Print Email Facebook Twitter Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae Title Membrane potential independent transport of NH3 in the absence of ammonium permeases in Saccharomyces cerevisiae Author Cueto Rojas, H.F. (TU Delft OLD BT/Cell Systems Engineering) Milne, Nicholas (Evolva Biotech A/S) van Helmond, W. (TU Delft OLD ChemE/Organic Materials and Interfaces; Netherlands Forensic Institute - NFI) Pieterse, M.M. (TU Delft OLD BT/Analytical Biotechnology) Maris, Antonius J A (AlbaNova University Center) Daran, J.G. (TU Delft BT/Industriele Microbiologie) Wahl, S.A. (TU Delft OLD BT/Cell Systems Engineering) Date 2017-04-17 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 Δmep strain was dependent on the extracellular NH3 concentrations. Metabolomic analysis revealed a significantly higher intracellular NHX 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 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 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. Subject Ammonia passive diffusionAmmonium transportCentral nitrogen metabolismIntracellular ammoniumMetabolomicsThermodynamics To reference this document use: http://resolver.tudelft.nl/uuid:bb299e17-4b17-4759-b153-8aa0b9cce732 DOI https://doi.org/10.1186/s12918-016-0381-1 ISSN 1752-0509 Source BMC Systems Biology, 11 (1) Part of collection Institutional Repository Document type journal article Rights © 2017 H.F. Cueto Rojas, Nicholas Milne, W. van Helmond, M.M. Pieterse, Antonius J A Maris, J.G. Daran, S.A. Wahl Files PDF s12918_016_0381_1.pdf 1.19 MB Close viewer /islandora/object/uuid:bb299e17-4b17-4759-b153-8aa0b9cce732/datastream/OBJ/view