Effect of Pyruvate Carboxylase Overexpression on the Physiology of
            <i>Corynebacterium glutamicum</i>

Koffas, Mattheos; Jung, Gyoo Yeol; Aon, Juan C.; Stephanopoulos, Gregory

doi:10.1128/aem.68.11.5422-5428.2002

Cited by 44 publications

(23 citation statements)

References 44 publications

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“…7). Such an effect has been previously observed in C. glutamicum (Koffas et al, 2002;Peters-Wendisch et al, 1998) and other organisms such as S. cerevisiae (Brewster et al, 1994). Our results indicate that certain carbon sources preferentially induce high levels of expression of one gene or a set of genes.…”

Section: Discussionsupporting

confidence: 70%

Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase

2007

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A basic pattern of gene expression and of relative expression levels during different growth phases was obtained for Corynebacterium glutamicum R grown on different carbon sources. The gapA-pgk-tpi-ppc gene cluster was transcribed as a mono-or polycistronic mRNA, depending on the growth phase. The 1.4 kb (gapA) and 2.3 kb (pgk-tip) mRNAs were expressed in the early through late exponential phases, whereas the 3.7 kb (gapA-pgk-tpi) and 5.4 kb (pgk-tpi-ppc) mRNAs were only detected in the mid-exponential phase. All other glycolytic genes except pps, glk and pgi were transcribed as monocistronic mRNAs under all tested conditions. Identification and alignment of the promoter regions of the transcriptional start sites of glycolytic genes revealed strong similarities to the s A consensus promoter sequences of Gram-positive bacteria.All genes involved in glycolysis were coordinately expressed in medium containing glucose. Growth in the presence of glucose gave rise to abundant expression of most glycolytic genes, with the level of gapA transcript being the highest. Glucose depletion led to a rapid repression of most glycolytic genes and a corresponding two-to fivefold increased expression of the gluconeogenic genes pps, pck and malE, which are induced by pyruvate, lactate, acetate and/or other organic acids.

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Section: Discussionsupporting

confidence: 70%

Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase

2007

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“…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.…”

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confidence: 99%

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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“…Analytic methods. The dry cell weight was estimated based on the correlation 1 OD 600 unit is equal to 0.28 g of dry cell weight/liter (17). The glucose concentration was determined using an SBA-40D biosensor automatic analyzer (Shandong, China).…”

Section: Figmentioning

confidence: 99%

Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum

Zhang

Shang

Lai

et al. 2012

Appl Environ Microbiol

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bCorynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the Larabinose regulator AraC, the P BAD promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P BAD -based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P BAD promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P BAD induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of ␣-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum. Corynebacterium glutamicum is one of the most important microorganisms for producing bulk amino acids and organic acids (18,44). The development of genetic tools has made it convenient to metabolically engineer specific traits in this bacterium (16,27). Through expressing exogenous gene clusters to construct new metabolic pathways, C. glutamicum has been engineered to produce a variety of biological materials, such as D-pantothenate, xylitol, trehalose, and polyhydroxybutyrate (2,15,19,32).As an important tool for molecular biology and metabolic engineering, an efficient inducible expression system should have several characteristics that include sensitivity to a nontoxic and inexpensive inducer, a wide dynamic rang regulation, and little or no leaky basal expression. To date, the P lac -derived promoter systems from Escherichia coli have been the most widely used controllable expression systems in corynebacteria; however, these expression systems exhibit a lower level of inducible expression in C. glutamicum and high basal expression under noninducing conditions (26). Although many attempts have been made to increase the expression and tight regulation of the P tac promoter, which is a hybrid promoter of P trp and P lacUV5 (45,46), the inducibility of these promoters remains relatively low as a result of the low isopropyl-␤-D-thiogalactopyranoside (IPTG) permeability of C. glutamicum strains (30). Moreover, the high cost and potential toxicity of IPTG are not ideal for industrial-scale protein expressio...

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Effect of Pyruvate Carboxylase Overexpression on the Physiology of Corynebacterium glutamicum

Cited by 44 publications

References 44 publications

Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase

Expression of Corynebacterium glutamicum glycolytic genes varies with carbon source and growth phase

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

Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum

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