In vivo analysis of NH4+ transport and central nitrogen metabolism in Saccharomyces cerevisiae during aerobic nitrogen-limited growth

Cueto-Rojas HF, Seifar RM, Ten Pierick A, Van Helmond W, Pieterse MM, Heijnen JJ, Wahl SA (2016)

Publication Type: Journal article

Publication year: 2016

Journal

Applied and Environmental Microbiology American Society for Microbiology

Book Volume: 82

Pages Range: 6831-6845

Journal Issue: 23

DOI: 10.1128/AEM.01547-16

Abstract

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.

Authors with CRIS profile

Sebastian Aljoscha Wahl

Involved external institutions

Delft University of Technology (TU Delft) NL Netherlands (NL)

How to cite

APA:

Cueto-Rojas, H.F., Seifar, R.M., Ten Pierick, A., Van Helmond, W., Pieterse, M.M., Heijnen, J.J., & Wahl, S.A. (2016). In vivo analysis of NH4+ transport and central nitrogen metabolism in Saccharomyces cerevisiae during aerobic nitrogen-limited growth. Applied and Environmental Microbiology, 82(23), 6831-6845. https://dx.doi.org/10.1128/AEM.01547-16

MLA:

Cueto-Rojas, H. F., et al. "In vivo analysis of NH4+ transport and central nitrogen metabolism in Saccharomyces cerevisiae during aerobic nitrogen-limited growth." Applied and Environmental Microbiology 82.23 (2016): 6831-6845.

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