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Proteins differentially expressed with (pink) or without (green) 50 mM acetic acid. (A) Strain W3110 ackA-pta was grown in LBK broth–50 mM MOPS–50 mM TES, pH 6.7, to an OD600 of 0.4. (B) Strain W3110 was grown in glycerol M63 salts–50 mM MOPS–50 mM TES, pH 6.7, to an OD600 of 0.2. Other growth and gel conditions were as for Fig. 2.
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Acetate and formate are major fermentation products ofEscherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate
or formate, and the protein profiles were compar...
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Citations
... In particular, during Test 3, AA increased from 2.00 ± 0.04 g/L to 2.72 ± 0.10 g/L and pH decreased from 7.08 ± 0.02 down to 5.79 ± 0.04 within 72 h, whereas for Test 2, the concentration of AA increased roughly by 0.50 g/L, while pH decreased to 6.59 ± 0.01 at t = 24 h. Afterwards, AA started to be consumed, which resulted to a pH increase from 6.59 ± 0.01 to 8.89 ± 0.06 at t = 72 h [29]. As growth under higher glucose concentrations was hindered likely due to the overflow metabolism of glucose, which results in AA excretion and the corresponding pH reduction, the initial concentration of 0.5 g/L for both glucose and AA was chosen as the optimum for further experimental set ups. ...
Background
A potential alternative to lactic acid production through sugar fermentation is its recovery from grass silage leachate. The separation and purification of lactic acid from fermentation broths remain a key issue, as it amounts to up to 80% of its industrial production cost. In this study, a genetically engineered E. coli strain (A1:ldhA), that cannot catabolize lactic acid, has been used to selectively remove impurities from a synthetic medium comprising typical components (i.e., glucose and acetic acid) of green grass silage leachate. A systematic approach has been followed to provide a proof-of-concept for a bio-purification process of lactic acid solutions in a membrane bioreactor operating in semi-continuous mode.
Results
The synthetic medium composition was initially optimized in shake-flasks experiments, followed by scale-up in bench-scale bioreactor. Complete (i.e., 100%) and 60.4% removal for glucose and acetic acid, respectively, has been achieved in batch bioreactor experiments with a synthetic medium comprising 0.5 g/L glucose and 0.5 g/L acetic acid as carbon sources, and 10 g/L lactic acid; no lactic acid catabolism was observed in all batch fermentation tests. Afterwards, a hybrid biotechnological process combining semi-continuous bioreactor fermentation and ultrafiltration membrane separation (membrane bioreactor) was applied to in-situ separate purified medium from the active cells. The process was assessed under different semi-continuous operating conditions, resulting in a bacteria-free effluent and 100% glucose and acetic acid depletion, with no lactic acid catabolism, thus increasing the purity of the synthetic lactic acid solution.
Conclusions
The study clearly demonstrated that a bio-purification process for lactic acid employing the engineered E. coli strain cultivated in a membrane bioreactor is a technically feasible concept, paving the way for further technological advancement.
... In particular, during Test 3, AA increased from 2.00 ± 0.04 g/L to 2.72 ± 0.10 g/L and pH decreased from 7.08 ± 0.02 down to 5.79 ± 0.04 within 72 h, whereas for Test 2, the concentration of AA increased roughly by 0.50 g/L, while pH decreased to 6.59 ± 0.01 at t = 2 4h. Afterwards, AA started to be consumed, which resulted to a pH increase from 6.59 ± 0.01 to 8.89 ± 0.06 at t = 72 h [29]. As growth under higher glucose concentrations was hindered likely due to the over ow metabolism of glucose, which results in AA excretion and the corresponding pH reduction, the initial concentration of 0.5 g/L for both glucose and AA was chosen as the optimum for further experimental set ups. ...
Background: A potential alternative to lactic acid production through sugar fermentation is its recovery from grass silage leachate. The separation and purification of lactic acid from fermentation broths remains a key issue, as it amounts to up to 80% of its industrial production cost. In this study, a genetically engineered E. coli strain (A1:ldhA), that cannot catabolize lactic acid, has been used to selectively remove impurities from a synthetic medium comprising typical components (i.e. glucose and acetic acid) of green grass silage leachate. A systematic approach has been followed to provide a proof-of-concept for a bio-purification process of lactic acid solutions in a membrane bioreactor operating in semi-continuous mode.
Results: The synthetic medium composition was initially optimized in shake-flasks experiments, followed by scale-up in bench-scale bioreactor. Complete (i.e. 100%) and 60.4% removal for glucose and acetic acid, respectively, has been achieved in batch bioreactor experiments with a synthetic medium comprising 0.5 g/L glucose and 0.5 g/L acetic acid as carbon sources, and 10 g/L lactic acid; no lactic acid catabolism was observed in all batch fermentation tests. Afterwards, a hybrid biotechnological process combining semi-continuous bioreactor fermentation and ultrafiltration membrane separation (membrane bioreactor) was applied to in-situ separate purified medium from the active cells. The process was assessed under different semi-continuous operating conditions, resulting in a bacteria-free effluent and 100% glucose and acetic acid depletion, with no lactic acid catabolism, thus increasing the purity of the synthetic lactic acid solution.
Conclusion: The study clearly demonstrated that a bio-purification process for lactic acid employing the engineered E. coli strain cultivated in a membrane bioreactor is a technically feasible concept, paving the way for further technological advancement.
... In addition, a 1.5-to 3-fold increase in ethanol and formate levels in p-CA and FA-treated cells, a 2-to 4-fold decrease in isocitrate levels in t-CA and FA-treated cells, and about a 3-fold increase in acetate and a 9-fold increase in succinate levels in FA-treated cells were also observed. These observations suggest that all three phenolic acids trigger a switch from aerobic to anaerobic metabolism since lactate, ethanol, acetate, formate, and succinate constitute the major fermentation products in E. coli (Kirkpatrick et al. 2001). In addition, the accumulation of some of the amino acids in response to phenolic acid treatments also points to a reduction in protein synthesis and cellular respiration. ...
Phenolic acids are derivatives of benzoic and cinnamic acids, which possess important biological activities at certain concentrations. Trans-cinnamic acid (t-CA) and its derivatives, such as p-coumaric acid (p-CA) and ferulic acid (FA) have been shown to have antibacterial activity against various Gram-positive and -negative bacteria. However, there is limited information available concerning the antibacterial mode of action of these phenolic acids. In this study, we aimed to ascertain metabolic alterations associated with exposure to t-CA, p-CA, and FA in Escherichia coli BW25113 using a nuclear magnetic resonance (NMR)-based metabolomics approach. The results showed that t-CA, p-CA, and FA treatments led to significant changes (p < 0.05) in the concentration of 42, 55, and 74% of the identified metabolites in E. coli, respectively. Partial least-squares discriminant analysis (PLS-DA) revealed a clear separation between control and phenolic acid groups with regard to metabolic response. Moreover, it was found that FA and p-CA treatment groups were clustered closely together but separated from the t-CA treatment group. Arginine, putrescine, cadaverine, galactose, and sucrose had the greatest impact on group differentiation. Quantitative pathway analysis demonstrated that arginine and proline, pyrimidine, glutathione, and galactose metabolisms, as well as aminoacyl-tRNA and arginine biosyntheses, were markedly affected by all phenolic acids. Finally, the H2O2 content of E. coli cells was significantly increased in response to t-CA and p-CA whereas all phenolic acids caused a dramatic increase in the number of apurinic/apyrimidinic sites. Overall, this study suggests that the metabolic response of E. coli cells to t-CA is relatively different from that to p-CA and FA. However, all phenolic acids had a certain impact on oxidative/antioxidant status, genomic stability, arginine-related pathways, and nucleic acid metabolism.
... This hypothesis predicts that conditions in which both carbon source and stress level change will yield growth and death rates that reside between the sugar and stress lines. Indeed, we find that combinations of glycerol+TMP and glycerol+NaCl lie between these lines, as does acetate, which is a poor carbon source known to induce a stress response (47,48) Fig. S9). ...
Antibiotic effectiveness depends on a variety of factors. While many mechanistic details of antibiotic action are known, the connection between death rate and bacterial physiology is poorly understood. A common observation is that death rate in antibiotics rises linearly with growth rate; however, it remains unclear how other factors, such as environmental conditions and whole-cell physiological properties, affect bactericidal activity. To address this, we developed a high-throughput assay to precisely measure antibiotic-mediated death. We found that death rate is linear in growth rate, but the slope depends on environmental conditions. Growth under stress lowers death rate compared to nonstressed environments with similar growth rate. To understand stress’s role, we developed a mathematical model of bacterial death based on resource allocation that includes a stress-response sector; we identify this sector using RNA-seq. Our model accurately predicts the minimal inhibitory concentration (MIC) with zero free parameters across a wide range of growth conditions. The model also quantitatively predicts death and MIC when sectors are experimentally modulated using cyclic adenosine monophosphate (cAMP), including protection from death at very low cAMP levels. The present study shows that different conditions with equal growth rate can have different death rates and establishes a quantitative relation between growth, death, and MIC that suggests approaches to improve antibiotic efficacy.
... Formic acid is a weak acid (pK a = 3.74), and its toxicity is associated with the diffusion across the cell membrane at low pH, thus acidifying the cytoplasm and impairing the proton motive force (50). Formate can also lead to oxidative stress and inhibit the activity of bacterial cytochrome c oxidases (51,52). To overcome the toxicity brought about by this C1 acid, microorganisms have evolved different dissimilation mechanisms. ...
The soil bacterium Pseudomonas putida is a robust biomanufacturing host that assimilates a broad range of substrates while efficiently coping with adverse environmental conditions. P. putida is equipped with functions related to one-carbon (C1) compounds (e.g. methanol, formaldehyde, and formate) oxidation—yet pathways to assimilate these carbon sources are largely absent. In this work, we adopted a systems-level approach to study the genetic and molecular basis of C1 metabolism in P. putida . RNA sequencing identified two oxidoreductases, encoded by PP_0256 and PP_4596 , transcriptionally active in the presence of formate. Quantitative physiology of deletion mutants revealed growth defects at high formate concentrations, pointing to an important role of these oxidoreductases in C1 tolerance. Moreover, we describe a concerted detoxification process for methanol and formaldehyde, the C1 intermediates upstream formate. Alcohol oxidation to highly-reactive formaldehyde by PedEH and other broad-substrate-range dehydrogenases underpinned the (apparent) suboptimal methanol tolerance of P. putida . Formaldehyde was mostly processed by a glutathione-dependent mechanism encoded in the frmAC operon, and thiol-independent FdhAB and AldB-II overtook detoxification at high aldehyde concentrations. Deletion strains were constructed and characterized towards unveiling these biochemical mechanisms, underscoring the worth of P. putida for emergent biotechnological applications—e.g. engineering synthetic formatotrophy and methylotrophy.
IMPORTANCE
C1 substrates continue to attract interest in biotechnology, as their use is both cost-effective and ultimately expected to mitigate the impact of greenhouse gas emissions. However, our current understanding of bacterial C1 metabolism remains relatively limited in species that cannot grow on (i.e., assimilate) these substrates. Pseudomonas putida , a model Gram-negative environmental bacterium, constitutes a prime example of this sort. The biochemical pathways active in response to methanol, formaldehyde, and formate have been largely overlooked—although the ability of P. putida to process C1 molecules has been previously alluded to in the literature. By using a systems-level strategy, this study bridges such knowledge gap through the identification and characterization of mechanisms underlying methanol, formaldehyde, and formate detoxification—including hitherto unknown enzymes that act on these substrates. The results reported herein both expand our understanding of microbial metabolism and lay a solid foundation for engineering efforts toward valorizing C1 feedstocks.
... This could point to mixed-acid fermentation actively reusing formate when it is present in high concentration to generate the high-energy metabolite pyruvate. A past study showed that adding formate to E. coli downregulated the expression of 6-phosphofructokinase pfkB (45). How mixed-acid fermentation and the upper part of glycolysis work together under high formate concentration is still elusive. ...
Shigellosis caused by Shigella is one of the most important foodborne illnesses in global health, but little is known about the metabolic cross talk between this bacterial pathogen and its host cells during the different stages of the infection process. A detailed understanding of the metabolism can potentially lead to new drug targets remedying the pressing problem of antibiotic resistance. Here, we use stable isotope-resolved metabolomics as an unbiased and fast method to investigate how Shigella metabolizes 13C-glucose in three different environments: inside the host cells, adhering to the host cells, and alone in suspension. We find that especially formate metabolism by bacteria is sensitive to these different environments. The role of formate in pathogen metabolism is sparsely described in the literature compared to the roles of acetate and butyrate. However, its metabolic pathway is regarded as a potential drug target due to its production in microorganisms and its absence in humans. Our study provides new knowledge about the regulatory effect of formate. Bacterial metabolism of formate is pH dependent when studied alone in culture medium, whereas this effect is less pronounced when the bacteria adhere to the host cells. Once the bacteria are inside the host cells, we find that formate accumulation is reduced. Formate also affects the host cells resulting in a reduced infection rate. This was correlated to an increased immune response. Thus, intriguingly formate plays a double role in pathogenesis by increasing the virulence of Shigella and at the same time stimulating the immune response of the host. IMPORTANCE Bacterial infection is a pressing societal concern due to development of resistance toward known antibiotics. Central carbon metabolism has been suggested as a potential new target for drug development, but metabolic changes upon infection remain incompletely understood. Here, we used a cellular infection model to study how the bacterial pathogen Shigella adapts its metabolism depending on the environment starting from the extracellular medium until Shigella successfully invaded and proliferated inside host cells. The mixed-acid fermentation of Shigella was the major metabolic pathway during the infectious process, and the glucose-derived metabolite formate surprisingly played a divergent role in the pathogen and in the host cell. Our data show reduced infection rate when both host cells and bacteria were treated with formate, which correlated with an upregulated immune response in the host cells. The formate metabolism in Shigella thus potentially provides a route toward alternative treatment strategies for Shigella prevention.
... RpoS is a major regulator of the Frontiers in Bioengineering and Biotechnology frontiersin.org general stress response in E. coli and positively affects resistance to acid stress (Kirkpatrick et al., 2001). Since, the deletion of ackA-pta in the naïve strain already improved growth performance to the level of the K4e2 strain, reverse engineering of rpoC and pdhR, that cannot directly be connected to carbon and energy metabolism, was not pursued. ...
Microbial C1 fixation has a vast potential to support a sustainable circular economy. Hence, several biotechnologically important microorganisms have been recently engineered for fixing C1 substrates. However, reports about C1-based bioproduction with these organisms are scarce. Here, we describe the optimization of a previously engineered formatotrophic Escherichia coli strain. Short-term adaptive laboratory evolution enhanced biomass yield and accelerated growth of formatotrophic E. coli to 3.3 g-CDW/mol-formate and 6 h doubling time, respectively. Genome sequence analysis revealed that manipulation of acetate metabolism is the reason for better growth performance, verified by subsequent reverse engineering of the parental E. coli strain. Moreover, the improved strain is capable of growing to an OD600 of 22 in bioreactor fed-batch experiments, highlighting its potential use for industrial bioprocesses. Finally, demonstrating the strain’s potential to support a sustainable, formate-based bioeconomy, lactate production from formate was engineered. The optimized strain generated 1.2 mM lactate —10% of the theoretical maximum— providing the first proof-of-concept application of the reductive glycine pathway for bioproduction.
... Decreased susceptibility of E. coli to Gm under anaerobic conditions. To investigate whether the type of metabolism used for growth influences the level of AG susceptibility, we measured MIC of the wild type (WT) strain in aerobiosis and anaerobiosis, in Luria-Bertani (LB) medium supplemented with fumarate, NO 3 -, or no electron acceptor, where in the latter instance we assumed mixed-acid fermentation was used (26). Under all anaerobic growth conditions tested, the MIC value of Gm was 4-fold higher than under oxygen, i.e., 8 mg/mL. ...
Antibiotic resistance is a major public health, social, and economic problem. Aminoglycosides (AG) are known to be highly effective against Gram-negative bacteria, but their use is limited to life-threatening infections because of their nephrotoxicity and ototoxicity at therapeutic dose.
... 10,11 Although naturally produced SCFAs have positive effects on the microbiome, there is evidence to suggest that acetate, when externally added in the acidic form, induced a stress response in Escherichia coli. 15 Kirkpatrick et al found that acetic acid at 50 mM induced multiple members of the RpoS regulon; Dps (a DNA-binding protein) was the most strongly induced, while seventeen proteins were repressed. 15 RpoS is a sigma factor responsible for the general stress response and regulating stationary phase in bacterial cells. ...
... 15 Kirkpatrick et al found that acetic acid at 50 mM induced multiple members of the RpoS regulon; Dps (a DNA-binding protein) was the most strongly induced, while seventeen proteins were repressed. 15 RpoS is a sigma factor responsible for the general stress response and regulating stationary phase in bacterial cells. 16 Kirkpatrick et al also found that formic acid prompted a stress response in E. coli like that of acetic acid. ...
... The addition of acetic acid after the stress response mitigated the activation of those proteins. 15 Further studies have shown that sodium acetate enhanced the resistance of E. coli to oxidative stress and heat killing, and all three SCFAs (acetate, butyrate, and propionate) at neutral pH increased the acid survival of E. coli. 6 Although there is substantive research exploring the effects of acetate on stress response in E. coli, there is a current gap in understanding the same effects of butyrate and propionate. ...
Regulation of microbial symbiosis in the human intestinal tract is imperative to maintain overall human health and prevent dysbiosis-related diseases, such as inflammatory bowel disease and obesity. Short-chain fatty acids (SCFA) in the intestine are produced by bacterial fermentation and aid in inflammation reduction, dietary fiber digestion, and metabolizing nutrients for the colon. SCFA, notably acetate, butyrate, and propionate, are starting to be used in clinical interventions for GI diseases. While acetate has been shown to mitigate a stress response in the proteome of Escherichia coli cells, little is known about the effects of butyrate and propionate on the same cells. This study aims to evaluate the effects that butyrate and propionate have on the activation of stress promoters in E. coli when induced with a known stressor. Three different strains of E. coli containing the pUCD615 plasmid were used, each with a different promoter fused to the structural genes of the lac operon on the plasmid. Each promoter detected a unique stress response: grpE’::lux fusion (heat shock), recA’::lux fusion (SOS response), and katG’::lux fusion (oxidative damage). Activation of these stress promoters by treatment groups resulted in the emission of bioluminescence which was quantified and compared across treatment groups. All three SCFAs at 25 mM added to cultures prior to stressing the bacteria caused significantly lower bioluminescence levels when compared to the stressed culture without prior addition of SCFA. This indicates that these SCFAs may reduce the stress response in E. coli. KEYWORDS: Short-chain fatty acids; acetate; butyrate; propionate; Escherichia coli; stress response; Vibrio fischeri luxCDABE; grpE; katG; recA
... The extended lag phases in three of the four bacteria in the presence of acetate suggest that an adaptive response was required to increase acid tolerance and exploit the new nutrient source. Energy is required to pump out hydrogen ions from the cell, and acetate is known to trigger the production of several proteins in E. coli [27], which means that energy is not available to initiate growth. * Significant (p < 0.05) difference between the mean maximum growth rates in standard or modified broth with and without Co-cit (column 2) or between modified NB and standard NB (column 4). ...
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