E1 Enzyme of the Pyruvate Dehydrogenase Complex in
            <i>Corynebacterium glutamicum</i>
            : Molecular Analysis of the Gene and Phylogenetic Aspects

Schreiner, Mark E.; Fiur, Diana; Holátko, Jiří; Pátek, Miroslav; Eikmanns, Bernhard J.

doi:10.1128/jb.187.17.6005-6018.2005

Cited by 78 publications

(75 citation statements)

References 77 publications

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“…We previously showed that the PQO activity in C. glutamicum WT, together with the activities of the acetate activating enzymes acetate kinase and phosphotransacetylase, is not able to compensate for PDHC activity (60). By overexpression of the pqo gene, we here demonstrate that PQO, when present in large amounts, can substitute at least partially for the PDHC in C. glutamicum.…”

Section: Discussionmentioning

confidence: 68%

“…The defined PDHC-negative mutant C. glutamicum WT ⌬aceE, however, showed negligible growth in TY medium and in minimal medium containing glucose, indicating that the PQO activity in the WT strain cannot compensate for the PDHC activity under the conditions tested (60). To test for the possibility that a high PQO activity can substitute for PDHC activity, we constructed the double mutant C. glutamicum WT ⌬aceE⌬pqo, transformed it with pEKEx-pqo or pEKEx2 (as a control), and assayed for the growth of the strains on different media.…”

Section: Analysis Of the Pqo Gene And Its Chromosomal Organizationmentioning

confidence: 99%

“…Total RNA from exponentially growing cells of C. glutamicum (optical density at 600 nm [OD 600 ] of about 3) was isolated as described recently by Schreiner et al (60). Aliquots of RNA were stored at Ϫ70°C.…”

Section: Methodsmentioning

confidence: 99%

“…This bacterium is an aerobic, "high-GC gram-positive" organism that grows on a variety of sugars and organic acids and is widely used in the industrial production of amino acids, particularly L-glutamate and L-lysine (35). Due to its importance for the distribution of the carbon flux within the metabolism and for the precursor supply for amino acid synthesis, the phosphoenolpyruvate-pyruvate node of this organism has been intensively studied (reviewed in reference 19), and much attention has been focused on pyruvate-converting enzymes, such as pyruvate carboxylase (34,49,50,51), pyruvate dehydrogenase complex (14,60,66), and PQO (59). Like the E. coli enzyme, the C. glutamicum PQO is a homotetrameric flavoprotein containing TPP, and it is activated by Triton X-100, phosphatidylglycerol and dipalmitoyl-phosphatidylglycerol.…”

mentioning

confidence: 99%

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Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum : Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

et al. 2006

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Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO 2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.Pyruvate:quinone oxidoreductase (PQO) (EC 1.2.2.2) catalyzes the oxidative decarboxylation of pyruvate to acetate and CO 2 with a quinone as the physiological electron acceptor. The Escherichia coli PQO enzyme, also designated pyruvate oxidase (POX), is a nonessential, peripheral membrane protein consisting of four identical subunits, each containing tightly bound flavin adenine dinucleotide (FAD) and loosely bound thiamine pyrophosphate (TPP) and Mg 2ϩ (4,22,37,38,75). This enzyme has been shown to be strongly activated by low concentrations of phospholipids and detergents (5,8,15,16,28), and the activation has been shown to be accompanied by conformational changes and alteration of various properties of the enzyme (11,12,74). Aside from extensive biochemical characterization of the E. coli PQO, the respective gene ( poxB) and its expression have been studied in detail (7, 9, 25). Expression of the poxB gene was dependent on sigma factor RpoS and thus induced in the stationary growth phase (7). Accordingly, the authors speculated that the enzyme might be responsible for oxidative pyruvate decarboxylation during the transition between the exponential aerobic growth phase and the stationary growth phase when cultures become anaerobic. Further studies with poxB inactivation mutants and with poxBoverexpressing strains of E. coli indicated that PQO activity contributes to the aerobic growth efficiency and that a high PQO activity, together with acetyl-coenzyme A (CoA) syn...

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Section: Discussionmentioning

confidence: 68%

Section: Analysis Of the Pqo Gene And Its Chromosomal Organizationmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum : Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

et al. 2006

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“…Chloramphenicol acetyltransferase (CAT) activity was determined as described by Schreiner et al (43).…”

Section: Promoter Binding Assays With His-tagged Rama and Ramb Fusionmentioning

confidence: 99%

The Alcohol Dehydrogenase Gene adhA in Corynebacterium glutamicum Is Subject to Carbon Catabolite Repression

Arndt

Eikmanns

2007

J Bacteriol

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Corynebacterium glutamicum has recently been shown to grow on ethanol as a carbon and energy source and to possess high alcohol dehydrogenase (ADH) activity when growing on this substrate and low ADH activity when growing on ethanol plus glucose or glucose alone. Here we identify the C. glutamicum ADH gene (adhA), analyze its transcriptional organization, and investigate the relevance of the transcriptional regulators of acetate metabolism RamA and RamB for adhA expression. Sequence analysis of adhA predicts a polypeptide of 345 amino acids showing up to 57% identity with zinc-dependent ADH enzymes of group I. Inactivation of the chromosomal adhA gene led to the inability to grow on ethanol and to the absence of ADH activity, indicating that only a single ethanol-oxidizing ADH enzyme is present in C. glutamicum. Transcriptional analysis revealed that the C. glutamicum adhA gene is monocistronic and that its expression is repressed in the presence of glucose and of acetate in the growth medium, i.e., that adhA expression is subject to catabolite repression. Further analyses revealed that RamA and RamB directly bind to the adhA promoter region, that RamA is essential for the expression of adhA, and that RamB exerts a negative control on adhA expression in the presence of glucose or acetate in the growth medium. However, since the glucose-and acetate-dependent down-regulation of adhA expression was only partially released in a RamB-deficient mutant, there might be an additional regulator involved in the catabolite repression of adhA.

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Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

Moxley

Brown

Eiteman

2023

Engineering in Life Sciences

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Several chromosomally expressed AceE variants were constructed in Escherichia coli ΔldhA ΔpoxB ΔppsA and compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of the budA and budB genes from Enterobacter cloacae ssp. dissolvens. The best acetoin‐producing strains were subsequently studied in controlled batch culture at the one‐liter scale. PDH variant strains attained up to four‐fold greater acetoin than the strain expressing the wild‐type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43 g/L of pyruvate‐derived products, acetoin (38.5 g/L) and 2R,3R‐butanediol (5.0 g/L), corresponding to an effective concentration of 59 g/L considering the dilution. The acetoin yield from glucose was 0.29 g/g with a volumetric productivity of 0.9 g/L·h (0.34 g/g and 1.0 g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network.

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E1 Enzyme of the Pyruvate Dehydrogenase Complex in Corynebacterium glutamicum : Molecular Analysis of the Gene and Phylogenetic Aspects

Cited by 78 publications

References 77 publications

Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum : Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum : Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

The Alcohol Dehydrogenase Gene adhA in Corynebacterium glutamicum Is Subject to Carbon Catabolite Repression

Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

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