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Escherichia coli Δglk ΔmanZ ΔptsG glucose - strains that lack the glucose phosphotransferase system (PTS) and the mannose PTS as well as glucokinase have been widely used by researchers studying the PTS. In this study we show that both fast- and slow-growing spontaneous glucose + revertants can be readily obtained from Δglk ΔmanZ ΔptsG glucose - strains. All of the fast-growing revertants either altered the N-acetylglucosamine PTS or caused its overproduction by inactivating the NagC repressor protein, which regulates the N-acetylglucosamine PTS, and these revertants could utilize either glucose or N-acetylglucosamine as a sole carbon source. When a ΔnagE deletion, which abolishes the N-acetylglucosamine PTS, was introduced into the Δglk ΔmanZ ΔptsG glucose - strains, fast-growing revertants could no longer be isolated. Based on our results and other studies, it is clear that the N-acetylglucosamine PTS is the most easily adaptable PTS for transporting and phosphorylating glucose, other than the glucose PTS and mannose PTS, which are the primary glucose transport systems. While the slow-growing glucose + revertants were not characterized, they were likely mutations that other researchers have observed before and affect other PTSs or sugar kinases.

Escherichia coli lacking the glucose phosphotransferase system (PTS), mannose PTS and glucokinase are supposedly unable to grow on glucose as the sole carbon source (Curtis SJ, Epstein W. J Bacteriol 1975;122:1189-1199). We report that W ptsG manZ glk (ALS1406) grows slowly on glucose in media containing glucose with a second carbon source: ALS1406 metabolizes glucose after that other carbon source, including arabinose, fructose, glycerol, succinate or xylose, is exhausted. Galactose is an exception to this rule, as ALS1406 simultaneously consumes both galactose and glucose. The ability of ALS1406 to metabolize glucose in a xylose-glucose mixture was unchanged by an additional knockout in any single gene involved in carbohydrate transport and utilization, including agp (periplasmic glucose-1-phosphatase), galP (galactose permease), xylA (xylose isomerase), alsK (allose kinase), crr (glucose PTS enzyme IIA), galK (galactose kinase), mak (mannokinase), malE (maltose transporter), malX (maltose PTS enzyme IIBC), mglB (methyl-galactose transporter subunit), nagE (N-acetyl glucosamine PTS enzyme IICBA), nanK (N-acetyl mannosamine kinase) or pgm (phosphoglucose mutase). Glucose metabolism was only blocked by the deletion of two metabolic genes, pgi (phosphoglucose isomerase) and zwf (glucose-6-phosphate 1-dehydrogenase), which prevents the entry of glucose-6-phosphate into the pentose phosphate and Embden-Meyerhof-Parnas pathways. Carbon-limited steady-state studies demonstrated that xylose must be sub-saturating for glucose to be metabolized, while nitrogen-limited studies showed that xylose is partly converted to glucose when xylose is in excess. Under transient conditions, ALS1406 converts almost 25 % (mass) xylose into glucose as a result of reversible transketolase and transaldolase and the re-entry of carbon into the pentose phosphate pathway via glucose-6-phosphate 1-dehydrogenase.

Eighteen strains of Escherichia coli were compared for maximum specific growth rate (μ MAX) on 85 mM acetate as the sole carbon source. The C strain ATCC8739 had the greatest growth rate (0.41 h(-1)) while SCS-1 had the slowest growth rate (0.15 h(-1)). Transcriptional analysis of three of the strains (ATCC8739, BL21, SMS-3-5) was conducted to elucidate why ATCC8739 had the greatest maximum growth rate. Seventy-one genes were upregulated 2-fold or greater in ATCC8739, while 128 genes were downregulated 2-fold or greater in ATCC8739 compared to BL21 and SMS-3-5. To generate a strain that could grow more quickly on acetate, ATCC8739 was cultured in a chemostat using a progressively increasing dilution rate. When the dilution rate reached 0.50 h(-1), three isolated colonies each grew faster than ATCC8739 on 85 mM acetate, with MEC136 growing the fastest with a growth rate of 0.51 h(-1), about 25 % greater than ATCC8739. Transcriptional analysis of MEC136 showed that eight genes were downregulated 2-fold or greater and one gene was upregulated 2-fold or greater compared to ATCC8739. Genomic sequencing revealed that MEC136 contained a single mutation, causing a serine to proline change in amino acid 266 of RpoA, the α subunit of the RNA polymerase core enzyme. The 260-270 amino acid region of RpoA has been shown to be a key region of the protein that affects the interaction of the α subunit of the RNA polymerase core enzyme with several global transcriptional activators, such as CRP and FNR.

Adaptive evolution was employed to generate sodium (Na(+))-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na(+) concentrations. The isolates also showed increased tolerance of K(+), although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na(+) tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kil, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na(+). Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na(+) tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate.

Background: The efficient microbial utilization of lignocellulosic hydrolysates has remained challenging because this material is composed of multiple sugars and also contains growth inhibitors such as acetic acid (acetate). Using an engineered consortium of strains derived from Escherichia coli C and a synthetic medium containing acetate, glucose, xylose and arabinose, we report on both the microbial removal of acetate and the subsequent simultaneous utilization of the sugars., Results: In a first stage, a strain unable to utilize glucose, xylose and arabinose (ALS1392, strain E. coli C ptsG manZ glk crr xylA araA) removed 3 g/L acetate within 30 hours. In a subsequent second stage, three E. coli strains (ALS1370, ALS1371, ALS1391), which are each engineered to utilize only one sugar, together simultaneously utilized glucose, xylose and arabinose. The effect of non-metabolizable sugars on the metabolism of the target sugar was minimal. Additionally the deletions necessary to prevent the consumption of one sugar only minimally affected the consumption of a desired sugar. For example, the crr deletion necessary to prevent glucose consumption reduced xylose and arabinose utilization by less than 15% compared to the wild-type. Similarly, the araA deletion used to exclude arabinose consumption did not affect xylose- and glucose-consumption., Conclusions: Despite the modest reduction in the overall rate of sugar consumption due to the various deletions that were required to generate the consortium of strains, the approach constitutes a significant improvement in any single-organism approach to utilize sugars found in lignocellulosic hydrolysate in the presence of acetate.

Acetic acid is an unavoidable constituent of the biomass hydrolysates generated from acetylated hemicellulose and lignin, and acetate affects the performance of microbes used to convert these hydrolysates into biofuels or other biochemicals. In this study, acetate was selectively removed from synthetic mixtures of glucose and xylose using metabolically engineered Escherichia coli strains having mutations in the glucose phosphotransferase system (PTS) genes (ptsG, manZ, crr), glucokinase (glk), and xylose (xylA). In batch culture, ALS1060 (ptsG manZ glk xylA) consumed exclusively acetate to depletion, and then consumed the two sugars only at a very slow rate (a growth rate of about 0.01 h(-1)). We also examined the effects of an additional knockout of either malX, fruA, fruB, bglF, or crr, genes that are involved in other PTSs, and a batch process using KD840 (ptsG manZ glk crr xylA) demonstrated a further reduction in glucose or xylose consumption by E. coli. These results demonstrate the feasibility of using a substrate-selective approach for the pre-treatment of biomass hydrolysate for microbial processes.

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h(-1) versus 0.44 g/g at 0.25 h(-1)), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h(-1) versus 0.59 g/g h at 0.10 h(-1)). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h(-1)). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h(-1), the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal.

Succinate production under different concentrations of carbon dioxide (CO(2)) was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). A series of two-phase fermentations were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using several constant concentrations of CO(2). As the concentration of CO(2) in the gas phase increased from 0% to 50%, the succinate specific productivity increased from 1.9 mg/gh to 225 mg/gh, and the succinate yield increased from 0.04 g/g to 0.75 g/g. Above 50% CO(2), succinate production did not increase further. Intracellular fluxes were determined at three different CO(2) concentrations (3%, 10%, and 50%) using (13)C-label tracing coupled with LC-MS analysis. The fraction of carbon flux into the pentose phosphate pathway increased from 0.04 at 3% CO(2) to 0.17 at 50% CO(2). Also, the fractional flux through anaplerotic carboxylation at the phosphoenolpyruvate (PEP) node increased slightly from 0.53 at 3% CO(2) to 0.63 at 50% CO(2). The increased flux into the pentose phosphate pathway is attributed to an increased demand for reduced cofactors with elevated CO(2). A four-process explicit model to describe the CO(2) transfer and utilization was proposed. The model predicted that at CO(2) concentrations below about 30-40% the system becomes limited by gas phase CO(2), while at higher CO(2) concentrations the system is limited by PEP carboxylase enzyme kinetics.

Succinate production was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). Two-phase fermentations using a defined medium at several controlled levels of pH were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using 100% (v/v) CO(2). A pH of 6.4 yielded the highest specific succinate productivity. A metabolic flux analysis at a pH of 6.4 using (13)C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate, while 93% of the succinate was formed via the reductive arm of the TCA cycle. The flux distribution at a pH of 6.8 was also analyzed and was not significantly different compared to that at a pH of 6.4. Ca(OH)(2) was superior to NaOH or KOH as the base for controlling the pH. By maintaining the pH at 6.4 using 25% (w/v) Ca(OH)(2), the process achieved an average succinate productivity of 1.42 g/l h with a yield of 0.61 g/g.

We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h(-1) to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h(-1). At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.

We describe a new approach for the simultaneous conversion of xylose and glucose sugar mixtures which potentially could be used for lignocellulosic biomass hydrolysate. In this study we used this approach to demonstrate the production of lactic acid. This process uses two substrate-selective strains of Escherichia coli, one which is unable to consume glucose and one which is unable to consume xylose. In addition to knockouts in pflB encoding for pyruvate formate lyase, the xylose-selective (glucose deficient) strain E. coli ALS1073 has deletions of the glk, ptsG, and manZ genes while the glucose-selective (xylose deficient) strain E. coli ALS1074 has a xylA deletion. By combining these two strains in a single process the xylose and glucose in a mixed sugar solution are simultaneously converted to lactate. Furthermore, the biomass concentrations of each strain can readily be adjusted in order to optimize the overall product formation. This approach to the utilization of mixed sugars eliminates the problem of diauxic growth, and provides great operational flexibility.

A two-phase, lactate fermentation by Escherichia coli ALS974 generates succinate and ethanol anaerobically from acetate. These by-products can be minimized by monitoring acetate concentration indirectly with dissolved O(2) (DO) during the initial aerobic cell-growth phase. Without DO monitoring, 3 g succinate/l and 1 g ethanol/l were generated while, with monitoring, less than 1 g succinate/l and no detectable ethanol were generated with 130 g lactate/l being produced. Furthermore, using a cell-recycle fermentation with ultrafiltration prolonged the anaerobic lactate production phase from 22 to 34 h, thereby achieving a lactate productivity of 4.2 g/l h, nearly 20% greater than the productivity of the fed-batch process.

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h(-1). Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g . h), pyruvate formation rate (1.11 g/g h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h(-1) achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter h and yield of 0.68 g/g.

Escherichia coli is the organism of choice for the expression of a wide variety of recombinant proteins for therapeutic, diagnostic and industrial applications. E. coli generates acetic acid (acetate) as an undesirable by-product that has several negative effects on protein production. Various strategies have been developed to limit acetate accumulation or reduce its negative effects to increase the productivity of recombinant proteins. This article reviews recent strategies for reducing or eliminating acetate, including approaches that optimize the protein production process as well as those that involve modifying the host organism itself.

DL-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6-10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at micro = 0.15 h(-1), only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.

Background: Like other bacteria, Escherichia coli must carefully regulate the intracellular concentration of sodium ion (Na+). During the bacterial production of any organic acid, cations like Na+ invariably accumulate during a process which must maintain a near neutral pH. In this study, the E. coli nhaA gene encoding the Na+/H+ antiporter membrane protein and the nhaR gene encoding the NhaA regulatory protein were overexpressed in wild-type E. coli MG1655 and in MG1655 pflB (ALS1317) which lacks pyruvate formate lyase activity and thus accumulates lactate under anaerobic conditions. Results: Expression of either the nhaA or nhaR gene on the high copy inducible expression vector pTrc99A caused a significant reduction in the growth rate of MG1655. No change in growth rate was observed for MG1655 or ALS1317 for Na+ concentrations of 0.75-0.90 M when the medium copy pBR322 plasmid was used to overexpress the two genes. In a fed-batch process to produce the model acid lactate with NaOH addition for pH control, lactate accumulation ceased in MG1655, MG1655/pBR322, MG1655/pBR322-nhaR and MG1655/pBR322-nhaA when the concentration reached 55-58 g/L. In an identical process lactate accumulation in MG1655/pBR322-nhaAR did not terminate until the concentration reached over 70 g/L. Conclusions: Although overexpression the genes did not improve growth rate at high Na+ concentrations, the overexpression of nhaA and nhaR together led to a 25% increase in lactate production. Thus, the observed (absence of) impact that these genetic modifications had on growth rate is a poor indicator of their effect on acid accumulation. The overexpression of nhaAR did not cause faster lactate production, but permitted the culture to continue accumulating lactate at 10% greater Na+ concentration. [ABSTRACT FROM AUTHOR]

Background: The efficient microbial utilization of lignocellulosic hydrolysates has remained challenging because this material is composed of multiple sugars and also contains growth inhibitors such as acetic acid (acetate). Using an engineered consortium of strains derived from Escherichia coli C and a synthetic medium containing acetate, glucose, xylose and arabinose, we report on both the microbial removal of acetate and the subsequent simultaneous utilization of the sugars. Results: In a first stage, a strain unable to utilize glucose, xylose and arabinose (ALS1392, strain E. coli C ptsG manZ glk crr xylA araA) removed 3 g/L acetate within 30 hours. In a subsequent second stage, three E. coli strains (ALS1370, ALS1371, ALS1391), which are each engineered to utilize only one sugar, together simultaneously utilized glucose, xylose and arabinose. The effect of non-metabolizable sugars on the metabolism of the target sugar was minimal. Additionally the deletions necessary to prevent the consumption of one sugar only minimally affected the consumption of a desired sugar. For example, the crr deletion necessary to prevent glucose consumption reduced xylose and arabinose utilization by less than 15% compared to the wild-type. Similarly, the araA deletion used to exclude arabinose consumption did not affect xylose- and glucose-consumption. Conclusions: Despite the modest reduction in the overall rate of sugar consumption due to the various deletions that were required to generate the consortium of strains, the approach constitutes a significant improvement in any single-organism approach to utilize sugars found in lignocellulosic hydrolysate in the presence of acetate. [ABSTRACT FROM AUTHOR]

We report the conversion of glycerol to pyruvate by E. coli ALS929 containing knockouts in the genes encoding for phosphoenolpyruvate synthase, lactate dehydrogenase, pyruvate formate lyase, the pyruvate dehydrogenase complex, and pyruvate oxidase. As a result of these knockouts, ALS929 has a growth requirement of acetate for the generation of acetyl CoA. In steady-state chemostat experiments using excess glycerol and limited by acetate, lower growth rates favored the formation of pyruvate from glycerol (0.60 g/g at 0.10 h−1 versus 0.44 g/g at 0.25 h−1), while higher growth rates resulted in the maximum specific glycerol consumption rate (0.85 g/g h at 0.25 h−1 versus 0.59 g/g h at 0.10 h−1). The presence of glucose significantly improved pyruvate productivity and yield from glycerol (0.72 g/g at 0.10 h−1). In fed-batch studies using exponential acetate/glucose-limited feeding at a constant growth rate of 0.10 h−1, the final pyruvate concentration achieved was about 40 g/L in 36 h. A derivative of ALS929 which additionally knocked out methylglyoxal synthase did not further increase pyruvate productivity or yield, indicating that pyruvate formation was not limited by accumulation of methylglyoxal. [ABSTRACT FROM AUTHOR]

Escherichia coli pfl ldhA ptsG (AFP111) was studied for succinate production in defined medium using trace gases typically found in flue gas; i.e. oxygen (O2), nitrogen dioxide (NO2), sulfur dioxide (SO2) and carbon monoxide (CO). Following aerobic cell growth, cells were exposed to 50% CO2 and 3-10% O2, or 50-300 ppm NO2, SO2 or CO during the succinate production phase. Although 3% O2 did not significantly affect succinate formation, 10% O2 reduced the final succinate concentration from 33 to 17 g/L, specific productivity from 1.90 to 1.13 mmol/g h and yield from 1.15 to 0.81 mol/mol glucose. The effect of O2 correlated with the culture redox potential (ORP) with more reducing conditions favoring succinate production. The trace gases NO2 and SO2 also reduced the rate of succinate formation by as much as 50%, and led to a greater than twofold increase in pyruvate formation. Similar concentrations of CO showed no effect on succinate production rate or yield. Using synthetic flue gas AFP111 generated 12 g/L succinate with a specific productivity of 0.73 mmol/g h and a yield of 0.65 mol/mol. [ABSTRACT FROM AUTHOR]

Succinate production was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase ( pfl), lactate dehydrogenase ( ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II ( ptsG). Two-phase fermentations using a defined medium at several controlled levels of pH were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using 100% (v/v) CO2. A pH of 6.4 yielded the highest specific succinate productivity. A metabolic flux analysis at a pH of 6.4 using 13C-labeled glucose showed that 61% of the PEP partitioned to oxaloacetate and 39% partitioned to pyruvate, while 93% of the succinate was formed via the reductive arm of the TCA cycle. The flux distribution at a pH of 6.8 was also analyzed and was not significantly different compared to that at a pH of 6.4. Ca(OH)2 was superior to NaOH or KOH as the base for controlling the pH. By maintaining the pH at 6.4 using 25% (w/v) Ca(OH)2, the process achieved an average succinate productivity of 1.42 g/l h with a yield of 0.61 g/g. [ABSTRACT FROM AUTHOR]

A correction is presented to the article "Transcriptional analysis and adaptive evolution of Escherichia coli strains growing on acetate," by Eashwar Rajaraman and others, which appeared in a 2016 issue.

Abstract: Succinate production under different concentrations of carbon dioxide (CO2) was studied in Escherichia coli AFP111, which contains mutations in pyruvate formate lyase (pfl), lactate dehydrogenase (ldhA) and the phosphotransferase system glucosephosphotransferase enzyme II (ptsG). A series of two-phase fermentations were conducted in which an aerobic cell growth phase was followed by an anaerobic succinate production phase using several constant concentrations of CO2. As the concentration of CO2 in the gas phase increased from 0% to 50%, the succinate specific productivity increased from 1.9mg/gh to 225mg/gh, and the succinate yield increased from 0.04g/g to 0.75g/g. Above 50% CO2, succinate production did not increase further. Intracellular fluxes were determined at three different CO2 concentrations (3%, 10%, and 50%) using 13C-label tracing coupled with LC–MS analysis. The fraction of carbon flux into the pentose phosphate pathway increased from 0.04 at 3% CO2 to 0.17 at 50% CO2. Also, the fractional flux through anaplerotic carboxylation at the phosphoenolpyruvate (PEP) node increased slightly from 0.53 at 3% CO2 to 0.63 at 50% CO2. The increased flux into the pentose phosphate pathway is attributed to an increased demand for reduced cofactors with elevated CO2. A four-process explicit model to describe the CO2 transfer and utilization was proposed. The model predicted that at CO2 concentrations below about 30–40% the system becomes limited by gas phase CO2, while at higher CO2 concentrations the system is limited by PEP carboxylase enzyme kinetics. [Copyright &y& Elsevier]

Adaptive evolution was employed to generate sodium (Na+)-tolerant mutants of Escherichia coli MG1655. Four mutants with elevated sodium tolerance, designated ALS1184, ALS1185, ALS1186, and ALS1187, were independently isolated after 73 days of serial transfer in medium containing progressively greater Na+ concentrations. The isolates also showed increased tolerance of K+, although this cation was not used for selective pressure. None of the adapted mutants showed increased tolerance to the nonionic osmolyte sucrose. Several physiological parameters of E. coli MG1655 and ALS1187, the isolate with the greatest Na+ tolerance, were calculated and compared using glucose-limited chemostats. Genome sequencing showed that the ALS1187 isolate contained mutations in five genes, emrR, hfq, kit, rpsG, and sspA, all of which could potentially affect the ability of E. coli to tolerate Na+. Two of these genes, hfq and sspA, are known to be involved in global regulatory processes that help cells endure a variety of cellular stresses. Pyruvate formate lyase knockouts were constructed in strains MG1655 and ALS1187 to determine whether increased Na+ tolerance afforded increased anaerobic generation of lactate. In fed-batch fermentations, E. coli ALS1187 pflB generated 76.2 g/liter lactate compared to MG1655 pflB, which generated only 56.3 g/liter lactate. [ABSTRACT FROM AUTHOR]

We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h-1. Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h-1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter h and yield of 0.68 g/g. [ABSTRACT FROM AUTHOR]