scholarly journals C1Metabolism in Corynebacterium glutamicum: an Endogenous Pathway for Oxidation of Methanol to Carbon Dioxide

2013 ◽  
Vol 79 (22) ◽  
pp. 6974-6983 ◽  
Author(s):  
Sabrina Witthoff ◽  
Alice Mühlroth ◽  
Jan Marienhagen ◽  
Michael Bott
Keyword(s):  
Corynebacterium Glutamicum ◽  
Methanol Oxidation ◽  
Transcriptional Regulator ◽  
Global Gene Expression ◽  
Industrial Biotechnology ◽  
Wild Type ◽  
Content Type ◽  
Oxidation Of Methanol ◽  
Dehydrogenase Gene ◽  
Important Host

ABSTRACTMethanol is considered an interesting carbon source in “bio-based” microbial production processes. SinceCorynebacterium glutamicumis an important host in industrial biotechnology, in particular for amino acid production, we performed studies of the response of this organism to methanol. TheC. glutamicumwild type was able to convert13C-labeled methanol to13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be upregulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase geneadhAshowed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The ΔaldΔadhEand ΔaldΔmshCdeletion mutants were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2was still possible. The oxidation of formate to CO2is catalyzed by the formate dehydrogenase FdhF, recently identified by us. Similar to the case with ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression ofadhAandald. In conclusion, we were able to show thatC. glutamicumpossesses an endogenous pathway for methanol oxidation to CO2and to identify the enzymes and a transcriptional regulator involved in this pathway.

scholarly journals MdoR Is a Novel Positive Transcriptional Regulator for the Oxidation of Methanol in Mycobacterium sp. Strain JC1

2011 ◽  
Vol 193 (22) ◽  
pp. 6288-6294 ◽  
Author(s):  
Hyuk Park ◽  
Young T. Ro ◽  
Young M. Kim
Keyword(s):  
Methanol Oxidation ◽  
Promoter Region ◽  
Transcriptional Regulator ◽  
Sole Source ◽  
Binding Motif ◽  
Mobility Shift ◽  
Content Type ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Oxidation Of Methanol

Mycobacteriumsp. strain JC1 is able to grow on methanol as a sole source of carbon and energy using methanol:N,N′-dimethyl-4-nitrosoaniline oxidoreductase (MDO) as a key enzyme for methanol oxidation. The second open reading frame (mdoR) upstream of, and running divergently from, themdogene was identified as a gene for a TetR family transcriptional regulator. The N-terminal region of MdoR contained a helix-turn-helix DNA-binding motif. An electrophoretic mobility shift assay (EMSA) indicated that MdoR could bind to amdopromoter region containing an inverted repeat. ThemdoRdeletion mutant did not grow on methanol, but growth on methanol was restored by a plasmid containing an intactmdoRgene. In DNase I footprinting and EMSA experiments, MdoR bound to two inverted repeats in the putativemdoRpromoter region. Reverse transcription-PCR indicated that themdoRgene was transcribed only in cells growing on methanol, whereas β-galactosidase assays showed that themdoRpromoter was activated in the presence of methanol. These results indicate that MdoR serves as a transcriptional activator for the expression ofmdoand its own gene. Also, MdoR is the first discovered member of the TetR family of transcriptional regulators to be involved in the regulation of the methanol oxidation, as well as to function as a positive autoregulator.

scholarly journals Metabolic Engineering of Corynebacterium glutamicum for Methanol Metabolism

2015 ◽  
Vol 81 (6) ◽  
pp. 2215-2225 ◽  
Author(s):  
Sabrina Witthoff ◽  
Katja Schmitz ◽  
Sebastian Niedenführ ◽  
Katharina Nöh ◽  
Stephan Noack ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biotechnological Production ◽  
Methanol Metabolism ◽  
Initial Oxidation ◽  
Content Type ◽  
Oxidation Of Methanol ◽  
Bacillus Methanolicus ◽  
Production Processes ◽  
Starting Point ◽  
Limited Application

ABSTRACTMethanol is already an important carbon feedstock in the chemical industry, but it has found only limited application in biotechnological production processes. This can be mostly attributed to the inability of most microbial platform organisms to utilize methanol as a carbon and energy source. With the aim to turn methanol into a suitable feedstock for microbial production processes, we engineered the industrially important but nonmethylotrophic bacteriumCorynebacterium glutamicumtoward the utilization of methanol as an auxiliary carbon source in a sugar-based medium. Initial oxidation of methanol to formaldehyde was achieved by heterologous expression of a methanol dehydrogenase fromBacillus methanolicus, whereas assimilation of formaldehyde was realized by implementing the two key enzymes of the ribulose monophosphate pathway ofBacillus subtilis: 3-hexulose-6-phosphate synthase and 6-phospho-3-hexuloisomerase. The recombinantC. glutamicumstrain showed an average methanol consumption rate of 1.7 ± 0.3 mM/h (mean ± standard deviation) in a glucose-methanol medium, and the culture grew to a higher cell density than in medium without methanol. In addition, [13C]methanol-labeling experiments revealed labeling fractions of 3 to 10% in the m + 1 mass isotopomers of various intracellular metabolites. In the background of aC. glutamicumΔaldΔadhEmutant being strongly impaired in its ability to oxidize formaldehyde to CO2, the m + 1 labeling of these intermediates was increased (8 to 25%), pointing toward higher formaldehyde assimilation capabilities of this strain. The engineeredC. glutamicumstrains represent a promising starting point for the development of sugar-based biotechnological production processes using methanol as an auxiliary substrate.

scholarly journals A Novel CO-Responsive Transcriptional Regulator and Enhanced H2Production by an Engineered Thermococcus onnurineus NA1 Strain

2014 ◽  
Vol 81 (5) ◽  
pp. 1708-1714 ◽  
Author(s):  
Min-Sik Kim ◽  
Ae Ran Choi ◽  
Seong Hyuk Lee ◽  
Hae-Chang Jung ◽  
Seung Seob Bae ◽  
...  
Keyword(s):  
Production Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory System ◽  
Transcriptional Regulator ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Content Type ◽  
Transcriptional Regulatory

ABSTRACTGenome analysis revealed the existence of a putative transcriptional regulatory system governing CO metabolism inThermococcus onnurineusNA1, a carboxydotrophic hydrogenogenic archaeon. The regulatory system is composed of CorQ with a 4-vinyl reductase domain and CorR with a DNA-binding domain of the LysR-type transcriptional regulator family in close proximity to the CO dehydrogenase (CODH) gene cluster. Homologous genes of the CorQR pair were also found in the genomes ofThermococcusspecies and “CandidatusKorarchaeum cryptofilum” OPF8. In-frame deletion of eithercorQorcorRcaused a severe impairment in CO-dependent growth and H2production. WhencorQandcorRdeletion mutants were complemented by introducing thecorQRgenes under the control of a strong promoter, the mRNA and protein levels of the CODH gene were significantly increased in a ΔCorR strain complemented with integratedcorQR(ΔCorR/corQR↑) compared with those in the wild-type strain. In addition, the ΔCorR/corQR↑strain exhibited a much higher H2production rate (5.8-fold) than the wild-type strain in a bioreactor culture. The H2production rate (191.9 mmol liter−1h−1) and the specific H2production rate (249.6 mmol g−1h−1) of this strain were extremely high compared with those of CO-dependent H2-producing prokaryotes reported so far. These results suggest that thecorQRgenes encode a positive regulatory protein pair for the expression of a CODH gene cluster. The study also illustrates that manipulation of the transcriptional regulatory system can improve biological H2production.

scholarly journals AraR, an l-Arabinose-Responsive Transcriptional Regulator in Corynebacterium glutamicum ATCC 31831, Exerts Different Degrees of Repression Depending on the Location of Its Binding Sites within the Three Target Promoter Regions

2015 ◽  
Vol 197 (24) ◽  
pp. 3788-3796 ◽  
Author(s):  
Takayuki Kuge ◽  
Haruhiko Teramoto ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Binding Sites ◽  
Large Scale ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Primary Role ◽  
Promoter Regions ◽  
Independent Manner ◽  
Content Type

ABSTRACTInCorynebacterium glutamicumATCC 31831, a LacI-type transcriptional regulator AraR, represses the expression ofl-arabinose catabolism (araBDA), uptake (araE), and the regulator (araR) genes clustered on the chromosome. AraR binds to three sites: one (BSB) between the divergent operons (araBDAandgalM-araR) and two (BSE1and BSE2) upstream ofaraE.l-Arabinose acts as an inducer of the AraR-mediated regulation. Here, we examined the roles of these AraR-binding sites in the expression of the AraR regulon. BSBmutation resulted in derepression of botharaBDAandgalM-araRoperons. The effects of BSE1and/or BSE2mutation onaraEexpression revealed that the two sites independently function as theciselements, but BSE1plays the primary role. However, AraR was shown to bind to these sites with almost the same affinityin vitro. Taken together, the expression ofaraBDAandaraEis strongly repressed by binding of AraR to a single site immediately downstream of the respective transcriptional start sites, whereas the binding site overlapping the −10 or −35 region of thegalM-araRandaraEpromoters is less effective in repression. Furthermore, downregulation ofaraBDAandaraEdependent onl-arabinose catabolism observed in the BSBmutant and the AraR-independentaraRpromoter identified withingalM-araRadd complexity to regulation of the AraR regulon derepressed byl-arabinose.IMPORTANCECorynebacterium glutamicumhas a long history as an industrial workhorse for large-scale production of amino acids. An important aspect of industrial microorganisms is the utilization of the broad range of sugars for cell growth and production process. MostC. glutamicumstrains are unable to use a pentose sugarl-arabinose as a carbon source. However, genes forl-arabinose utilization and its regulation have been recently identified inC. glutamicumATCC 31831. This study elucidates the roles of the multiple binding sites of the transcriptional repressor AraR in the derepression byl-arabinose and thereby highlights the complex regulatory feedback loops in combination withl-arabinose catabolism-dependent repression of the AraR regulon in an AraR-independent manner.

scholarly journals Identification and Functional Analysis of the Gene Cluster for l-Arabinose Utilization in Corynebacterium glutamicum

2009 ◽  
Vol 75 (11) ◽  
pp. 3419-3429 ◽  
Author(s):  
Hideo Kawaguchi ◽  
Miho Sasaki ◽  
Alain A. Vertès ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Growth Rate ◽  
Corynebacterium Glutamicum ◽  
Gene Cluster ◽  
Wild Type Strain ◽  
Transcriptional Regulator ◽  
Transcriptional Unit ◽  
Gene Products ◽  
Wild Type ◽  
Mutant Cells ◽  
Arabinose Isomerase

ABSTRACT Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter−1) but not at a higher concentration of l-arabinose (40 g liter−1). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

scholarly journals A Gain-of-Function Mutation in Gating of Corynebacterium glutamicum NCgl1221 Causes Constitutive Glutamate Secretion

2012 ◽  
Vol 78 (15) ◽  
pp. 5432-5434 ◽  
Author(s):  
Yoshitaka Nakayama ◽  
Kenjiro Yoshimura ◽  
Hidetoshi Iida
Keyword(s):  
Patch Clamp ◽  
Corynebacterium Glutamicum ◽  
Mechanosensitive Channel ◽  
Wild Type ◽  
Gain Of Function ◽  
Content Type ◽  
Wild Type Counterpart

ABSTRACTThe A-to-V mutation at position 111 (A111V) in the mechanosensitive channel NCgl1221 (MscCG) causes constitutive glutamate secretion inCorynebacterium glutamicum. Patch clamp experiments revealed that NCgl1221 (A111V) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of NCgl1221 leads to the oversecretion of glutamate.

scholarly journals Development of Biotin-Prototrophic and -Hyperauxotrophic Corynebacterium glutamicum Strains

2013 ◽  
Vol 79 (15) ◽  
pp. 4586-4594 ◽  
Author(s):  
Masato Ikeda ◽  
Aya Miyamoto ◽  
Sumire Mutoh ◽  
Yuko Kitano ◽  
Mei Tajima ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Type Strain ◽  
Wild Type Strain ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Pimelic Acid ◽  
Wild Type ◽  
Biotin Synthase ◽  
Content Type ◽  
Carbon Carbon Bond

ABSTRACTTo develop the infrastructure for biotin production through naturally biotin-auxotrophicCorynebacterium glutamicum, we attempted to engineer the organism into a biotin prototroph and a biotin hyperauxotroph. To confer biotin prototrophy on the organism, the cotranscribedbioBFgenes ofEscherichia coliwere introduced into theC. glutamicumgenome, which originally lacked thebioFgene. The resulting strain still required biotin for growth, but it could be replaced by exogenous pimelic acid, a source of the biotin precursor pimelate thioester linked to either coenzyme A (CoA) or acyl carrier protein (ACP). To bridge the gap between the pimelate thioester and its dedicated precursor acyl-CoA (or -ACP), thebioIgene ofBacillus subtilis, which encoded a P450 protein that cleaves a carbon-carbon bond of an acyl-ACP to generate pimeloyl-ACP, was further expressed in the engineered strain by using a plasmid system. This resulted in a biotin prototroph that is capable of thede novosynthesis of biotin. On the other hand, thebioYgene responsible for biotin uptake was disrupted in wild-typeC. glutamicum. Whereas the wild-type strain required approximately 1 μg of biotin per liter for normal growth, thebioYdisruptant (ΔbioY) required approximately 1 mg of biotin per liter, almost 3 orders of magnitude higher than the wild-type level. The ΔbioYstrain showed a similar high requirement for the precursor dethiobiotin, a substrate forbioB-encoded biotin synthase. To eliminate the dependency on dethiobiotin, thebioBgene was further disrupted in both the wild-type strain and the ΔbioYstrain. By selectively using the resulting two strains (ΔbioBand ΔbioBY) as indicator strains, we developed a practical biotin bioassay system that can quantify biotin in the seven-digit range, from approximately 0.1 μg to 1 g per liter. This bioassay proved that the engineered biotin prototroph ofC. glutamicumproduced biotin directly from glucose, albeit at a marginally detectable level (approximately 0.3 μg per liter).

scholarly journals Transcriptome Sequencing Data of Bacillus anthracis Vollum ΔhtrA and Its Parental Strain, Isolated under Oxidative Stress

2020 ◽  
Vol 9 (35) ◽  
Author(s):  
Theodor Chitlaru ◽  
Inbar Cohen-Gihon ◽  
Ofir Israeli ◽  
Uri Elia ◽  
Galia Zaide ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Bacillus Anthracis ◽  
Transcriptome Sequencing ◽  
Global Gene Expression ◽  
Data Sets ◽  
Transcriptome Data ◽  
Wild Type ◽  
Sequencing Data ◽  
Content Type

ABSTRACT The high-temperature requirement chaperone/protease (HtrA) is involved in the stress response of the anthrax-causing pathogen Bacillus anthracis. Resilience to oxidative stress is essential for the manifestation of B. anthracis pathogenicity. Here, we announce transcriptome data sets detailing global gene expression in B. anthracis wild-type and htrA-disrupted strains following H2O2-induced oxidative stress.

scholarly journals The LysR-Type Transcriptional Regulator CrgA Negatively Regulates the Flagellar Master Regulator flhDC in Ralstonia solanacearum GMI1000

2020 ◽  
Vol 203 (1) ◽  
Author(s):  
Xiaojing Fan ◽  
Zhiwen Zhao ◽  
Tingyan Sun ◽  
Wei Rou ◽  
Caiying Gui ◽  
...  
Keyword(s):  
Cell Motility ◽  
Ralstonia Solanacearum ◽  
Host Plants ◽  
Transcriptional Regulator ◽  
Growth Defect ◽  
Wild Type ◽  
Mobility Shift ◽  
Content Type ◽  
Gus Reporter ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC. In addition, the ΔcrgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC. Despite these alterations, the ΔcrgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato (Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants. IMPORTANCE Ralstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum because it allows this pathogen to efficiently invade and colonize host plants. In R. solanacearum, motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum.

scholarly journals Construction of a Prophage-Free Variant of Corynebacterium glutamicum ATCC 13032 for Use as a Platform Strain for Basic Research and Industrial Biotechnology

2013 ◽  
Vol 79 (19) ◽  
pp. 6006-6015 ◽  
Author(s):  
Meike Baumgart ◽  
Simon Unthan ◽  
Christian Rückert ◽  
Jasintha Sivalingam ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Stress Conditions ◽  
Phenotypic Characterization ◽  
Industrial Biotechnology ◽  
Plasmid Copy ◽  
Enhanced Yellow Fluorescent Protein ◽  
Modification System ◽  
Content Type

ABSTRACTThe activity of bacteriophages and phage-related mobile elements is a major source for genome rearrangements and genetic instability of their bacterial hosts. The genome of the industrial amino acid producerCorynebacterium glutamicumATCC 13032 contains three prophages (CGP1, CGP2, and CGP3) of so far unknown functionality. Several phage genes are regularly expressed, and the large prophage CGP3 (∼190 kbp) has recently been shown to be induced under certain stress conditions. Here, we present the construction of MB001, a prophage-free variant ofC. glutamicumATCC 13032 with a 6% reduced genome. This strain does not show any unfavorable properties during extensive phenotypic characterization under various standard and stress conditions. As expected, we observed improved growth and fitness of MB001 under SOS-response-inducing conditions that trigger CGP3 induction in the wild-type strain. Further studies revealed that MB001 has a significantly increased transformation efficiency and produced about 30% more of the heterologous model protein enhanced yellow fluorescent protein (eYFP), presumably as a consequence of an increased plasmid copy number. These effects were attributed to the loss of the restriction-modification system (cg1996-cg1998) located within CGP3. The deletion of the prophages without any negative effect results in a novel platform strain for metabolic engineering and represents a useful step toward the construction of aC. glutamicumchassis genome of strain ATCC 13032 for biotechnological applications and synthetic biology.

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