Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions

Fukui, Keita; Koseki, Chie; Yamamoto, Yoko; Nakamura, Jun; Sasahara, Ayako; Yuji, Reiko; Hashiguchi, Ken-ichi; Usuda, Yoshihiro; Matsui, Kunihiko; Kojima, Hiroyuki; Abe, Keietsu

doi:10.1016/j.jbiotec.2011.03.010

Cited by 40 publications

(28 citation statements)

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“…5). Succinate exporters have been identified in several C. glutamicum strains, but their detailed characterization is lacking (49,50). We speculate that, although cultivated at neutral pH, the organism is well adjusted to sustain the observed production fluxes at pH 5.7; moreover, the ability to maintain a high concentration gradient across the cell membrane creates a favorable contribution to the energetics of the efflux processes.…”

Section: Discussionmentioning

confidence: 94%

Carbon Flux Analysis by ¹³ C Nuclear Magnetic Resonance To Determine the Effect of CO ₂ on Anaerobic Succinate Production by Corynebacterium glutamicum

Radoš

Turner

Fonseca

et al. 2014

Appl Environ Microbiol

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c Wild-type Corynebacterium glutamicum produces a mixture of lactic, succinic, and acetic acids from glucose under oxygen deprivation. We investigated the effect of CO 2 on the production of organic acids in a two-stage process: cells were grown aerobically in glucose, and subsequently, organic acid production by nongrowing cells was studied under anaerobic conditions. The presence of CO 2 caused up to a 3-fold increase in the succinate yield (1 mol per mol of glucose) and about 2-fold increase in acetate, both at the expense of L-lactate production; moreover, dihydroxyacetone formation was abolished. The redistribution of carbon fluxes in response to CO 2 was estimated by using 13 C-labeled glucose and 13 C nuclear magnetic resonance (NMR) analysis of the labeling patterns in end products. The flux analysis showed that 97% of succinate was produced via the reductive part of the tricarboxylic acid cycle, with the low activity of the oxidative branch being sufficient to provide the reducing equivalents needed for the redox balance. The flux via the pentose phosphate pathway was low (ϳ5%) regardless of the presence or absence of CO 2 . Moreover, there was significant channeling of carbon to storage compounds (glycogen and trehalose) and concomitant catabolism of these reserves. The intracellular and extracellular pools of lactate and succinate were measured by in vivo NMR, and the stoichiometry (H ؉ :organic acid) of the respective exporters was calculated. This study shows that it is feasible to take advantage of natural cellular regulation mechanisms to obtain high yields of succinate with C. glutamicum without genetic manipulation.

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Section: Discussionmentioning

confidence: 94%

Carbon Flux Analysis by ¹³ C Nuclear Magnetic Resonance To Determine the Effect of CO ₂ on Anaerobic Succinate Production by Corynebacterium glutamicum

Radoš

Turner

Fonseca

et al. 2014

Appl Environ Microbiol

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“…The recent identification of the succinate exporter SucE in C. glutamicum (10,18) offers the interesting opportunity to test the effect of increased sucE expression on the succinate production of the reported strains. As the reaction catalyzed by GAPDH was shown to be critical, its optimization may improve the substrate consumption rates and thus further optimize the current producers.…”

Section: Discussionmentioning

confidence: 99%

“…This conversion is not coupled to growth (22). Transport of succinate into the medium was shown to involve the product of cg2425, the first succinate exporter to be identified (10,18).…”

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confidence: 99%

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

Litsanov

Brocker

Bott

2012

Appl Environ Microbiol

194

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ABSTRACTPrevious studies have demonstrated the capability ofCorynebacterium glutamicumfor anaerobic succinate production from glucose under nongrowing conditions. In this work, we have addressed two shortfalls of this process, the formation of significant amounts of by-products and the limitation of the yield by the redox balance. To eliminate acetate formation, a derivative of the type strain ATCC 13032 (strain BOL-1), which lacked all known pathways for acetate and lactate synthesis (ΔcatΔpqoΔpta-ackAΔldhA), was constructed. Chromosomal integration of the pyruvate carboxylase genepycP458Sinto BOL-1 resulted in strain BOL-2, which catalyzed fast succinate production from glucose with a yield of 1 mol/mol and showed only little acetate formation. In order to provide additional reducing equivalents derived from the cosubstrate formate, thefdhgene fromMycobacterium vaccae, coding for an NAD+-coupled formate dehydrogenase (FDH), was chromosomally integrated into BOL-2, leading to strain BOL-3. In an anaerobic batch process with strain BOL-3, a 20% higher succinate yield from glucose was obtained in the presence of formate. A temporary metabolic blockage of strain BOL-3 was prevented by plasmid-borne overexpression of the glyceraldehyde 3-phosphate dehydrogenase genegapA. In an anaerobic fed-batch process with glucose and formate, strain BOL-3/pAN6-gapaccumulated 1,134 mM succinate in 53 h with an average succinate production rate of 1.59 mmol per g cells (dry weight) (cdw) per h. The succinate yield of 1.67 mol/mol glucose is one of the highest currently described for anaerobic succinate producers and was accompanied by a very low level of by-products (0.10 mol/mol glucose).

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“…Recently, the results of a BLAST (7) search of the nucleotide sequence of the aspT gene and the amino acid sequence of the AspT protein against current nucleotide and protein data bases, respectively, suggested that the aspartate:alanine exchanger transporters are conserved in many bacterial species (8,9). Very recently, Fukui et al (10) found SucE1, an aspartate:alanine exchanger transporter from Corynebacterium glutamicum, plays a role in the export of succinate generated during fermentation. The putative broad distribution of AspT orthologs and paralogs in bacteria suggests that further biochemical study of AspT could provide valuable insights into membrane transport.…”

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confidence: 99%

Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes

Sasahara

Nanatani

Enomoto

et al. 2011

Journal of Biological Chemistry

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In some strains of the lactic acid bacterium Tetragenococcus halophilus, a proton-motive force is generated by the combined action of an intracellular L-aspartate decarboxylation reaction, catalyzed by an L-aspartate-4-decarboxylase (AspD, EC 4.1.1.12), and an electrogenic aspartate 1Ϫ :alanine 0 exchange reaction, catalyzed by an aspartate:alanine antiporter (AspT, TC number 2.A.81.1.1):The proton-motive force generated is sufficiently high to drive ATP synthesis via the bacterial F 0 F 1 -ATPase. This combination of proton-motive force and ATP synthesis has been proposed as a protonmotive metabolic cycle, the prototype model of which is found in Oxalobacter formigenes (1-3). Such decarboxylation reactions are thought to be advantageous for cells because they generate metabolic energy and regulate intracellular pH (4, 5).In previous work with proteoliposomes, we found that the aspartate:alanine exchange catalyzed by AspT is electrogenic (4, 6). AspT is classified as a conventional secondary transport protein and belongs to the newly classified aspartate:alanine exchanger family (TC number 2.A.81) of transporters in the system developed by Saier et al. (25,26). Recently, the results of a BLAST (7) search of the nucleotide sequence of the aspT gene and the amino acid sequence of the AspT protein against current nucleotide and protein data bases, respectively, suggested that the aspartate:alanine exchanger transporters are conserved in many bacterial species (8, 9). Very recently, Fukui et al. (10) found SucE1, an aspartate:alanine exchanger transporter from Corynebacterium glutamicum, plays a role in the export of succinate generated during fermentation. The putative broad distribution of AspT orthologs and paralogs in bacteria suggests that further biochemical study of AspT could provide valuable insights into membrane transport.AspT is a membrane protein containing 543 amino acids (57.2 kDa). Its membrane topology has been studied by using alkaline phosphatase and ␤-lactamase fusion methods (11); AspT has also been studied by using the cysteine-substituted accessibility method (12, 13), which uses the impermeant, fluorescent thiol-specific probe Oregon Green 488 maleimide and the impermeant, nonfluorescent thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. These analyses revealed that AspT has a unique topology and that transmembrane domain 3 participates in the formation of a hydrophilic cleft in the membrane, implicating the transmembrane domain in the ligand-induced conformational changes (14). In previous work, we developed a solubilization and purification scheme by using n-dodecyl-␤-D-maltoside (DDM), 3 and we studied the oligomerization of AspT by means of a

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Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions

Cited by 40 publications

References 50 publications

Carbon Flux Analysis by ¹³ C Nuclear Magnetic Resonance To Determine the Effect of CO ₂ on Anaerobic Succinate Production by Corynebacterium glutamicum

Carbon Flux Analysis by ¹³ C Nuclear Magnetic Resonance To Determine the Effect of CO ₂ on Anaerobic Succinate Production by Corynebacterium glutamicum

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes

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Identification of succinate exporter in Corynebacterium glutamicum and its physiological roles under anaerobic conditions

Cited by 40 publications

References 50 publications

Carbon Flux Analysis by 13 C Nuclear Magnetic Resonance To Determine the Effect of CO 2 on Anaerobic Succinate Production by Corynebacterium glutamicum

Carbon Flux Analysis by 13 C Nuclear Magnetic Resonance To Determine the Effect of CO 2 on Anaerobic Succinate Production by Corynebacterium glutamicum

Toward Homosuccinate Fermentation: Metabolic Engineering of Corynebacterium glutamicum for Anaerobic Production of Succinate from Glucose and Formate

Substrate Specificity of the Aspartate:Alanine Antiporter (AspT) of Tetragenococcus halophilus in Reconstituted Liposomes

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Carbon Flux Analysis by ¹³ C Nuclear Magnetic Resonance To Determine the Effect of CO ₂ on Anaerobic Succinate Production by Corynebacterium glutamicum

Carbon Flux Analysis by ¹³ C Nuclear Magnetic Resonance To Determine the Effect of CO ₂ on Anaerobic Succinate Production by Corynebacterium glutamicum