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 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 4-Chlorobenzoate Uptake in Comamonas sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter

2006 ◽  
Vol 188 (24) ◽  
pp. 8407-8412 ◽  
Author(s):  
Jong-Chan Chae ◽  
Gerben J. Zylstra
Keyword(s):  
Growth Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proton Motive Force ◽  
Significant Inhibition ◽  
Motive Force ◽  
Wild Type ◽  
Knockout Mutant

ABSTRACT The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

scholarly journals Exopolyphosphatases PPX1 and PPX2 from Corynebacterium glutamicum

2009 ◽  
Vol 75 (10) ◽  
pp. 3161-3170 ◽  
Author(s):  
Steffen N. Lindner ◽  
Sandra Knebel ◽  
Hendrik Wesseling ◽  
Siegfried M. Schoberth ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Deletion Mutants ◽  
Gene Products ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Deletion Mutations ◽  
In The Wild ◽  
Growth Experiments

ABSTRACT Corynebacterium glutamicum accumulates up to 300 mM of inorganic polyphosphate (PolyP) in the cytosol or in granules. The gene products of cg0488 (ppx1) and cg1115 (ppx2) were shown to be active as exopolyphosphatases (PPX), as overexpression of either gene resulted in higher exopolyphosphatase activities in crude extracts and deletion of either gene with lower activities than those of the wild-type strain. PPX1 and PPX2 from C. glutamicum share only 25% identical amino acids and belong to different protein groups, which are distinct from enterobacterial, archaeal, and yeast exopolyphosphatases. In comparison to that in the wild type, more intracellular PolyP accumulated in the Δppx1 and Δppx2 deletion mutations but less when either ppx1 or ppx2 was overexpressed. When C. glutamicum was shifted from phosphate-rich to phosphate-limiting conditions, a growth advantage of the deletion mutants and a growth disadvantage of the overexpression strains compared to the wild type were observed. Growth experiments, exopolyphosphatase activities, and intracellular PolyP concentrations revealed PPX2 as being a major exopolyphosphatase from C. glutamicum. PPX2His was purified to homogeneity and shown to be active as a monomer. The enzyme required Mg2+ or Mn2+ cations but was inhibited by millimolar concentrations of Mg2+, Mn2+, and Ca2+. PPX2 from C. glutamicum was active with short-chain polyphosphates, even accepting pyrophosphate, and was inhibited by nucleoside triphosphates.

Collismycin A biosynthesis in Streptomyces sp. CS40 is regulated by iron levels through two pathway-specific regulators

Microbiology ◽  
2014 ◽  
Vol 160 (3) ◽  
pp. 467-478 ◽  
Author(s):  
Natalia M. Vior ◽  
Carlos Olano ◽  
Ignacio García ◽  
Carmen Méndez ◽  
José A. Salas
Keyword(s):  
Gene Cluster ◽  
Culture Medium ◽  
Wild Type Strain ◽  
Transcriptional Regulator ◽  
Moderate Increase ◽  
Wild Type ◽  
Environmental Stimulus ◽  
Fourfold Increase ◽  
In The Wild ◽  
Regulatory Model

Two putative pathway-specific regulators have been identified in the collismycin A gene cluster: ClmR1, belonging to the TetR-family, and the LuxR-family transcriptional regulator ClmR2. Inactivation of clmR1 led to a moderate increase of collismycin A yields along with an early onset of its production, suggesting an inhibitory role for the product of this gene. Inactivation of clmR2 abolished collismycin A biosynthesis, whereas overexpression of ClmR2 led to a fourfold increase in production yields, indicating that ClmR2 is an activator of collismycin A biosynthesis. Expression analyses of the collismycin gene cluster in the wild-type strain and in ΔclmR1 and ΔclmR2 mutants confirmed the role proposed for both regulatory genes, revealing that ClmR2 positively controls the expression of most of the genes in the cluster and ClmR1 negatively regulates both its own expression and that of clmR2. Additionally, production assays and further transcription analyses confirmed the existence of a higher regulatory level modulating collismycin A biosynthesis in response to iron concentrations in the culture medium. Thus, high iron levels inhibit collismycin A biosynthesis through the repression of clmR2 transcription. These results have allowed us to propose a regulatory model that integrates the effect of iron as the main environmental stimulus controlling collismycin A biosynthesis.

scholarly journals Discovery and Engineered Overproduction of Antimicrobial Nucleoside Antibiotic A201A from the Deep-Sea Marine Actinomycete Marinactinospora thermotolerans SCSIO 00652

2011 ◽  
Vol 56 (1) ◽  
pp. 110-114 ◽  
Author(s):  
Qinghua Zhu ◽  
Jun Li ◽  
Junying Ma ◽  
Minghe Luo ◽  
Bo Wang ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Deep Sea ◽  
Type Strain ◽  
Large Scale ◽  
Wild Type Strain ◽  
Production Capacity ◽  
Transcriptional Regulator ◽  
Scale Production ◽  
Wild Type ◽  
Content Type

ABSTRACTMarinactinospora thermotoleransSCSIO 00652, originating from a deep-sea marine sediment of the South China Sea, was discovered to produce antimicrobial nucleoside antibiotic A201A. Whole-genome scanning and annotation strategies enabled us to localize the genes responsible for A201A biosynthesis and to experimentally identify the gene cluster; inactivation ofmtdF, an oxidoreductase gene within the suspected gene cluster, abolished A201A production. Bioinformatics analysis revealed that a gene designatedmtdAfurthest upstream within the A201A biosynthetic gene cluster encodes a GntR family transcriptional regulator. To determine the role of MtdA in regulating A201A production, themtdAgene was inactivated in frame and the resulting ΔmtdAmutant was fermented alongside the wild-type strain as a control. High-performance liquid chromatography (HPLC) analyses of fermentation extracts revealed that the ΔmtdAmutant produced A201A in a yield ∼25-fold superior to that of the wild-type strain, thereby demonstrating thatMtdAis a negative transcriptional regulator governing A201A biosynthesis. By virtue of its high production capacity, the ΔmtdAmutant constitutes an ideal host for the efficient large-scale production of A201A. These results validateM. thermotoleransas an emerging source of antibacterial agents and highlight the efficiency of metabolic engineering for antibiotic titer improvement.

scholarly journals Glycerol Utilization Gene Cluster in Streptomyces clavuligerus

2009 ◽  
Vol 75 (9) ◽  
pp. 2991-2995 ◽  
Author(s):  
Sonia Baños ◽  
Rosario Pérez-Redondo ◽  
Bert Koekman ◽  
Paloma Liras
Keyword(s):  
Clavulanic Acid ◽  
Gene Cluster ◽  
Acid Production ◽  
Type Strain ◽  
Wild Type Strain ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Glycerol Utilization ◽  
In The Wild

ABSTRACT The Streptomyces clavuligerus ATCC 27064 glycerol cluster gylR-glpF1K1D1 is induced by glycerol but is not affected by glucose. S. clavuligerus growth and clavulanic acid production are stimulated by glycerol, but this does not occur in a glpK1-deleted mutant. Amplification of glpK1D1 results in transformants yielding larger amounts of clavulanic acid in the wild-type strain and in overproducer S. clavuligerus Gap15-7-30 or S. clavuligerus ΔrelA strains.

scholarly journals Light-inducible carotenoid production controlled by a MarR-type regulator in Corynebacterium glutamicum

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Satoru Sumi ◽  
Yuto Suzuki ◽  
Tetsuro Matsuki ◽  
Takahiro Yamamoto ◽  
Yudai Tsuruta ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Gene Cluster ◽  
Transcriptional Start Site ◽  
Transcriptional Regulator ◽  
Carotenoid Biosynthesis ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Biosynthesis Gene Cluster ◽  
Carotenoid Production

Abstract Carotenoid production in some non-phototropic bacteria occurs in a light-dependent manner to protect cells from photo-oxidants. Knowledge regarding the transcriptional regulator involved in the light-dependent production of carotenoids of non-phototrophic bacteria has been mainly confined to coenzyme B12-based photo-sensitive regulator CarH/LitR family proteins belonging to a MerR family transcriptional regulator. In this study, we found that bacteria belonging to Micrococcales and Corynebacteriales exhibit light-dependent carotenoid-like pigment production including an amino acid-producer Corynebacterium glutamicum AJ1511. CrtR is a putative MarR family transcriptional regulator located in the divergent region of a carotenoid biosynthesis gene cluster in the genome of those bacteria. A null mutant for crtR of C. glutamicum AJ1511 exhibited constitutive production of carotenoids independent of light. A complemented strain of the crtR mutant produced carotenoids in a light-dependent manner. Transcriptional analysis revealed that the expression of carotenoid biosynthesis genes is regulated in a light-dependent manner in the wild type, while the transcription was upregulated in the crtR mutant irrespective of light. In vitro experiments demonstrated that a recombinant CrtR protein binds to the specific sequences within the intergenic region of crtR and crtE, which corresponds to −58 to −7 for crtE, and +26 to −28 for crtR with respect to the transcriptional start site, and serves as a repressor for crtE transcription directed by RNA polymerase containing SigA. Taken together, the results indicate that CrtR light-dependently controls the expression of the carotenoid gene cluster in C. glutamicum and probably closely related Actinobacteria.

scholarly journals Expression of the Escherichia coli Catabolic Threonine Dehydratase in Corynebacterium glutamicum and Its Effect on Isoleucine Production

1999 ◽  
Vol 65 (7) ◽  
pp. 3100-3107 ◽  
Author(s):  
S. Guillouet ◽  
A. A. Rodal ◽  
G.-H. An ◽  
P. A. Lessard ◽  
A. J. Sinskey
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Carbon Balance ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Original Activity ◽  
Threonine Dehydratase ◽  
Fourfold Increase ◽  
Overexpressing Strain

ABSTRACT The catabolic or biodegradative threonine dehydratase (E.C. 4.2.1.16) of Escherichia coli is an isoleucine feedback-resistant enzyme that catalyzes the degradation of threonine to α-ketobutyrate, the first reaction of the isoleucine pathway. We cloned and expressed this enzyme in Corynebacterium glutamicum. We found that while the native threonine dehydratase of C. glutamicum was totally inhibited by 15 mM isoleucine, the heterologous catabolic threonine dehydratase expressed in the same strain was much less sensitive to isoleucine; i.e., it retained 60% of its original activity even in the presence of 200 mM isoleucine. To determine whether expressing the catabolic threonine dehydratase (encoded by the tdcB gene) provided any benefit for isoleucine production compared to the native enzyme (encoded by theilvA gene), fermentations were performed with the wild-type strain, an ilvA-overexpressing strain, and atdcB-expressing strain. By expressing the heterologous catabolic threonine dehydratase in C. glutamicum, we were able to increase the production of isoleucine 50-fold, whereas overexpression of the native threonine dehydratase resulted in only a fourfold increase in isoleucine production. Carbon balance data showed that when just one enzyme, the catabolic threonine dehydratase, was overexpressed, 70% of the carbon available for the lysine pathway was redirected into the isoleucine pathway.

scholarly journals CO-Dependent H2Production by Genetically Engineered Thermococcus onnurineus NA1

2013 ◽  
Vol 79 (6) ◽  
pp. 2048-2053 ◽  
Author(s):  
Min-Sik Kim ◽  
Seung Seob Bae ◽  
Yun Jae Kim ◽  
Tae Wan Kim ◽  
Jae Kyu Lim ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Steel Production ◽  
Transcriptional Analysis ◽  
Bioreactor Culture ◽  
Wild Type ◽  
Steel Mill ◽  
Engineered Strain

ABSTRACTHydrogenogenic CO oxidation (CO + H2O → CO2+ H2) has the potential for H2production as a clean renewable fuel.Thermococcus onnurineusNA1, which grows on CO and produces H2, has a unique gene cluster encoding the carbon monoxide dehydrogenase (CODH) and the hydrogenase. The gene cluster was identified as essential for carboxydotrophic hydrogenogenic metabolism by gene disruption and transcriptional analysis. To develop a strain producing high levels of H2, the gene cluster was placed under the control of a strong promoter. The resulting mutant, MC01, showed 30-fold-higher transcription of the mRNA encoding CODH, hydrogenase, and Na+/H+antiporter and a 1.8-fold-higher specific activity for CO-dependent H2production than did the wild-type strain. The H2production potential of the MC01 mutant in a bioreactor culture was 3.8-fold higher than that of the wild-type strain. The H2production rate of the engineered strain was severalfold higher than those of any other CO-dependent H2-producing prokaryotes studied to date. The engineered strain also possessed high activity for the bioconversion of industrial waste gases created as a by-product during steel production. This work represents the first demonstration of H2production from steel mill waste gas using a carboxydotrophic hydrogenogenic microbe.

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).

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