Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum. Quantification by 13C- and 1H-NMR spectroscopy

Abstract

The Gram-positive Corynebacterium glutamicum has the potential to synthesize L-lysine via a split pathway, where amino-ketopimelate is converted to the ultimate lysine precursor diaminopimelate either by reactions involving succinylated intermediates, or by one single reaction catalysed by D-diaminopimelate dehydrogenase. To quantify the flux distribution via both pathways, 13C-enriched glucose was used and specific enrichments in lysine and in pyruvate-derived metabolites were determined by 13C- and 1H-NMR spectroscopy. Using a system of linear equations, the contribution of the D-diaminopimelate dehydrogenase pathway was determined to be about 30% for the total lysine synthesized. This was irrespective of whether lysine-accumulating mutants or the wild-type strain were analysed. However, when the distribution was determined at various cultivation times, the flux partitioning over the dehydrogenase pathway in a producing strain decreased from 72% at the beginning to 0% at the end of lysine accumulation. When ammonium sulphate was replaced by the organic nitrogen source glutamate, the ammonium-dependent D-diaminopimelate dehydrogenase pathway did not contribute to total 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 governed by the ammonium availability. This is thus an example where an anabolic pathway is directly influenced by an extracellular medium component, probably via the kinetic characteristics of D-diaminopimelate dehydrogenase.