scholarly journals Lysine secretion by wild-type Corynebacterium glutamicum triggered by dipeptide uptake

1993 ◽  
Vol 139 (12) ◽  
pp. 3115-3122 ◽  
Author(s):  
A. Erdmann ◽  
B. Weil ◽  
R. Kramer
Keyword(s):  
Corynebacterium Glutamicum ◽  
Wild Type ◽  
Dipeptide Uptake

scholarly journals Global Expression Profiling and Physiological Characterization of Corynebacterium glutamicum Grown in the Presence of l-Valine

2003 ◽  
Vol 69 (5) ◽  
pp. 2521-2532 ◽  
Author(s):  
C. Lange ◽  
D. Rittmann ◽  
V. F. Wendisch ◽  
M. Bott ◽  
H. Sahm
Keyword(s):  
Corynebacterium Glutamicum ◽  
Expression Profiling ◽  
Large Subunit ◽  
Mrna Level ◽  
Acetohydroxyacid Synthase ◽  
Limiting Factor ◽  
Unexpected Result ◽  
Dependent Manner ◽  
Wild Type ◽  
Concentration Dependent Manner

ABSTRACT Addition of l-valine (50 to 200 mM) to glucose minimal medium had no effect on the growth of wild-type Corynebacterium glutamicum ATCC 13032 but inhibited the growth of the derived valine production strain VAL1 [13032 ΔilvA ΔpanBC(pJC1ilvBNCD)] in a concentration-dependent manner. In order to explore this strain-specific valine effect, genomewide expression profiling was performed using DNA microarrays, which showed that valine caused an increased ilvBN mRNA level in VAL1 but not in the wild type. This unexpected result was confirmed by an increased cellular level of the ilvB protein product, i.e., the large subunit of acetohydroxyacid synthase (AHAS), and by an increased AHAS activity of valine-treated VAL1 cells. The conclusion that valine caused the limitation of another branched-chain amino acid was confirmed by showing that high concentrations of l-isoleucine could relieve the valine effect on VAL1 whereas l-leucine had the same effect as valine. The valine-caused isoleucine limitation was supported by the finding that the inhibitory valine effect was linked to the ilvA deletion that results in isoleucine auxotrophy. Taken together, these results implied that the valine effect is caused by competition for uptake of isoleucine by the carrier BrnQ, which transports all branched-chained amino acids. Indeed, valine inhibition could also be relieved by supplementing VAL1 with the dipeptide isoleucyl-isoleucine, which is taken up by a dipeptide transport system rather than by BrnQ. Interestingly, addition of external valine stimulated valine production by VAL1. This effect is most probably due to a reduced carbon usage for biomass production and to the increased expression of ilvBN, indicating that AHAS activity may still be a limiting factor for valine production in the VAL1 strain.

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 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 Role of the ssu and seu Genes of Corynebacterium glutamicum ATCC 13032 in Utilization of Sulfonates and Sulfonate Esters as Sulfur Sources

2005 ◽  
Vol 71 (10) ◽  
pp. 6104-6114 ◽  
Author(s):  
D. J. Koch ◽  
C. Rückert ◽  
D. A. Rey ◽  
A. Mix ◽  
A. Pühler ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Sequence Similarity ◽  
Gene Clusters ◽  
Flavin Mononucleotide ◽  
Gene Products ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Mutant Strains ◽  
Similarity Searches

ABSTRACT Corynebacterium glutamicum ATCC 13032 was found to be able to utilize a broad range of sulfonates and sulfonate esters as sulfur sources. The two gene clusters potentially involved in sulfonate utilization, ssuD1CBA and ssuI-seuABC-ssuD2, were identified in the genome of C. glutamicum ATCC 13032 by similarity searches. While the ssu genes encode proteins resembling Ssu proteins from Escherichia coli or Bacillus subtilis, the seu gene products exhibited similarity to the dibenzothiophene-degrading Dsz monooxygenases of Rhodococcus strain IGTS8. Growth tests with the C. glutamicum wild-type and appropriate mutant strains showed that the clustered genes ssuC, ssuB, and ssuA, putatively encoding the components of an ABC-type transporter system, are required for the utilization of aliphatic sulfonates. In C. glutamicum sulfonates are apparently degraded by sulfonatases encoded by ssuD1 and ssuD2. It was also found that the seu genes seuA, seuB, and seuC can effectively replace ssuD1 and ssuD2 for the degradation of sulfonate esters. The utilization of all sulfonates and sulfonate esters tested is dependent on a novel putative reductase encoded by ssuI. Obviously, all monooxygenases encoded by the ssu and seu genes, including SsuD1, SsuD2, SeuA, SeuB, and SeuC, which are reduced flavin mononucleotide dependent according to sequence similarity, have SsuI as an essential component. Using real-time reverse transcription-PCR, the ssu and seu gene cluster was found to be expressed considerably more strongly during growth on sulfonates and sulfonate esters than during growth on sulfate.

scholarly journals Heat Shock Proteome Analysis of Wild-Type Corynebacterium glutamicum ATCC 13032 and a Spontaneous Mutant Lacking GroEL1, a Dispensable Chaperone

2013 ◽  
Vol 195 (11) ◽  
pp. 2707-2707
Author(s):  
C. Barreiro ◽  
E. Gonzalez-Lavado ◽  
S. Brand ◽  
A. Tauch ◽  
J. F. Martin
Keyword(s):  
Heat Shock ◽  
Corynebacterium Glutamicum ◽  
Proteome Analysis ◽  
Spontaneous Mutant ◽  
Wild Type

scholarly journals Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

2005 ◽  
Vol 71 (1) ◽  
pp. 207-213 ◽  
Author(s):  
Veronika ElišÃ¯Â¿Â½kov� ◽  
Miroslav P�tek ◽  
Jiř� Hol�tko ◽  
Jan Nešvera ◽  
Damien Leyval ◽  
...  
Keyword(s):  
Amino Acids ◽  
Corynebacterium Glutamicum ◽  
Shuttle Vector ◽  
Branched Chain Amino Acids ◽  
Site Directed Mutagenesis ◽  
Enzyme Molecule ◽  
Acetohydroxy Acid Synthase ◽  
Wild Type ◽  
Allosteric Site ◽  
Branched Chain

ABSTRACT Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Kmr). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.

scholarly journals Identification and Characterization of the Dicarboxylate Uptake System DccT in Corynebacterium glutamicum

2008 ◽  
Vol 190 (19) ◽  
pp. 6458-6466 ◽  
Author(s):  
Jung-Won Youn ◽  
Elena Jolkver ◽  
Reinhard Krämer ◽  
Kay Marin ◽  
Volker F. Wendisch
Keyword(s):  
Corynebacterium Glutamicum ◽  
Tricarboxylic Acid Cycle ◽  
Carbon Sources ◽  
Mrna Levels ◽  
Uptake System ◽  
Dependent Manner ◽  
Wild Type ◽  
Prolonged Incubation ◽  
In The Wild ◽  
Biochemical Analyses

ABSTRACT Many bacteria can utilize C4-carboxylates as carbon and energy sources. However, Corynebacterium glutamicum ATCC 13032 is not able to use tricarboxylic acid cycle intermediates such as succinate, fumarate, and l-malate as sole carbon sources. Upon prolonged incubation, spontaneous mutants which had gained the ability to grow on succinate, fumarate, and l-malate could be isolated. DNA microarray analysis showed higher mRNA levels of cg0277, which subsequently was named dccT, in the mutants than in the wild type, and transcriptional fusion analysis revealed that a point mutation in the promoter region of dccT was responsible for increased expression. The overexpression of dccT was sufficient to enable the C. glutamicum wild type to grow on succinate, fumarate, and l-malate as the sole carbon sources. Biochemical analyses revealed that DccT, which is a member of the divalent anion/Na+ symporter family, catalyzes the effective uptake of dicarboxylates like succinate, fumarate, l-malate, and likely also oxaloacetate in a sodium-dependent manner.

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