is not equally distributed, indicating a high degree of compartmentalization in the vacuole. Additionally, metabolomic analysis results were used to calculate the thermodynamic driving forces in the central N metabolism reactions, revealing that the main reactions in the central N metabolism are far from equilibrium. Using proteomics approaches, we were able to identify major changes, not only in N metabolism, but also in C metabolism and regulation.
铵是酵母发酵最常见的氮源。尽管其运输和同化机制已有充分记载，但仅有少数研究尝试测量细胞内铵的浓度并评估其对基因表达的影响。我们采用基于同位素稀释质谱法（IDMS）的方法，能够在相同生长速率（0.05 h⁻¹）下，使用三种不同氮源（铵、尿素和谷氨酸），测量在氮限制的好氧恒化器培养中的细胞内铵浓度。实验结果表明，在此生长速率下，无论氮源如何，中央氮代谢中的反应都需要相似浓度的细胞内（IC）铵，约为3.6 mmol NH₄⁺/升。基于实验结果和不同假设，估算了液泡和细胞质中的铵浓度。此外，我们还发现了由于铵泄漏到细胞外空间而导致的无效循环，在氮限制条件下，这一过程消耗的ATP最多可达细胞ATP产量的30%，以及Gdh1和Gdh2反应之间的无效氧化还原循环。最后，使用鸟枪法蛋白质组学，通过相对于标记参考物确定蛋白质表达，识别了各种环境条件之间的差异，并将其与先前确定的氮化合物传感机制相关联。在我们的工作中，我们使用定量方法研究中央氮代谢。首先，在不同氮源下测量细胞内铵。结果表明，细胞维持恒定的NH₄⁺浓度（约3 mmol NH₄⁺/升），与所施加的氮源无关。我们推测，需要这一数量的细胞内铵来获得足够的热力学驱动力。此外，我们基于铵运输机制的热力学分析计算表明，铵的分布并不均匀，这表明液泡中存在高度的区室化。此外，代谢组学分析结果用于计算中央氮代谢反应中的热力学驱动力，结果表明中央氮代谢中的主要反应远未达到平衡。使用蛋白质组学方法，我们不仅能够识别氮代谢中的主要变化，还能识别碳代谢和调节方面的主要变化。
