Role of Cytochrome
            <i>bd</i>
            Oxidase from
            <i>Corynebacterium glutamicum</i>
            in Growth and Lysine Production

Kabus, Armin; Niebisch, Axel; Bott, Michael

doi:10.1128/aem.01818-06

Cited by 55 publications

(37 citation statements)

References 39 publications

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“…In contrast, Gram-positive bacteria usually contain cydABDC operons. For example, the cydABCD operon is polycistronic in C. glutamicum (24) and B. subtilis (9). Mapping of the cydAB and cydDC promoters.…”

Section: Resultsmentioning

confidence: 99%

Hypoxia-Activated Cytochrome bd Expression in Mycobacterium smegmatis Is Cyclic AMP Receptor Protein Dependent

2014

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Mycobacteria are obligate aerobes and respire using two terminal respiratory oxidases, an aa 3 -type cytochrome c oxidase and a cytochrome bd-type menaquinol oxidase. Cytochrome bd is encoded by cydAB from the cydABDC gene cluster that is conserved throughout the mycobacterial genus. Here we report that cydAB and cydDC in Mycobacterium smegmatis constitute two separate operons under hypoxic growth conditions. The transcriptional start sites of both operons were mapped, and a series of cydA-lacZ and cydD-lacZ transcriptional reporter fusions were made to identify regulatory promoter elements. A 51-bp region was identified in the cydAB promoter that was required for maximal cydA-lacZ expression in response to hypoxia. A cyclic AMP receptor protein (CRP)-binding site (viz. GTGAN 6 CCACC) was identified in this region, and mutation of this site to CCCAN 6 CTTTC abolished cydA-lacZ expression in response to hypoxia. Binding of purified CRP (MSMEG_0539) to the cydAB promoter DNA was analyzed using electrophoretic mobility shift assays. CRP binding was dependent on GTGAN 6 CCACC and showed cyclic AMP (cAMP) dependency. No CRP site was present in the cydDC promoter, and a 10-bp inverted repeat (CGGTG GTACCGGTACCACCG) was required for maximal cydD-lacZ expression. Taken together, the data indicate that CRP is a direct regulator of cydAB expression in response to hypoxia and that the regulation of cydDC expression is CRP independent and under the control of an unknown regulator.T he electron transport chain in mycobacteria plays a vital role in growth by generating a proton motive force across the bacterial inner membrane to fuel energetic processes in the cell membrane. Electrons enter the electron transport chain primarily via NADH dehydrogenase and succinate dehydrogenase complexes with the menaquinone-menaquinol pool, and the chain terminates through the actions of two terminal oxidases: an aa 3 -type cytochrome c oxidase, encoded by ctaBCDEF (1), and cytochrome bd oxidase, encoded by the cydABDC gene cluster (2). In vitro studies have shown that the cydABDC gene cluster in Mycobacterium smegmatis is upregulated Ͼ50-fold in response to 0.6% air saturation at a low growth rate (3) and that cydA-lacZ expression in M. smegmatis increases 2-to 3-fold between 5 and 0.5% air saturation (2), suggesting that cytochrome bd oxidase is adapted to function at low oxygen tensions in mycobacteria. However, no studies have reported the oxygen affinity of cytochrome bd in mycobacteria.In vivo analyses have revealed that the copy numbers of the cydA and cydC transcripts from Mycobacterium tuberculosis increase during chronic infection in mouse lungs (4) and that mutations in cydC result in impaired growth in mice (4). Additionally, M. tuberculosis cydC mutants are impaired in persistence in mice treated with isoniazid (INH) (5), suggesting discrete functions for cydAB and cydDC in this obligate human pathogen. Despite the importance of cydAB and cydDC in mycobacterial physiology and disease, the transcriptional organization of this cl...

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Section: Resultsmentioning

confidence: 99%

Hypoxia-Activated Cytochrome bd Expression in Mycobacterium smegmatis Is Cyclic AMP Receptor Protein Dependent

2014

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“…Growth was followed by measuring the optical density at 600 nm (OD 600 ) with an Ultrospec 500 pro spectrophotometer (Amersham Biotech, Freiburg, Germany). The biomass concentration was calculated from OD 600 values using an experimentally determined correlation factor of 0.25 g cells (dry weight) (cdw)/ liter for an OD 600 of 1 (26).…”

Section: Methodsmentioning

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

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189

131

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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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“…In addition, C. glutamicum can synthesize ATP by SLP in the tricarboxylic acid (TCA) cycle and by the conversion of acetyl coenzyme A (acetyl-CoA) to acetate by acetate kinase (27). Since oxygen (and nitrate) serves as a terminal electron acceptor in its respiratory energy metabolism, C. glutamicum generates ATP by ETP (28). By SLP and ETP with the cytochrome bc 1 -aa 3 branch, complete aerobic oxidation of glucose and acetate yields 26.7 and 7.3 mol of ATP, respectively (29,30).…”

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confidence: 99%

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

Reddy

Lindner

Wendisch

2015

Appl Environ Microbiol

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Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADPdependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154 -7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-II D213G and thus by coupling to electron transport phosphorylation (ETP).A TP generation occurs naturally by substrate-level phosphorylation (SLP), electron transport phosphorylation (ETP), photophosphorylation, and decarboxylation phosphorylation. The Embden-Meyerhof-Parnas (EMP) pathway of glycolysis supplies ATP by SLP in the absence of terminal electron acceptors or anaerobic conditions, as well as supplying direct precursors for biomass formation (1). Bacteria and archaea possess one or more of multiple biologically feasible routes for glucose catabolism, such as the EMP pathway, the Entner-Doudoroff (ED) pathway, and the phosphoketolase pathway, which yield zero to 3 ATP molecules per glucose molecule and exist in different variants. In natural habitats, a trade-off between the rate and the yield of ATP production is observed for heterotrophic organisms; faster but less efficient ATP production may be advantageous under conditions characterized by a low abundance of growth substrates (2, 3). For example, Zymomonas mobilis ferments suga...

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Role of Cytochrome bd Oxidase from Corynebacterium glutamicum in Growth and Lysine Production

Cited by 55 publications

References 39 publications

Hypoxia-Activated Cytochrome bd Expression in Mycobacterium smegmatis Is Cyclic AMP Receptor Protein Dependent

Hypoxia-Activated Cytochrome bd Expression in Mycobacterium smegmatis Is Cyclic AMP Receptor Protein Dependent

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

Metabolic Engineering of an ATP-Neutral Embden-Meyerhof-Parnas Pathway in Corynebacterium glutamicum: Growth Restoration by an Adaptive Point Mutation in NADH Dehydrogenase

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