Studies on ammonium assimilation by 'Saccharomyces cerevisiae'
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Saccharomyces cerevisiae can assimilate ammonium by NADP-GDH or by GS-GOGAT. The aim of this project was to improve the efficiency of ammonium assimilation, and therefore substrate utilisation, of S. cerevisiae by elimination of the energy inefficient pathway (GS-GOGAT). GOGAT- mutants were isolated from a GDH- parent strain by their inability to use ammonium as sole nitrogen source. Two structural gene mutants were identified, one in each of the two structural genes encoding GOGAT. Constructs with different combinations of GDH- and GOGAT- mutations and corresponding wild type alleles were made, and their growth studied in medium supplemented with different levels of ammonium. The growth properties (as final culture density and growth rate) of GOGAT- and GOGAT+ strains transformed with the GDH1 gene, and grown with excess ammonium were very similar. It was concluded that, under the conditions used in this study, the loss of GOGAT does not improve the growth properties of the strain. Non-transformed constructs were grown with excess and limiting ammonium. Growth properties of the GDH- and GOGAT- strains suggest that GS-GOGAT functions in ammonium assimilation at very low ammonium levels. This conclusion needs further investigation because the GDH+ GOGAT- construct had lower NADP-GDH activity than the wild type. The physiology of ammonium assimilation by two industrial strains was compared to that of a laboratory wild type at different ammonium concentrations using shake-flask culture. All three strains possessed the three activities in MM+20mM NH4+, and the profiles of appearance/disappearance of activity were very similar. At lower ammonium concentrations, important differences between the strains became apparent. It is unclear if it is due to simple strain heterogeneity or represents significant differences between industrial and laboratory strains. On the basis of the enzyme data, GS-GOGAT appears to be important in ammonium assimilation by DCL1 at limiting concentrations.
Thesis, PhD Doctor of Philosophy
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