Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum

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, '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...

In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

“…It is known that two alternative pathways are present in C. glutamicum for lysine synthesis. As shown in Table 5, enzymes of both pathways were expressed at all examined time points, confirming previous findings that both pathways contribute to lysine production (33,45). In addition to the lysine export genes, different genes of lysine biosynthesis were subjected to changes in transcript level during the phase shift.…”

Characterization of myo -Inositol Utilization by Corynebacterium glutamicum : the Stimulon, Identification of Transporters, and Influence on l -Lysine Formation

“…Although myoinositol contributes about 8% of the total carbon in a mixture with glucose, the resulting yield is reduced by as much as 29% (Table 3). The effect of cells grown in different complex media and used as inoculum on the final yield is well established (25), and the present study, as well as a proteome study (15), shows that iol genes are expressed on complex media usually used to derive the inoculum. However, in our experiments the preculture medium for deriving the inoculum was identical to the culture medium where the yields were determined.…”

Application of MALDI‐TOF MS to lysine‐producing Corynebacterium glutamicum

“…This might be caused by the different growth conditions. It is known that the flux partitioning ratio between dehydrogenase and succinylase pathway responds to parameters such as ammonium concentration or cultivation stage [25].…”