Response of the Central Metabolism inCorynebacterium glutamicumto the use of an NADH-Dependent Glutamate Dehydrogenase

Marx, Achim; Eikmanns, Bernhard J.; Sahm, Hermann; Graaf, Albert A. de; Eggeling, Lothar

doi:10.1006/mben.1998.0106

Cited by 116 publications

(83 citation statements)

References 40 publications

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“…Attempts to engineer microbial metabolic fluxes through manipulations at the level of NADP-GDH (Marx et al, 1999;Nissen et al, 2000) clearly suggest the significance of this enzyme at the carbon-nitrogen interface of metabolism. It is interesting that, although NADP-GDH is central to aspergillus nitrogen metabolism (and ammonia assimilation), its activity is amenable to regulation by 2-oxoglutarate.…”

Section: Discussionmentioning

confidence: 99%

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Noor

Punekar

2005

Microbiology

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NADP-dependent glutamate dehydrogenase (NADP-GDH) mediates fungal ammonium assimilation through reductive synthesis of glutamate from 2-oxoglutarate. By virtue of its position at the interface of carbon and nitrogen metabolism, biosynthetic NADP-GDH is a potential candidate for metabolic control. In order to facilitate characterization, a new and effective dye-affinity method was devised to purify NADP-GDH from two aspergilli, Aspergillus niger and Aspergillus nidulans. The A. niger NADP-GDH was characterized at length and its kinetic interaction constants with glutamate (K m 34?7 mM) and ammonium (K m 1?05 mM; K i 0?4 mM) were consistent with an anabolic role. Isophthalate, 2-methyleneglutarate and 2,4-pyridinedicarboxylate were significant inhibitors, with respective K i values of 6?9, 9?2 and 202?0 mM. The A. niger enzyme showed allosteric properties and a sigmoid response (n H =2?5) towards 2-oxoglutarate saturation. The co-operative behaviour was a feature common to NADP-GDH from Aspergillus awamori, A. nidulans and Aspergillus oryzae. NADP-GDH may therefore be a crucial determinant in adjusting 2-oxoglutarate flux between the tricarboxylic acid cycle and glutamate biosynthesis in aspergilli.

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Section: Discussionmentioning

confidence: 99%

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Noor

Punekar

2005

Microbiology

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“…There are previous observations with glyceraldehyde 3-phosphate dehydrogenase, glutamate dehydrogenase, and isocitrate dehydrogenase indicating that cofactor specificity is a key factor in determining the distribution of metabolic fluxes and/or growth in bacteria (Martínez et al, 2008;Marx et al, 1999;Zhu et al, 2005). In spite of its clear contribution to redox balance and growth, in E. coli, the oxPPP has not been studied in this regard.…”

Section: Introductionmentioning

confidence: 99%

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

et al. 2014

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In Escherichia coli, the oxidative branch of the pentose phosphate pathway (oxPPP) is one of the major sources of NADPH when glucose is the sole carbon nutrient. However, unbalanced NADPH production causes growth impairment as observed in a strain lacking phosphoglucoisomerase (Dpgi). In this work, we studied the metabolic response of this bacterium to the replacement of its glucose-6-phosphate dehydrogenase (G6PDH) by an NADH-producing variant. The homologous enzyme from Leuconostoc mesenteroides was studied by molecular dynamics and site-directed mutagenesis to obtain the NAD-preferring LmG6PDH R46E,Q47E . Through homologous recombination, the zwf loci (encoding G6PDH) in the chromosomes of WT and Dpgi E. coli strains were replaced by DNA encoding LmG6PDH R46E,Q47E . Contrary to some predictions performed with flux balance analysis, the replacements caused a substantial effect on the growth rates, increasing 59 % in the Dpgi strain, while falling 44 % in the WT. Quantitative PCR (qPCR) analysis of the zwf locus showed that the expression level of the mutant enzyme was similar to the native enzyme and the expression of genes encoding key enzymes of the central pathways also showed moderate changes among the studied strains. The phenotypic and qPCR data were integrated into in silico modelling, showing an operative G6PDH flux contributing to the NADH pool. Our results indicated that, in vivo, the generation of NADH by G6PDH is beneficial or disadvantageous for growth depending on the operation of the upper Embden-Meyerhof pathway. Interestingly, a genomic database search suggested that in bacteria lacking phosphofructokinase, the G6PDHs tend to have similar preferences for NAD and NADP. The importance of the generation of NADPH in a pathway such as the oxPPP is discussed.

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“…In principle, NADPH can be synthesized in C. glutamicum by the enzymes of the oxidative pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase (Moritz et al 2000), by isocitrate dehydrogenase (Eikmanns et al 1995) and by malic enzyme (Gourdon et al 2000). Carbon flux analysis revealed that during growth on glucose, NADPH for lysine biosynthesis is provided by the PPP, whereas isocitrate dehydrogenase and malic enzyme are of minor importance (Marx et al 1996(Marx et al , 1997(Marx et al , 1999. During growth of C. glutamicum on fructose or on fructose/glucose mixtures, a low carbon flux through the oxidative PPP was demonstrated (Dominguez et al 1998;Kiefer et al 2004;Pons et al 1996).…”

Section: Introductionmentioning

confidence: 99%

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

Kabus

Georgi

Wendisch

et al. 2007

Appl Microbiol Biotechnol

124

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A critical factor in the biotechnological production of L-lysine with Corynebacterium glutamicum is the sufficient supply of NADPH. The membrane-integral nicotinamide nucleotide transhydrogenase PntAB of Escherichia coli can use the electrochemical proton gradient across the cytoplasmic membrane to drive the reduction of NADP + via the oxidation of NADH. As C. glutamicum does not possess such an enzyme, we expressed the E. coli pntAB genes in the genetically defined C. glutamicum lysineproducing strain DM1730, resulting in membrane-associated transhydrogenase activity of 0.7 U/mg protein. When cultivated in minimal medium with 10% (w/v) carbon source, the presence of transhydrogenase slightly reduced glucose consumption, whereas the consumption of fructose, glucose plus fructose, and, in particular, sucrose was stimulated. Biomass was increased by pntAB expression between 10 and 30% on all carbon sources tested. Most importantly, the lysine concentration was increased in the presence of transhydrogenase by ∼10% on glucose, ∼70% on fructose, ∼50% on glucose plus fructose, and even by ∼300% on sucrose. Thus, the presence of a proton-coupled transhydrogenase was shown to be an efficient way to improve lysine production by C. glutamicum. In contrast, pntAB expression had a negative effect on growth and glutamate production of C. glutamicum wild type.

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Response of the Central Metabolism inCorynebacterium glutamicumto the use of an NADH-Dependent Glutamate Dehydrogenase

Cited by 116 publications

References 40 publications

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon–nitrogen interface

Metabolic impact of an NADH-producing glucose-6-phosphate dehydrogenase in Escherichia coli

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

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