Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

Kelle, Ralf; Laufer, Birgit; Brunzema, Carsten; Weuster‐Botz, Dirk; Krämer, Reinhard; Wandrey, Christian

doi:10.1002/(sici)1097-0290(19960705)51:1<40::aid-bit5>3.0.co;2-0

Cited by 20 publications

(7 citation statements)

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“…The design of the minimal medium used in this study was based on minimal media for Corynebacterium described in other studies (17,18), and on the Neidhardt minimal medium for enterobacteria (23). It was modified to optimize growth rate, to guarantee pH stability over a long period of growth, and to prevent the formation of precipitates, thereby correcting three major disadvantages of minimal media previously used for growth of C. glutamicum.…”

Section: Methodsmentioning

confidence: 99%

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

et al. 2000

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Like many other bacteria, Corynebacterium glutamicum possesses two types of L-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in the mqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicum obscure.Recently, we discovered the gene for a relatively unknown type of malate dehydrogenase called malate:quinone oxidoreductase (MQO; (EC 1.1.99.16) [also called "malate dehydrogenase (acceptor)"] (22). Like the NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37), MQO catalyzes the oxidation of malate to oxaloacetate. The enzyme is membrane associated, probably through weak ionic or hydrophobic interactions. Tightly bound flavin adenine dinucleotide serves as a prosthetic group, and quinones instead of NAD are the electron acceptors of the enzyme. The quinones are subsequently oxidized by the electron transfer chain. These properties place MQO, like succinate dehydrogenase (SDH), both in the electron transfer chain and in the citric acid cycle. The existence of MQO as an enzymatic entity was first proven in 1956 (8). MQO activity was since then observed in several bacteria, both gram positive and gram negative (see references cited in reference 22) but not in archaebacteria or eucaryotes. After identification (for the first time) of a DNA sequence encoding an MQO in Corynebacterium glutamicum, it was found t...

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Section: Methodsmentioning

confidence: 99%

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

et al. 2000

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“…The experiments were performed with C. glutamicum wild type (ATCC 13032) and a GDH-negative mutant C. glutamicum EB1 (4). Continuous cultures were run with a fully synthetic medium as described elsewhere (20), modified for the glucose concentration (100 g/liter) and the (NH 4 ) 2 SO 4 concentration, which was varied between 10 and 15 g/liter by separate dosage. The medium was inoculated with washed cells from an overnight culture in BHI medium (Difco) to give an initial optical density at 600 nm (OD 600 ) of about 5.…”

Section: Methodsmentioning

confidence: 99%

In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by ¹⁵ N Nuclear Magnetic Resonance

Tesch

Graaf

Sahm

1999

Appl Environ Microbiol

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Glutamate dehydrogenase (GDH) and glutamine synthetase (GS)–glutamine 2-oxoglutarate-aminotransferase (GOGAT) represent the two main pathways of ammonium assimilation in Corynebacterium glutamicum. In this study, the ammonium assimilating fluxes in vivo in the wild-type ATCC 13032 strain and its GDH mutant were quantitated in continuous cultures. To do this, the incorporation of15N label from [15N]ammonium in glutamate and glutamine was monitored with a time resolution of about 10 min with in vivo 15N nuclear magnetic resonance (NMR) used in combination with a recently developed high-cell-density membrane-cyclone NMR bioreactor system. The data were used to tune a standard differential equation model of ammonium assimilation that comprised ammonia transmembrane diffusion, GDH, GS, GOGAT, and glutamine amidotransferases, as well as the anabolic incorporation of glutamate and glutamine into biomass. The results provided a detailed picture of the fluxes involved in ammonium assimilation in the two different C. glutamicumstrains in vivo. In both strains, transmembrane equilibration of 100 mM [15N]ammonium took less than 2 min. In the wild type, an unexpectedly high fraction of 28% of the NH4 + was assimilated via the GS reaction in glutamine, while 72% were assimilated by the reversible GDH reaction via glutamate. GOGAT was inactive. The analysis identified glutamine as an important nitrogen donor in amidotransferase reactions. The experimentally determined amount of 28% of nitrogen assimilated via glutamine is close to a theoretical 21% calculated from the high peptidoglycan content of C. glutamicum. In the GDH mutant, glutamate was exclusively synthesized over the GS/GOGAT pathway. Its level was threefold reduced compared to the wild type.

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“…Soon enough, however, it was recognised that this was not due to a general membrane-leakiness, because it was very selective for glutamate (and was even against a glutamate concentration gradient), but that it was due to a change in membrane tension that activated a mechanosensitive glutamate efflux pump encoded by a gene called NCgl1221 (a homologue of the E. coli yggB gene, now known as mscS, the mechanosensitive channel of small conductance) (Hashimoto et al, 2012, Hashimoto et al, 2010, Nakamura et al, 2007, Nakayama et al, 2012, Yamashita et al, 2013, Yao et al, 2009. Similar efflux pumps are now known to be involved in the export of product during a variety of other amino acid fermentations Sahm, 2003, Mitsuhashi, 2014), such as those for lysine (Bellmann et al, 2001, Bröer and Krämer, 1991a, Bröer and Krämer, 1991b, Kelle et al, 1996, Vrljic et al, 1996, isoleucine (Hermann andKrämer, 1996, Xie et al, 2012) threonine (Lee et al, 2007), methionine (Trötschel et al, 2005) and others (Van Dyk, 2008).…”

Section: Some Useful Case Histories From Classical Fermentationsmentioning

confidence: 99%

Metabolic Control Analysis

Mulquiney¹,

Kuchel²

2003

Modelling Metabolism With Mathematica

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Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

Cited by 20 publications

References 20 publications

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by ¹⁵ N Nuclear Magnetic Resonance

Metabolic Control Analysis

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Reaction engineering analysis of L-lysine transport by Corynebacterium glutamicum

Cited by 20 publications

References 20 publications

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by 15 N Nuclear Magnetic Resonance

Metabolic Control Analysis

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In Vivo Fluxes in the Ammonium-Assimilatory Pathways in Corynebacterium glutamicum Studied by ¹⁵ N Nuclear Magnetic Resonance