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Characterization of Pyruvate Uptake in Escherichia coli K-12  [PDF]
Jens Kreth, Joseph W. Lengeler, Knut Jahreis
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0067125
Abstract: The monocarboxylate pyruvate is an important metabolite and can serve as sole carbon source for Escherichia coli. Although specific pyruvate transporters have been identified in two bacterial species, pyruvate transport is not well understood in E. coli. In the present study, pyruvate transport was investigated under different growth conditions. The transport of pyruvate shows specific activities depending on the growth substrate used as sole carbon source, suggesting the existence of at least two systems for pyruvate uptake: i) one inducible system and probably highly specific for pyruvate and ii) one system active under non-induced conditions. Using the toxic pyruvate analog 3-fluoropyruvate, a mutant was isolated unable to grow on and transport pyruvate. Further investigation revealed that a revertant selected for growth on pyruvate regained the inducible pyruvate transport activity. Characterization of pyruvate excretion showed that the pyruvate transport negative mutant accumulated pyruvate in the growth medium suggesting an additional transport system for pyruvate excretion. The here presented data give valuable insight into the pyruvate metabolism and transport of E. coli suggesting the presence of at least two uptake systems and one excretion system to balance the intracellular level of pyruvate.
New insights into Escherichia coli metabolism: carbon scavenging, acetate metabolism and carbon recycling responses during growth on glycerol
Karla Martínez-Gómez, Noemí Flores, Héctor M Casta?eda, Gabriel Martínez-Batallar, Georgina Hernández-Chávez, Octavio T Ramírez, Guillermo Gosset, Sergio Encarnación, Francisco Bolivar
Microbial Cell Factories , 2012, DOI: 10.1186/1475-2859-11-46
Abstract: Transcriptional and proteomic analysis of metabolic central genes of strain JM101 growing on glycerol, revealed important changes not only in the synthesis of MglB, LamB and MalE proteins, but also in the overexpression of carbon scavenging genes: lamB, malE, mglB, mglC, galP and glk and some members of the RpoS regulon (pfkA, pfkB, fbaA, fbaB, pgi, poxB, acs, actP and acnA). Inactivation of rpoS had an important effect on stoichiometric parameters and growth adaptation on glycerol. The observed overexpression of poxB, pta, acs genes, glyoxylate shunt genes (aceA, aceB, glcB and glcC) and actP, suggested a possible carbon flux deviation into the PoxB, Acs and glyoxylate shunt. In this scenario acetate synthesized from pyruvate with PoxB was apparently reutilized via Acs and the glyoxylate shunt enzymes. In agreement, no acetate was detected when growing on glycerol, this strain was also capable of glycerol and acetate coutilization when growing in mineral media and derivatives carrying inactivated poxB or pckA genes, accumulated acetate. Tryptophanase A (TnaA) was synthesized at high levels and indole was produced by this enzyme, in strain JM101 growing on glycerol. Additionally, in the isogenic derivative with the inactivated tnaA gene, no indole was detected and acetate and lactate were accumulated. A high efficiency aromatic compounds production capability was detected in JM101 carrying pJLBaroGfbrtktA, when growing on glycerol, as compared to glucose.The overexpression of several carbon scavenging, acetate metabolism genes and the absence of acetate accumulation occurred in JM101 cultures growing on glycerol. To explain these results it is proposed that in addition to the glycolytic metabolism, a gluconeogenic carbon recycling process that involves acetate is occurring simultaneously in this strain when growing on glycerol. Carbon flux from glycerol can be efficiently redirected in JM101 strain into the aromatic pathway using appropriate tools.Escherichia coli i
Glycerol Modulates Water Permeation through Escherichia coli Aquaglyceroporin GlpF  [PDF]
Liao Y. Chen
Quantitative Biology , 2012, DOI: 10.1016/j.bbamem.2013.03.008
Abstract: Among aquaglyceroporins that transport both water and glycerol across the cell membrane, Escherichia coli glycerol uptake facilitator (GlpF) is the most thoroughly studied. However, one question remains: Does glycerol modulate water permeation? This study answers this fundamental question by determining the chemical-potential profile of glycerol along the permeation path through GlpF's conducting pore. There is a deep well near the Asn-Pro-Ala (NPA) motifs (dissociation constant 14 microM) and a barrier near the selectivity filter (10.1 kcal/mol above the well bottom). This profile owes its existence to GlpF's perfect steric arrangement: The glycerol-protein van der Waals interactions are attractive near the NPA but repulsive elsewhere in the conducting pore. In light of the single-file nature of waters and glycerols lining up in GlpF's amphipathic pore, it leads to the following conclusion: Glycerol modulates water permeation in the microM range. At mM concentrations, GlpF is glycerol-saturated and a glycerol dwelling in the well occludes the conducting pore. Therefore, water permeation is fully correlated to glycerol dissociation that has an Arrhenius activation barrier of 6.5 kcal/mol. Validation of this theory is based on the existent in vitro data, some of which have not been given the proper attention they deserved: The Arrhenius activation barriers were found to be 7 kcal/mol for water permeation and 9.6 kcal/mol for glycerol permeation; The presence of up to 100 mM glycerol did not affect the kinetics of water transport with very low permeability, in apparent contradiction with the existent theories that predicted high permeability (0 M glycerol).
Elementary Mode Analysis for the Rational Design of Efficient Succinate Conversion from Glycerol by Escherichia coli
Zhen Chen,Hongjuan Liu,Jianan Zhang,Dehua Liu
Journal of Biomedicine and Biotechnology , 2010, DOI: 10.1155/2010/518743
Abstract: By integrating the restriction of oxygen and redox sensing/regulatory system, elementary mode analysis was used to predict the metabolic potential of glycerol for succinate production by E. coli under either anaerobic or aerobic conditions. It was found that although the theoretical maximum succinate yields under both anaerobic and aerobic conditions are 1.0 mol/mol glycerol, the aerobic condition was considered to be more favorable for succinate production. Although increase of the oxygen concentration would reduce the succinate yield, the calculation suggests that controlling the molar fraction of oxygen to be under 0.65 mol/mol would be beneficial for increasing the succinate productivity. Based on the elementary mode analysis, the rational genetic modification strategies for efficient succinate production under aerobic and anaerobic conditions were obtained, respectively. Overexpressing the phosphoenolpyruvate carboxylase or heterogonous pyruvate carboxylase is considered to be the most efficient strategy to increase the succinate yield.
Biosynthesis of Ethanol and Hydrogen by Glycerol Fermentation Using Escherichia coli  [PDF]
Nida Chaudhary, Michael O. Ngadi, Benjamin K. Simpson, Lamin S. Kassama
Advances in Chemical Engineering and Science (ACES) , 2011, DOI: 10.4236/aces.2011.13014
Abstract: Production of high value products from glycerol via anaerobic fermentation is of utmost importance for the biodiesel industry. The microorganism Escherichia coli (E. coli) K12 was used for fermentation of glycerol. The effects of glycerol concentration and headspace conditions on the cell growth, ethanol and hydrogen production were investigated. A full factorial experimental design with 3 replicates was conducted in order to test these factors. Under the three headspace conditions tested, the increase of glycerol concentration accelerated glycerol fermentation. The yields of hydrogen and ethanol were the lowest when glycerol concentration of 10 g/L was used. The maximum production of hydrogen was observed with an initial glycerol concentration of 25 g/L at a final concentration of hydrogen was 32.15 mmol/L. This study demonstrated that hydrogen production negatively affects cell growth. Maximum ethanol yield was obtained with a glycerol concentration of 10 g/L and was up to 0.40 g/g glycerol under membrane condition headspace. Statistical optimization showed that optimal conditions for hydrogen production are 20 g/L initial glycerol with initial sparging of the reactor headspace. The optimal conditions for ethanol production are 10 g/L initial glycerol with membrane.
Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli  [cached]
Meza Eugenio,Becker Judith,Bolivar Francisco,Gosset Guillermo
Microbial Cell Factories , 2012, DOI: 10.1186/1475-2859-11-127
Abstract: Background In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined. Results When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation. Conclusions Metabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS-pykA-) and VH35 (PTS-pykF-) have useful prop
Coutilization of glucose and glycerol enhances the production of aromatic compounds in an Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system
Karla Martínez, Ramón de Anda, Georgina Hernández, Adelfo Escalante, Guillermo Gosset, Octavio T Ramírez, Francisco G Bolívar
Microbial Cell Factories , 2008, DOI: 10.1186/1475-2859-7-1
Abstract: PB12 strain is capable of coutilizing mixtures of glucose-arabinose, glucose-gluconate and glucose-glycerol. This capacity increases its specific growth rate (μ) given that this strain metabolizes more moles of carbon source per unit time. The presence of plasmids pRW300aroGfbr and pCLtktA reduces the μ of strain PB12 in all mixtures of carbon sources, but enhances the productivity and yield of aromatic compounds, especially in the glucose-glycerol mixture, as compared to glucose or glycerol cultures. No acetate was detected in the glycerol and the glucose-glycerol batch fermentations.Due to the lack of catabolite repression, PB12 strain carrying multicopy plasmids containing tktA and aroGfbr genes is capable of coutilizing glucose and other carbon sources; this capacity, reduces its μ but increases the production of aromatic compounds.Glucose is the preferred carbon source for Escherichia coli (E. coli); the presence of this monosaccharide inhibits the utilization of secondary carbon sources. This process is known as carbon catabolite repression, and the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) is the main regulator of this response. The PTS components EI, HPr, EIIAGlc, EIICBGlc encoded by the ptsHIcrr operon and the ptsG gene, as well as other important proteins like the adenylate cyclase (Cya) and the cAMP-CRP complex, are members of the carbon catabolite modulon regulating the activities of genes involved in carbon transport [1-5]. The inactivation of PTS has pleiotropic effects in E. coli. It has been demonstrated that the deletion of the ptsHIcrr operon drastically reduces the specific growth rate (μ) of the cell (from 0.7 to 0.1 hr-1) when growing on glucose as the only carbon source. In this type of strains, the overexpression of several genes involved in carbon utilization occurs as a response to carbon limited conditions. Accordingly, the cell is capable of coutilizing glucose with several other carbon sources that are not only car
Expression of heterogenous pyruvate carboxylase in Escherichia coli with lactose as inducer and its effect on succinate production

Dan Wang,Yu Mao,Lan M,Qiang Li,Wangliang Li,Jianmin Xing,Zhiguo Su,

生物工程学报 , 2009,
Abstract: Escherichia coli strain DC1515,deficient in glucose phosphotransferase(ptsG),lactate dehydrogenase(ldhA) and pyruvate:formate lyase(pflA),is a promising candidate for the fermentative production of succinate.To further improve the succinate producing capability of DC1515,we constructed plasmid pTrchisA-pyc with heterogenous pyruvate carboxylase(pyc) from Bacillus subtilis 168 under the Trc promoter and introduced it into DC1515.We used lactose as a substitute of IPTG to induce pyc.We optimized the culture c...
Expression of glycerol dehydrogenase gene in Escherichia coli by Codon Optimization

Longpan Tang,Jincong Yu,Danfeng Dai,Baishan Fang,

微生物学报 , 2011,
Abstract: Objective]To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization.Methods]For immediately downstream region of initiation codon in gldA,we designed optimized sequence by choosing higher AT-content synonymous,in order that this region's AT-content was increased without changing the corresponding amino acids.Then we had wild gene gldA-WT site-directed mutagenesis depending on mega-primers PCR,so that physically optimized gene gldA-4 was acquired.We ...
Metabolic Erosion Primarily Through Mutation Accumulation, and Not Tradeoffs, Drives Limited Evolution of Substrate Specificity in Escherichia coli  [PDF]
Nicholas Leiby,Christopher J. Marx
PLOS Biology , 2014, DOI: 10.1371/journal.pbio.1001789
Abstract: Evolutionary adaptation to a constant environment is often accompanied by specialization and a reduction of fitness in other environments. We assayed the ability of the Lenski Escherichia coli populations to grow on a range of carbon sources after 50,000 generations of adaptation on glucose. Using direct measurements of growth rates, we demonstrated that declines in performance were much less widespread than suggested by previous results from Biolog assays of cellular respiration. Surprisingly, there were many performance increases on a variety of substrates. In addition to the now famous example of citrate, we observed several other novel gains of function for organic acids that the ancestral strain only marginally utilized. Quantitative growth data also showed that strains with a higher mutation rate exhibited significantly more declines, suggesting that most metabolic erosion was driven by mutation accumulation and not by physiological tradeoffs. These reductions in growth by mutator strains were ameliorated by growth at lower temperature, consistent with the hypothesis that this metabolic erosion is largely caused by destabilizing mutations to the associated enzymes. We further hypothesized that reductions in growth rate would be greatest for substrates used most differently from glucose, and we used flux balance analysis to formulate this question quantitatively. To our surprise, we found no significant relationship between decreases in growth and dissimilarity to glucose metabolism. Taken as a whole, these data suggest that in a single resource environment, specialization does not mainly result as an inevitable consequence of adaptive tradeoffs, but rather due to the gradual accumulation of disabling mutations in unused portions of the genome.
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