Mutational analysis of the pentose phosphate pathway and the Entner-Doudoroff pathway in Gluconobacter oxydans reveals improved growth of an edd-eda deletion mutant on mannitol
Ontology highlight
ABSTRACT:
ORGANISM(S): Gluconobacter oxydans 621H
PROVIDER: GSE38933 | GEO | 2012/08/01
SECONDARY ACCESSION(S): PRJNA169427
REPOSITORIES: GEO
Similar Datasets
Project description:The obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H oxidizes sugars and sugar alcohols primarily in the periplasm and only a small fraction is metabolized in the cytoplasm. The latter can occur either via the Entner-Doudoroff pathway (EDP) or via the pentose phosphate pathway (PPP). The Embden-Meyerhof pathway is non-functional and a cyclic operation of the tricarboxylic acid cycle is prevented by the absence of succinate dehydrogenase. In this work, the cytoplasmic catabolism of fructose formed by oxidation of mannitol was analyzed with a M-NM-^Tgnd mutant lacking the oxidative PPP and a M-NM-^Tedd-eda mutant devoid of the EDP. The growth characteristics of the two mutants under controlled conditions with mannitol as carbon source and enzyme activities showed that the PPP is the main route for cytoplasmic fructose catabolism, whereas the EDP is dispensable and even unfavorable. The M-NM-^Tedd-eda mutant (lacking 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) formed 24% more cell mass than the reference strain. In contrast, deletion of gnd (6-phosphogluconate dehydrogenase) severely inhibited growth and caused a strong selection pressure for secondary mutations inactivating glucose-6-phosphate dehydrogenase, thus preventing fructose catabolism via the EDP, too. These M-NM-^Tgnd zwf* mutants were almost totally disabled in fructose catabolism, but still produced about 14% of the carbon dioxide of the reference strain, possibly by catabolizing substrates from the yeast extract. Overexpression of gnd in the reference strain improved biomass formation in a similar manner as deletion of edd-eda, further confirming the importance of the PPP for cytoplasmic fructose catabolism. The three transcriptome comparisons of M-NM-^Tupp M-NM-^Tgnd zwf* vs. M-NM-^Tupp and of M-NM-^Tupp M-NM-^Tedd-eda vs. M-NM-^Tupp, and of M-NM-^Tupp M-NM-^Tgnd versus M-NM-^Tupp, were repeated independently three times in biological replicates resulting in 3 x 3 hybridizations as termed by sample 1 to 9.
2012-07-31 | E-GEOD-38933 | biostudies-arrayexpress
Project description:In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by 13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For 13C-MFA, cells were cultivated with specifically 13C-labeled glucose and intracellular metabolites were analyzed for their labeling pattern by LC-MS. In growth phase I, 90% of the glucose was oxidized periplasmatically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can only be metabolized via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP). 13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phase I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II. The transcriptome comparisons of G. oxydans growth phase II vs. growth phase I were repeated independently three times in biological replicates resulting in 3 hybridizations as termed by sample 1 to 3.
2013-02-13 | E-GEOD-42223 | biostudies-arrayexpress
Project description:In this study, the distribution and regulation of periplasmic and cytoplasmic carbon fluxes in Gluconobacter oxydans 621H with glucose were studied by 13C-based metabolic flux analysis (13C-MFA) in combination with transcriptomics and enzyme assays. For 13C-MFA, cells were cultivated with specifically 13C-labeled glucose and intracellular metabolites were analyzed for their labeling pattern by LC-MS. In growth phase I, 90% of the glucose was oxidized periplasmatically to gluconate and partially further oxidized to 2-ketogluconate. Of the glucose taken up by the cells, 9% was phosphorylated to glucose 6-phosphate, whereas 91% was oxidized by cytoplasmic glucose dehydrogenase to gluconate. Additional gluconate was taken up into the cells by transport. Of the cytoplasmic gluconate, 70% was oxidized to 5-ketogluconate and 30% was phosphorylated to 6-phosphogluconate. In growth phase II, 87% of gluconate was oxidized to 2-ketogluconate in the periplasm and 13% was taken up by the cells and almost completely converted to 6-phosphogluconate. Since G. oxydans lacks phosphofructokinase, glucose 6-phosphate can only be metabolized via the oxidative pentose phosphate pathway (PPP) or the Entner-Doudoroff pathway (EDP). 13C-MFA showed that 6-phosphogluconate is catabolized primarily via the oxidative PPP in both phase I and II (62% and 93%) and demonstrated a cyclic carbon flux through the oxidative PPP. The transcriptome comparison revealed an increased expression of PPP genes in growth phase II, which was supported by enzyme activity measurements and correlated with the increased PPP flux in phase II. Moreover, genes possibly related to a general stress response displayed increased expression in growth phase II.
2013-02-13 | GSE42223 | GEO
Project description:Mathematical Definition
symbol/abbreviation
Mathematical symbols
Keq Equilibrium constant
Ki Inhibition constant
Km Affinity constant
v Predicted enzyme activities
Vf Maximal enzyme activities under saturating substrate and activator
conditions, and in the absence of inhibitors
h Hill coefficient
k Rate constant
Abbreviations
ACET Acetaldehyde
ADH Alcohol dehydrogenase
AK Adenylate kinase
ATPcons ATP consuming reactions
bPG 1,3-bisphosphoglycerate
ENO Enolase
ETOHcy Cytoplasmic ethanol
ETOHex Extracellular ethanol
ETOHexp Ethanol transport
GAP Glyceraldehyde 3-phosphate
GAPD Glyceraldehyde 3-phosphate dehydrogenase
GF Glucose facilitator
GK Glucokinase
GLUCcy Cytoplasmic glucose
GLUCex Extracellular glucose
GLUC6P Glucose 6-phosphate
GPD Glucose 6-phosphate dehydrogenase
KDPG 2-keto-3-deoxy-6-phosphogluconate
KDPGA 2-keto-3-deoxy-6-phosphogluconate aldolase
PDC Pyruvate decarboxylase
PEP Phosphoenolpyruvate
PGD 6-phosphogluconate dehydratase
PGK 3-phosphoglycerate kinase
PGL 6-phosphogluconolactonase
PGLACTON 6-phosphogluconolactone
PGLUCONATE 6-phosphogluconate
PGM Phosphoglycerate mutase
P3G 3-phosphoglycerate
P2G 2-phosphoglycerate
PYK Pyruvate kinase
PYR Pyruvate
2020-08-06 | MODEL2008060001 | BioModels
Project description:The model corresponds to the third columns (model simulations) in tables 3 and 5 of publication (PMID: 24085837, DOI: 10.1099/mic.0.071340-0)
Mathematical Definition
symbol/abbreviation
Mathematical symbols
Keq Equilibrium constant
Ki Inhibition constant
Km Affinity constant
v Predicted enzyme activities
Vf Maximal enzyme activities under saturating substrate and activator
conditions, and in the absence of inhibitors
h Hill coefficient
k Rate constant
Abbreviations
ACET Acetaldehyde
ADH Alcohol dehydrogenase
AK Adenylate kinase
ATPcons ATP consuming reactions
bPG 1,3-bisphosphoglycerate
ENO Enolase
ETOHcy Cytoplasmic ethanol
ETOHex Extracellular ethanol
ETOHexp Ethanol transport
GAP Glyceraldehyde 3-phosphate
GAPD Glyceraldehyde 3-phosphate dehydrogenase
GF Glucose facilitator
GK Glucokinase
GLUCcy Cytoplasmic glucose
GLUCex Extracellular glucose
GLUC6P Glucose 6-phosphate
GPD Glucose 6-phosphate dehydrogenase
KDPG 2-keto-3-deoxy-6-phosphogluconate
KDPGA 2-keto-3-deoxy-6-phosphogluconate aldolase
PDC Pyruvate decarboxylase
PEP Phosphoenolpyruvate
PGD 6-phosphogluconate dehydratase
PGK 3-phosphoglycerate kinase
PGL 6-phosphogluconolactonase
PGLACTON 6-phosphogluconolactone
PGLUCONATE 6-phosphogluconate
PGM Phosphoglycerate mutase
P3G 3-phosphoglycerate
P2G 2-phosphoglycerate
PYK Pyruvate kinase
PYR Pyruvate
2014-09-15 | MODEL1409050000 | BioModels
Project description:The model corresponds to the Table 4 and the last column of table 5 of publication (PMID: 24085837, DOI: 10.1099/mic.0.071340-0).
Mathematical Definition
symbol/abbreviation
Mathematical symbols
Keq Equilibrium constant
Ki Inhibition constant
Km Affinity constant
v Predicted enzyme activities
Vf Maximal enzyme activities under saturating substrate and activator
conditions, and in the absence of inhibitors
h Hill coefficient
k Rate constant
Abbreviations
ACET Acetaldehyde
ADH Alcohol dehydrogenase
AK Adenylate kinase
ATPcons ATP consuming reactions
bPG 1,3-bisphosphoglycerate
ENO Enolase
ETOHcy Cytoplasmic ethanol
ETOHex Extracellular ethanol
ETOHexp Ethanol transport
GAP Glyceraldehyde 3-phosphate
GAPD Glyceraldehyde 3-phosphate dehydrogenase
GF Glucose facilitator
GK Glucokinase
GLUCcy Cytoplasmic glucose
GLUCex Extracellular glucose
GLUC6P Glucose 6-phosphate
GPD Glucose 6-phosphate dehydrogenase
KDPG 2-keto-3-deoxy-6-phosphogluconate
KDPGA 2-keto-3-deoxy-6-phosphogluconate aldolase
PDC Pyruvate decarboxylase
PEP Phosphoenolpyruvate
PGD 6-phosphogluconate dehydratase
PGK 3-phosphoglycerate kinase
PGL 6-phosphogluconolactonase
PGLACTON 6-phosphogluconolactone
PGLUCONATE 6-phosphogluconate
PGM Phosphoglycerate mutase
P3G 3-phosphoglycerate
P2G 2-phosphoglycerate
PYK Pyruvate kinase
PYR Pyruvate
2014-09-15 | MODEL1409050001 | BioModels
Project description:GAPDHs from human pathogens S. aureus and P. aeruginosa can be readily inhibited by ROS-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment of H2O2 induces rapid metabolic reroute from glycolysis to pentose phosphate pathway (PPP). We conducted RNA-seq analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which reveals profound transcriptional changes including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces a metabolic reroute from glycolysis to PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Given that H2O2 can be produced constitutively by the host immune response, exposure to the steady-state stress of H2O2 recapitulates more accurately bacterial responses to host immune system in vivo. RNA-seq in Pseudomonas aeruginosa and Staphylococus aureus under steady state of H2O2
2014-05-08 | E-GEOD-55528 | biostudies-arrayexpress
Project description:Previous findings have demonstrated that the NADH/NAD+ ratio has a strong impact on the glycolytic flux in C. glutamicum under anaerobic conditions in the absence of external electron acceptors. During an attempt to rewire anaerobic metabolism to achieve high yield formation of ethanol, we inactivated the malate dehydrogenase and lactate dehydrogenase in a C. glutamicum strain expressing pyruvate decarboxylase and alcohol dehydrogenase, to eliminate formation of the by-products succinate and lactate, respectively. This modification increased the yield of ethanol but had a negative effect on glycolysis, which we found to correlate with an elevated NADH/NAD+ ratio. The pyruvate dehydrogenase (PDH) of C. glutamicum is active under anaerobic conditions, and can potentially exacerbate the negative effect on glycolysis, due to NADH formation. To reduce PDH activity under anaerobic conditions, we decided to replace the gene encoding the E3 subunit of PDH with its Escherichia coli counterpart, as E. coli PDH has been reported to be functional under aerobic conditions only. The resultant strain JS133 produced far less acetate with a further increased ethanol production, however, the glycolytic flux was still low. After observing differences in glycolytic flux for JS133 on glucose and fructose, we speculated that the pentose phosphate pathway (PPP) might be involved in the reduced flux on glucose. To prove this, we deleted the zwf gene, encoding glucose-6-phosphate dehydrogenase, which is the entry point into PPP, and immediately observed a stimulating effect on glycolysis. Subsequent characterization revealed a direct correlation between the intracellular NADH/NAD+ and NADPH/NADP+ ratios under anoxic conditions. Based on these findings we managed to re-channel the metabolism of C. glutamicum successfully towards either to ethanol or D-lactate with 92% and 98% of the theoretical yield respectively, which is the highest yields for D-lactate production thus far reported in the literature.
2020-10-02 | GSE143977 | GEO
Project description:Saccharomyces cerevisiae normally cannot assimilate mannitol, a promising brown macroalgal carbon source for bioethanol production. To date, the molecular mechanisms underlying this inability remain unknown. Here, we found that cells acquiring mannitol-assimilating ability appeared from wild-type S. cerevisiae strain during prolonged culture in mannitol medium. Our microarray analysis revealed that genes for putative mannitol dehydrogenase and hexose transporters were up-regulated in cells acquiring mannitol-assimilating ability. Take account of our other results including complementation analysis and cell growth data, we demonstrated that this acquisition of mannitol-assimilating ability was due to the spontaneous mutation in the gene encoding Tup1 or Cyc8. Tup1-Cyc8 is the general corepressor complex involved in the repression of many kinds of genes. Thus, it is suggested that the inability of wild-type S. cerevisiae to assimilate mannitol can be attributed to the transcriptional repression of a set of genes involved in mannitol utilization by Tup1-Cyc8 corepressor. In other words, Tup1-Cyc8 is a key regulator of mannitol metabolism in S. cerevisiae. We also showed that S. cerevisiae strain which carries mutant allele of TUP1 or CYC8 produced ethanol from mannitol efficiently. Especially, strain carrying mutant allele of CYC8 showed high tolerance to salt, which is superior to other ethanologenic microorganisms. This characteristic is highly beneficial to produce bioethanol from marine biomass. Taken together, Tup1-Cyc8 can be an ideal target to develop a yeast-algal bioethanol production system. To figure out how Mtl+ strains (cells acquiring ability to grow in mannitol medium) had acquired the ability to assimilate mannitol, we performed genome-wide analysis by using Nimblegen microarrays.
2014-12-04 | GSE55755 | GEO
Project description:Costa2014 - Computational Model of L. lactis
Metabolism
This model is described in the article:
An extended dynamic model of
Lactococcus lactis metabolism for mannitol and 2,3-butanediol
production.
Costa RS, Hartmann A, Gaspar P,
Neves AR, Vinga S.
Mol Biosyst 2014 Mar; 10(3):
628-639
Abstract:
Biomedical research and biotechnological production are
greatly benefiting from the results provided by the development
of dynamic models of microbial metabolism. Although several
kinetic models of Lactococcus lactis (a Lactic Acid Bacterium
(LAB) commonly used in the dairy industry) have been developed
so far, most of them are simplified and focus only on specific
metabolic pathways. Therefore, the application of mathematical
models in the design of an engineering strategy for the
production of industrially important products by L. lactis has
been very limited. In this work, we extend the existing kinetic
model of L. lactis central metabolism to include industrially
relevant production pathways such as mannitol and
2,3-butanediol. In this way, we expect to study the dynamics of
metabolite production and make predictive simulations in L.
lactis. We used a system of ordinary differential equations
(ODEs) with approximate Michaelis-Menten-like kinetics for each
reaction, where the parameters were estimated from multivariate
time-series metabolite concentrations obtained by our team
through in vivo Nuclear Magnetic Resonance (NMR). The results
show that the model captures observed transient dynamics when
validated under a wide range of experimental conditions.
Furthermore, we analyzed the model using global perturbations,
which corroborate experimental evidence about metabolic
responses upon enzymatic changes. These include that mannitol
production is very sensitive to lactate dehydrogenase (LDH) in
the wild type (W.T.) strain, and to mannitol
phosphoenolpyruvate: a phosphotransferase system (PTS(Mtl)) in
a LDH mutant strain. LDH reduction has also a positive control
on 2,3-butanediol levels. Furthermore, it was found that
overproduction of mannitol-1-phosphate dehydrogenase (MPD) in a
LDH/PTS(Mtl) deficient strain can increase the mannitol levels.
The results show that this model has prediction capability over
new experimental conditions and offers promising possibilities
to elucidate the effect of alterations in the main metabolism
of L. lactis, with application in strain optimization.
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2015-03-26 | BIOMD0000000572 | BioModels