Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Menkel, E; Thierbach, Georg; Eggeling, Lothar; Sahm, Hermann

doi:10.1128/aem.55.3.684-688.1989

Cited by 84 publications

(23 citation statements)

References 18 publications

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“…[257,267,273]). Menkel et al [284] already showed that addition of fumarate to the growth medium and thus increasing the oxaloacetate and aspartate availability led to an about 30% higher lysine yield with a producer strain. From this result it was concluded that the supply of the precursors oxaloacetate or aspartate is rate limiting for optimal lysine production.…”

Section: Metabolic Engineering Of the Node For Amino Acid Productionmentioning

confidence: 99%

The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

2005

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In many organisms, metabolite interconversion at the phosphoenolpyruvate (PEP)-pyruvate-oxaloacetate node involves a structurally entangled set of reactions that interconnects the major pathways of carbon metabolism and thus, is responsible for the distribution of the carbon flux among catabolism, anabolism and energy supply of the cell. While sugar catabolism proceeds mainly via oxidative or non-oxidative decarboxylation of pyruvate to acetyl-CoA, anaplerosis and the initial steps of gluconeogenesis are accomplished by C3- (PEP- and/or pyruvate-) carboxylation and C4- (oxaloacetate- and/or malate-) decarboxylation, respectively. In contrast to the relatively uniform central metabolic pathways in bacteria, the set of enzymes at the PEP-pyruvate-oxaloacetate node represents a surprising diversity of reactions. Variable combinations are used in different bacteria and the question of the significance of all these reactions for growth and for biotechnological fermentation processes arises. This review summarizes what is known about the enzymes and the metabolic fluxes at the PEP-pyruvate-oxaloacetate node in bacteria, with a particular focus on the C3-carboxylation and C4-decarboxylation reactions in Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum. We discuss the activities of the enzymes, their regulation and their specific contribution to growth under a given condition or to biotechnological metabolite production. The present knowledge unequivocally reveals the PEP-pyruvate-oxaloacetate nodes of bacteria to be a fascinating target of metabolic engineering in order to achieve optimized metabolite production.

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Section: Metabolic Engineering Of the Node For Amino Acid Productionmentioning

confidence: 99%

The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

2005

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“…The wild type of Corynebacterium glutamicum (ATCC 13032), its two lysine-producing mutants MH 20-22B [8] and DG 52-5 [9] and AS72 (derived from DG 52-5 lacking diaminopimelate dehydrogenase (Ddh-) [2] were used. They were inoculated into 100 ml brain-heart infusion (Difco, Detroit, USA) as a preculture medium.…”

Section: Strains and Growth Conditionsmentioning

confidence: 99%

“…Another prerequisite is the determination of I3C enrichments in the carbon atoms of either aspartate or pymvate. Since these intermediates are only found in limited amounts in culture fluids [9], we undertook label determination in alanine derived directly from pyruvate. In a culture of strain DG 52-5, 12 mM alanine was found, with 3.0% 13C enrichment at C-2 and 24.6% at C-3 determined.…”

Section: Assessment Of the Validity Of The Flux Distribution Modelmentioning

confidence: 99%

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

Sonntag

Eggeling

Graaf

et al. 1993

European Journal of Biochemistry

147

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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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“…1a) and introduced into C. glutamicum ATCC 13032 by electroporation. Cells in which integration had occurred by a single cross-over cell were isolated by selection for kanamycin resistance (Km R ) on CGIII agar (Menkel et al, 1989), and confirmed by PCR with two primer pairs, one specific for integration upstream of the gene of interest (PKTHYX1 and PKTHYX2), and the other specific for integration downstream (THYXPK1 and THYXPK2). Single cross-over cells were grown on LB agar plates containing 10% w/v sucrose to resolve the suicide plasmid, in the absence of NaCl and kanamycin.…”

Section: Deletion Mutagenesis and Complementation Of C Glutamicum Thyxmentioning

confidence: 99%

Alternative thymidylate synthase, ThyX, involved in Corynebacterium glutamicum ATCC 13032 survival during stationary growth phase

Park

Cho

Lee

et al. 2010

FEMS Microbiology Letters

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A BLASTP search has shown the presence of a gene homologous to an alternative thymidylate synthase (TS), thyX, in Corynebacterium glutamicum ATCC 13032. To determine if thyX is functionally analogous to thyA, thyX was cloned in a plasmid and the resulting construct was transferred by transformation into a thyA mutant of Escherichia coli. The ThyX from C. glutamicum compensated for the defect in TS-deficient E. coli. A functional knockout of the thyX gene was constructed by allelic replacement using a sucrose counter-selectable suicide plasmid and confirmed by PCR and reverse transcriptase-PCR analyses. This mutant was viable without thymidine supplementation, suggesting that thyX is not an essential gene in C. glutamicum. Growth of the thyX mutant was dependent upon coupling activity of dihydrofolate reductase (DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210. This indicates that thymidine synthesis was at least partially dependent on thyX expression. As it approached stationary phase, the thyX mutant lost viability much more rapidly than the parental wild type and the mutant complemented the thyX gene, suggesting that the activity of the ThyX enzyme is important in that phase of the growth cycle.

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Influence of increased aspartate availability on lysine formation by a recombinant strain of Corynebacterium glutamicum and utilization of fumarate

Cited by 84 publications

References 18 publications

The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

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

Alternative thymidylate synthase, ThyX, involved in Corynebacterium glutamicum ATCC 13032 survival during stationary growth phase

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