scholarly journals The ldhA Gene Encoding Fermentative L-Lactate Dehydrogenase in Corynebacterium glutamicum Is Positively Regulated by the Global Regulator GlxR

2021 ◽  
Vol 9 (3) ◽  
pp. 550
Author(s):  
Koichi Toyoda ◽  
Masayuki Inui
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Corynebacterium Glutamicum ◽  
Lactate Production ◽  
Exponential Phase ◽  
Aerobic Bacterium ◽  
Global Regulator ◽  
Gene Encoding ◽  
Fermentation Products ◽  
Ldha Gene

Bacterial metabolism shifts from aerobic respiration to fermentation at the transition from exponential to stationary growth phases in response to limited oxygen availability. Corynebacterium glutamicum, a Gram-positive, facultative aerobic bacterium used for industrial amino acid production, excretes L-lactate, acetate, and succinate as fermentation products. The ldhA gene encoding L-lactate dehydrogenase is solely responsible for L-lactate production. Its expression is repressed at the exponential phase and prominently induced at the transition phase. ldhA is transcriptionally repressed by the sugar-phosphate-responsive regulator SugR and L-lactate-responsive regulator LldR. Although ldhA expression is derepressed even at the exponential phase in the sugR and lldR double deletion mutant, a further increase in its expression is still observed at the stationary phase, implicating the action of additional transcription regulators. In this study, involvement of the cAMP receptor protein-type global regulator GlxR in the regulation of ldhA expression was investigated. The GlxR-binding site found in the ldhA promoter was modified to inhibit or enhance binding of GlxR. The ldhA promoter activity and expression of ldhA were altered in proportion to the binding affinity of GlxR. Similarly, L-lactate production was also affected by the binding site modification. Thus, GlxR was demonstrated to act as a transcriptional activator of ldhA.

scholarly journals The ldhA Gene, Encoding Fermentative l-Lactate Dehydrogenase of Corynebacterium glutamicum, Is under the Control of Positive Feedback Regulation Mediated by LldR

2009 ◽  
Vol 191 (13) ◽  
pp. 4251-4258 ◽  
Author(s):  
Koichi Toyoda ◽  
Haruhiko Teramoto ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Lactate Dehydrogenase ◽  
Corynebacterium Glutamicum ◽  
Promoter Region ◽  
Lactate Production ◽  
Feedback Regulation ◽  
Gene Encoding ◽  
Key Enzyme ◽  
In The Wild ◽  
Lactate Utilization ◽  
Ldha Gene

ABSTRACT Corynebacterium glutamicum ldhA encodes l-lactate dehydrogenase, a key enzyme that couples l-lactate production to reoxidation of NADH formed during glycolysis. We previously showed that in the absence of sugar, SugR binds to the ldhA promoter region, thereby repressing ldhA expression. In this study we show that LldR is another protein that binds to the ldhA promoter region, thus regulating ldhA expression. LldR has hitherto been characterized as an l-lactate-responsive transcriptional repressor of l-lactate utilization genes. Transposon mutagenesis of a reporter strain carrying a chromosomal ldhA promoter-lacZ fusion (PldhA-lacZ) revealed that ldhA disruption drastically decreased expression of PldhA-lacZ. PldhA-lacZ expression in the ldhA mutant was restored by deletion of lldR, suggesting that LldR acts as a repressor of ldhA in the absence of l-lactate and the LldR-mediated repression is not relieved in the ldhA mutant due to its inability to produce l-lactate. lldR deletion did not affect PldhA-lacZ expression in the wild-type background during growth on either glucose, acetate, or l-lactate. However, it upregulated PldhA-lacZ expression in the sugR mutant background during growth on acetate. The binding sites of LldR and SugR are located around the −35 and −10 regions of the ldhA promoter, respectively. C. glutamicum ldhA expression is therefore primarily repressed by SugR in the absence of sugar. In the presence of sugar, SugR-mediated repression of ldhA is alleviated, and ldhA expression is additionally enhanced by LldR inactivation in response to l-lactate produced by LdhA.

scholarly journals The Global Repressor SugR Controls Expression of Genes of Glycolysis and of the l-Lactate Dehydrogenase LdhA in Corynebacterium glutamicum

2008 ◽  
Vol 190 (24) ◽  
pp. 8033-8044 ◽  
Author(s):  
Verena Engels ◽  
Steffen N. Lindner ◽  
Volker F. Wendisch
Keyword(s):  
Lactate Dehydrogenase ◽  
Corynebacterium Glutamicum ◽  
Target Genes ◽  
Lactate Production ◽  
Oxygen Deprivation ◽  
Mrna Levels ◽  
Phosphotransferase System ◽  
Gel Retardation ◽  
Activity Measurements

ABSTRACT The transcriptional regulator SugR from Corynebacterium glutamicum represses genes of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). Growth experiments revealed that the overexpression of sugR not only perturbed the growth of C. glutamicum on the PTS sugars glucose, fructose, and sucrose but also led to a significant growth inhibition on ribose, which is not taken up via the PTS. Chromatin immunoprecipitation combined with DNA microarray analysis and gel retardation experiments were performed to identify further target genes of SugR. Gel retardation analysis confirmed that SugR bound to the promoter regions of genes of the glycolytic enzymes 6-phosphofructokinase (pfkA), fructose-1,6-bisphosphate aldolase (fba), enolase (eno), pyruvate kinase (pyk), and NAD-dependent l-lactate dehydrogenase (ldhA). The deletion of sugR resulted in increased mRNA levels of eno, pyk, and ldhA in acetate medium. Enzyme activity measurements revealed that SugR-mediated repression affects the activities of PfkA, Fba, and LdhA in vivo. As the deletion of sugR led to increased LdhA activity under aerobic and under oxygen deprivation conditions, l-lactate production by C. glutamicum was determined. The overexpression of sugR reduced l-lactate production by about 25%, and sugR deletion increased l-lactate formation under oxygen deprivation conditions by threefold. Thus, SugR functions as a global repressor of genes of the PTS, glycolysis, and fermentative l-lactate dehydrogenase in C. glutamicum.

Regulation of the ldhA gene, encoding the fermentative lactate dehydrogenase of Escherichia coli

Microbiology ◽  
2001 ◽  
Vol 147 (9) ◽  
pp. 2437-2446 ◽  
Author(s):  
Gene Ruijun Jiang ◽  
Sonia Nikolova ◽  
David P Clark
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Gene Encoding ◽  
Ldha Gene

scholarly journals Involvement of the Global Regulator GlxR in 3-Hydroxybenzoate and Gentisate Utilization by Corynebacterium glutamicum

2014 ◽  
Vol 80 (14) ◽  
pp. 4215-4225 ◽  
Author(s):  
Hongjun Chao ◽  
Ning-Yi Zhou
Keyword(s):  
Binding Site ◽  
Corynebacterium Glutamicum ◽  
Promoter Sequence ◽  
Negative Regulation ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Global Regulator ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Content Type

ABSTRACTCorynebacterium glutamicumis an industrially important producer of amino acids and organic acids, as well as an emerging model system for aromatic assimilation. An IclR-type regulator GenR has been characterized to activate the transcription ofgenDFMandgenKHoperons for 3-hydroxybenzoate and gentisate catabolism and represses its own expression. On the other hand, GlxR, a global regulator of the cyclic AMP (cAMP) receptor protein-fumarate nitrate reductase regulator (CRP-FNR) type, was also predicted to be involved in this pathway. In this study, electrophoretic mobility shift assays and footprinting analyses demonstrated that GlxR bound to three sites in the promoter regions of threegenoperons. A combination of site-directed mutagenesis of the biding sites, promoter activity assay, and GlxR overexpression demonstrated that GlxR repressed their expression by binding these sites. One GlxR binding site (DFMx) was found to be located −13 to +8 bp upstream of thegenDFMpromoter, which was involved in negative regulation ofgenDFMtranscription. The GlxR binding site R-KHx01 (located between positions −11 to +5) was upstream of thegenKHpromoter sequence and involved in negative regulation of its transcription. The binding site R-KHx02, at which GlxR binds togenRpromoter to repress its expression, was found within a footprint extending from positions −71 to −91 bp. These results reveal that GlxR represses the transcription of all threegenoperons and then contributes to the synchronization of their expression for 3-hydroxybenzoate and gentisate catabolism in collaboration with the specific regulator GenR.

scholarly journals Identification of a Gene Encoding a Transporter Essential for Utilization of C4 Dicarboxylates in Corynebacterium glutamicum

2008 ◽  
Vol 74 (17) ◽  
pp. 5290-5296 ◽  
Author(s):  
Haruhiko Teramoto ◽  
Tomokazu Shirai ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Exponential Phase ◽  
Wild Type ◽  
Aerobic Growth ◽  
Early Exponential Phase ◽  
Gene Encoding ◽  
Transporter Family ◽  
Genes Encoding

ABSTRACT The Corynebacterium glutamicum R genome contains a total of eight genes encoding proteins with sequence similarity to C4-dicarboxylate transporters identified from other bacteria. Three of the genes encode proteins within the dicarboxylate/amino acid:cation symporter (DAACS) family, another three encode proteins within the tripartite ATP-independent periplasmic transporter family, and two encode proteins within the divalent anion:Na+ symporter (DASS) family. We observed that a mutant strain deficient in one of these genes, designated dcsT, of the DASS family did not aerobically grow on the C4 dicarboxylates succinate, fumarate, and malate as the sole carbon sources. Mutant strains deficient in each of the other seven genes grew as well as the wild-type strain under the same conditions, although one of these genes is a homologue of dctA of the DAACS family, involved in aerobic growth on C4 dicarboxylates in various bacteria. The utilization of C4 dicarboxylates was markedly enhanced by overexpression of the dcsT gene. We confirmed that the uptake of [13C]labeled succinate observed for the wild-type cells was hardly detected in the dcsT-deficient mutant but was markedly enhanced in a dcsT-overexpressing strain. These results suggested that in C. glutamicum, the uptake of C4 dicarboxylates for aerobic growth was mainly mediated by the DASS transporter encoded by dcsT. The expression level of the dcsT gene transiently increased in the early exponential phase during growth on nutrient-rich medium. This expression was enhanced by the addition of succinate in the mid-exponential phase and was repressed by the addition of glucose in the early exponential phase.

scholarly journals Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1185-1195 ◽  
Author(s):  
Taiho Kambe ◽  
Junko Tada ◽  
Mariko Chikuma ◽  
Seiji Masuda ◽  
Masaya Nagao ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Embryonal Carcinoma ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Dependent Manner ◽  
P19 Cells ◽  
Independent Manner ◽  
Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

Removal of Anionic Metalloid/Metals by PEI-Modified Corynebacterium glutamicum

2013 ◽  
Vol 825 ◽  
pp. 568-571
Author(s):  
Namgyu Kim ◽  
Munsik Park ◽  
Jongmoon Park ◽  
Donghee Park
Keyword(s):  
Aqueous Solution ◽  
Binding Site ◽  
Corynebacterium Glutamicum ◽  
Adsorption Mechanism ◽  
Competitive Inhibition ◽  
Aqueous System ◽  
Reduction Mechanism ◽  
Removal Mechanism ◽  
Sulfate Ion ◽  
Industrial Fermentation

An anionic biosorbent was derived from an industrial fermentation biowate, Corynebacterium glutamicum, by being cross-linked with polyethylenimine (PEI). A fiber form of the biosorbent was used to examine its potentiality of removing anionic metals such as As (V), Cr (VI) and Mn (VII) in aqueous system. As (V) and Cr (VI) were efficiently removed by the biosorbent through anionic adsorption mechanism. Sulfate ion seriously inhibited adsorption of the anionic metals through competitive inhibition with respect to the binding site of the biosorbent. In the case of Mn (VII), its removal mechanism by the biosorbent was not anionic adsorption. Mn (VII) was completely removed in aqueous phase, meanwhile, Mn (II) appeared and increased in proportion to the Mn (VII) depletion. As a result, adsorption coupled reduction was chosen as the mechanism of Mn (VII) removal by the biosorbent. In conclusion, the anionic biosorbent could be used to remove various anionic metals from aqueous solution through anionic adsorption or reduction mechanism.

scholarly journals Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
Author(s):  
Jens Buchholz ◽  
Andreas Schwentner ◽  
Britta Brunnenkan ◽  
Christina Gabris ◽  
Simon Grimm ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Fed Batch ◽  
Complex Activity ◽  
Gene Encoding ◽  
Content Type ◽  
Genes Encoding ◽  
Cell Densities ◽  
Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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