Abstract
BackgroundCorynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate. It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. In this study, we quantitatively analyzed which anaplerotic pathway flux most markedly changes the glutamate overproduction induced by Tween 40 addition.ResultsWe performed a metabolic flux analysis (MFA) with [1-13C]- and [U-13C]-labeled glucose in the glutamate production phase of C. glutamicum, based on the analysis of the time courses of 13C incorporation into proteinogenic amino acids by gas chromatography-mass spectrometry (GC-MS). The flux from phosphoenolpyruvate (PEP) to oxaloacetate (Oxa) catalyzed by phosphoenolpyruvate carboxylase (PEPc) was active in the growth phase not producing glutamate, whereas that from pyruvate to Oxa catalyzed by pyruvate carboxylase (Pc) was inactive. In the glutamate overproduction phase induced by the addition of the detergent material Tween 40, the reaction catalyzed by Pc also became active in addition to the reaction catalyzed by PEPc.ConclusionIt was clarified by a quantitative 13C MFA that the reaction catalyzed by Pc is most markedly increased, whereas other fluxes of PEPc and PEPck remain constant in the glutamate overproduction induced by Tween 40. This result is consistent with the previous results obtained in a comparative study on the glutamate productions of genetically recombinant Pc- and PEPc-overexpressing strains. The importance of a specific reaction in an anaplerotic pathway was elucidated at a metabolic level by MFA.
Highlights
Corynebacterium glutamicum has several anaplerotic pathways, which are essential for the productions of amino acids, such as lysine and glutamate
C. glutamicum has a complex set of anaplerotic pathways for supplying oxaloacetate, i.e., reactions from phosphoenolpyruvate (PEP) to oxaloacetate catalyzed by phosphoenolpyruvate carboxylase (PEPc) [11,12], from pyruvate (Pyr) to oxaloacetate catalyzed by pyruvate carboxylase (Pc) [13], from oxaloacetate to PEP catalyzed by PEP carboxykinase (PEPck) [14], and from malate to Pyr catalyzed by malic enzymes [15,16]
It is found that the flux of Pc proportionally increases to glutamate production; in contrast, the flux of PEPc remains constant throughout fermentation (Fig. 4). This result directly indicates that the flux of Pc is the most important of those in the anaplerotic pathways in the glutamate overproduction induced by Tween 40 addition
Summary
Corynebacterium glutamicum has several anaplerotic pathways (anaplerosis), which are essential for the productions of amino acids, such as lysine and glutamate It is still not clear how flux changes in anaplerotic pathways happen when glutamate production is induced by triggers, such as biotin depletion and the addition of the detergent material, Tween 40. It is well known that there are some triggers of glutamate overproduction in C. glutamicum: the depletion of biotin, which is required for cell growth [2], the addition of detergent [3], the addition of lactam antibiotics, such as penicillin [4], and the temperature upshift of fermentation [5] With these triggers a marked change at the final branch point, 2-oxoglutarate in the TCA cycle, was observed owing to a decrease in 2-oxoglutarate dehydrogenase complex (ODHC) activity [6,7,8]. The metabolic fluxes of anaplerotic pathways under glutamate-overproducing conditions have not yet directly been measured
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