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

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

Cueto Rojas, H.F. (TU Delft OLD BT/Cell Systems Engineering)
Maleki Seifar, R. (TU Delft OLD BT/Cell Systems Engineering)
ten Pierick, A. (TU Delft OLD BT/Cell Systems Engineering)
van Helmond, W. (TU Delft OLD ChemE/Organic Materials and Interfaces)
Pieterse, M.M. (TU Delft OLD BT/Analytical Biotechnology)
Heijnen, J.J. (TU Delft OLD BT/Cell Systems Engineering)
Wahl, S.A. (TU Delft OLD BT/Cell Systems Engineering)

2016

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 ⁺/liter_IC, 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.

http://resolver.tudelft.nl/uuid:c7151f9a-e95b-4496-84a7-b2824f533dbd

https://doi.org/10.1128/AEM.01547-16

0099-2240

Applied and Environmental Microbiology, 82 (23), 6831-6845

Institutional Repository

journal article

© 2016 H.F. Cueto Rojas, R. Maleki Seifar, A. ten Pierick, W. van Helmond, M.M. Pieterse, J.J. Heijnen, S.A. Wahl