FLUX PARTITIONING IN THE SPLIT PATHWAY OF LYSINE SYNTHESIS IN CORYNEBACTERIUM-GLUTAMICUM QUANTIFICATION BY C-13-NMR AND H-1-NMR SPECTROSCOPY

The Gram-positive Corynebacterium glutamicum has the potential to synt hesize L-lysine via a split pathway, where amino-ketopimelate is conve rted to the ultimate lysine precursor diaminopimelate either by reacti ons involving succinylated intermediates, or by one single reaction ca talysed by D-diaminopimelate dehydrogenase. To quantify the flux distr ibution via both pathways, C-13-enriched glucose was used and specific enrichments in lysine and in pyruvate-derived metabolites were determ ined by C-13- and H-1-NMR spectroscopy. Using a system of linear equat ions, the contribution of the D-diaminopimelatc dehydrogenase pathway was determined to be about 30% for the total lysine synthesized. This was irrespective of whether lysine-accumulating mutants or the wild-ty pe strain were analysed. However, when the distribution was determined at various cultivation times, the flux partitioning over the dehydrog enase pathway in a producing strain decreased from 72% at the beginnin g to 0% at the end of lysine accumulation. When ammonium sulphate was replaced by the organic nitrogen source glutamate, the ammonium-depend ent D-diaminopimelate dehydrogenase pathway did not contribute to tota l lysine synthesis at all. Additional experiments with varying initial ammonium concentrations showed that in Corynebacterium glutamicum the flux distribution over the two pathways of lysine synthesis is govern ed by the ammonium availability. This is thus an example where an anab olic pathway is directly influenced by an extracellular medium compone nt, probably via the kinetic characteristics Of D-diaminopimelate dehy drogenase.