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, 'T-enriched glucose was used and specific enrichments in lysine and in pyruvate-derived metabolites were determined by 'jC-and 'H-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.In Corynebacterium glutamicum lysine is synthesized either via the dehydrogenase variant or the succinylase variant of the diaminopimelate pathway ( Fig. 1) [l, 21. This is thus an unusual example of a split anabolic route. Split and convergent routes are usually reserved to catabolic processes where they occur to yield central metabolites like, for example, pyruvate or oxaloacetate [3]. Due to this peculiar pathway of lysine synthesis in C. glutamicum and also the fact that lysine is produced with this bacterium on an industrial scale [4], quantification of the use of both pathways has been attempted. With ['3C]glucose as substrate, and several assumptions on total carbon flow in the cell, the use of the dehydrogenase variant was calculated to contribute 30-40% to total lysine synthesis [5]. In the second study devoted to this question, strains with altered metabolism were constructed and analysed [2], and it was concluded that the dehydrogenase variant contributes 55 -75 % to lysine synthesis. The two values are apparently in contradiction. They were calculated from indirect measurements and for different strains. Therefore, we here quantify the flux distribution by the use of I3C-and 'H-NMR spectroscopy. In similar studies with C. glutamicum, both on lysine and on glutamate pro- duction, "C enrichments were determined only in the end products (lysine or glutamate) and then used for flux modelling [6, 71. The essential element...

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