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BMC Syst Biol. 2017 Apr 17;11(1):49. doi: 10.1186/s12918-016-0381-1.

Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae.

Author information

1
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, The Netherlands.
2
Present Address: Evolva Biotech A/S, Lersø Parkallé 42, 2100, København Ø, Denmark.
3
Present Address: Nederlands Forensisch Instituut (NFI), Laan van Ypenburg 6, 2497 GB, Den Haag, The Netherlands.
4
Division of Industrial Biotechnology, School of Biotechnology, KTH Royal Institute of Technology, AlbaNova University Center, SE 106 91, Stockholm, Sweden.
5
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, The Netherlands. J.G.Daran@tudelft.nl.
6
Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, The Netherlands. S.A.Wahl@tudelft.nl.

Abstract

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 NHX 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 NHX ratios under aerobic nitrogen-limiting conditions were consistent with this hypothesis when NHx 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.

KEYWORDS:

Ammonia passive diffusion; Ammonium transport; Central nitrogen metabolism; Intracellular ammonium; Metabolomics; Thermodynamics

PMID:
28412970
PMCID:
PMC5392931
DOI:
10.1186/s12918-016-0381-1
[Indexed for MEDLINE]
Free PMC Article

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