Regulatory O 2 tensions for the synthesis of fermentation products in Escherichia coli and relation to aerobic respiration

1997 ◽  
Vol 168 (4) ◽  
pp. 290-296 ◽  
Author(s):  
Sabine Becker ◽  
Dorina Vlad ◽  
Sylvia Schuster ◽  
Peter Pfeiffer ◽  
G. Unden
Keyword(s):  
Escherichia Coli ◽  
Aerobic Respiration ◽  
Fermentation Products

scholarly journals Metabolic Engineering of Escherichia coli for Enhanced Production of Succinic Acid, Based on Genome Comparison and In Silico Gene Knockout Simulation

2005 ◽  
Vol 71 (12) ◽  
pp. 7880-7887 ◽  
Author(s):  
Sang Jun Lee ◽  
Dong-Yup Lee ◽  
Tae Yong Kim ◽  
Byung Hun Kim ◽  
Jinwon Lee ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Succinic Acid ◽  
In Silico ◽  
Acid Production ◽  
Gene Knockout ◽  
Fermentation Products ◽  
E Coli ◽  
Metabolic Analysis ◽  
Enhanced Production ◽  
Succinic Acid Production

ABSTRACT Comparative analysis of the genomes of mixed-acid-fermenting Escherichia coli and succinic acid-overproducing Mannheimia succiniciproducens was carried out to identify candidate genes to be manipulated for overproducing succinic acid in E. coli. This resulted in the identification of five genes or operons, including ptsG, pykF, sdhA, mqo, and aceBA, which may drive metabolic fluxes away from succinic acid formation in the central metabolic pathway of E. coli. However, combinatorial disruption of these rationally selected genes did not allow enhanced succinic acid production in E. coli. Therefore, in silico metabolic analysis based on linear programming was carried out to evaluate the correlation between the maximum biomass and succinic acid production for various combinatorial knockout strains. This in silico analysis predicted that disrupting the genes for three pyruvate forming enzymes, ptsG, pykF, and pykA, allows enhanced succinic acid production. Indeed, this triple mutation increased the succinic acid production by more than sevenfold and the ratio of succinic acid to fermentation products by ninefold. It could be concluded that reducing the metabolic flux to pyruvate is crucial to achieve efficient succinic acid production in E. coli. These results suggest that the comparative genome analysis combined with in silico metabolic analysis can be an efficient way of developing strategies for strain improvement.

scholarly journals Acetate and Formate Stress: Opposite Responses in the Proteome of Escherichia coli

2001 ◽  
Vol 183 (21) ◽  
pp. 6466-6477 ◽  
Author(s):  
Christopher Kirkpatrick ◽  
Lisa M. Maurer ◽  
Nikki E. Oyelakin ◽  
Yuliya N. Yoncheva ◽  
Russell Maurer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Opposite Effect ◽  
Stress Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acid Resistance ◽  
Luria Broth ◽  
Acetyl Coa ◽  
Fermentation Products ◽  
E Coli

ABSTRACT 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 compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-ptastrain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of theackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

scholarly journals Enhanced nutrient uptake is sufficient to drive emergent cross-feeding between bacteria in a synthetic community

2019 ◽  
Author(s):  
Ryan K Fritts ◽  
Jordan T Bird ◽  
Megan G Behringer ◽  
Anna Lipzen ◽  
Joel Martin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Nutrient Uptake ◽  
Rhodopseudomonas Palustris ◽  
Biogeochemical Cycles ◽  
Wild Type ◽  
Fermentation Products ◽  
E Coli ◽  
Constitutive Activation ◽  
Host Health ◽  
Nitrogen Scavenging

ABSTRACTInteractive microbial communities are ubiquitous, influencing biogeochemical cycles and host health. One widespread interaction is nutrient exchange, or cross-feeding, wherein metabolites are transferred between microbes. Some cross-fed metabolites, such as vitamins, amino acids, and ammonium (NH4+), are communally valuable and impose a cost on the producer. The mechanisms that enforce cross-feeding of communally valuable metabolites are not fully understood. Previously we engineered mutualistic cross-feeding between N2-fixing Rhodopseudomonas palustris and fermentative Escherichia coli. Engineered R. palustris excreted essential nitrogen as NH4+ to E. coli while E. coli excreted essential carbon as fermentation products to R. palustris. Here, we enriched for nascent cross-feeding in cocultures with wild-type R. palustris, not known to excrete NH4+. Emergent NH4+ cross-feeding was driven by adaptation of E. coli alone. A missense mutation in E. coli NtrC, a regulator of nitrogen scavenging, resulted in constitutive activation of an NH4+ transporter. This activity likely allowed E. coli to subsist on the small amount of leaked NH4+ and better reciprocate through elevated excretion of organic acids from a larger E. coli population. Our results indicate that enhanced nutrient uptake by recipients, rather than increased excretion by producers, is an underappreciated yet possibly prevalent mechanism by which cross-feeding can emerge.

scholarly journals Dihydrolipoamide Dehydrogenase Mutation Alters the NADH Sensitivity of Pyruvate Dehydrogenase Complex of Escherichia coli K-12

2008 ◽  
Vol 190 (11) ◽  
pp. 3851-3858 ◽  
Author(s):  
Youngnyun Kim ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Double Mutant ◽  
Pyruvate Dehydrogenase Complex ◽  
Anaerobic Growth ◽  
Lower Sensitivity ◽  
Dihydrolipoamide Dehydrogenase ◽  
Fermentation Products ◽  
E Coli ◽  
K 12

ABSTRACT Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher Ki for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.

Inhibitory Activity of Cheese Whey Fermented with Kefir Grains

2011 ◽  
Vol 74 (1) ◽  
pp. 94-100 ◽  
Author(s):  
A. LONDERO ◽  
R. QUINTA ◽  
A. G. ABRAHAM ◽  
R. SERENO ◽  
G. DE ANTONI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lactic Acid ◽  
Acid Production ◽  
Cheese Whey ◽  
Lactic Acid Production ◽  
Reaction Products ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Fermentation Products ◽  
E Coli ◽  
Kefir Grains

We investigated the chemical and microbiological compositions of three types of whey to be used for kefir fermentation as well as the inhibitory capacity of their subsequent fermentation products against 100 Salmonella sp. and 100 Escherichia coli pathogenic isolates. All the wheys after fermentation with 10% (wt/vol) kefir grains showed inhibition against all 200 isolates. The content of lactic acid bacteria in fermented whey ranged from 1.04 × 107 to 1.17 × 107 CFU/ml and the level of yeasts from 2.05 × 106 to 4.23 × 106 CFU/ml. The main changes in the chemical composition during fermentation were a decrease in lactose content by 41 to 48% along with a corresponding lactic acid production to a final level of 0.84 to 1.20% of the total reaction products. The MIC was a 30% dilution of the fermentation products for most of the isolates, while the MBC varied between 40 and 70%, depending on the isolate. The pathogenic isolates Salmonella enterica serovar Enteritidis 2713 and E. coli 2710 in the fermented whey lost their viability after 2 to 7 h of incubation. When pathogens were deliberately inoculated into whey before fermentation, the CFU were reduced by 2 log cycles for E. coli and 4 log cycles for Salmonella sp. after 24 h of incubation. The inhibition was mainly related to lactic acid production. This work demonstrated the possibility of using kefir grains to ferment an industrial by-product in order to obtain a natural acidic preparation with strong bacterial inhibitory properties that also contains potentially probiotic microorganisms.

scholarly journals Metabolic Flux Control at the Pyruvate Node in an Anaerobic Escherichia coli Strain with an Active Pyruvate Dehydrogenase

2010 ◽  
Vol 76 (7) ◽  
pp. 2107-2114 ◽  
Author(s):  
Qingzhao Wang ◽  
Mark S. Ou ◽  
Y. Kim ◽  
L. O. Ingram ◽  
K. T. Shanmugam
Keyword(s):  
Escherichia Coli ◽  
Mutant Strain ◽  
Pyruvate Dehydrogenase ◽  
Metabolic Flux ◽  
Anaerobic Growth ◽  
Kinetic Properties ◽  
Fermentation Products ◽  
E Coli ◽  
Pyruvate Node

ABSTRACT During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y ATP) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y ATP suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

scholarly journals Fermentation of Glycerol to Succinate by Metabolically Engineered Strains of Escherichia coli

2010 ◽  
Vol 76 (8) ◽  
pp. 2397-2401 ◽  
Author(s):  
Xueli Zhang ◽  
K. T. Shanmugam ◽  
Lonnie O. Ingram
Keyword(s):  
Escherichia Coli ◽  
Electron Acceptors ◽  
Carbon Flow ◽  
Glycerol Dehydrogenase ◽  
Glycerol Metabolism ◽  
Wild Type ◽  
Anaerobic Conditions ◽  
Mineral Salts ◽  
Succinate Production ◽  
Fermentation Products

ABSTRACT The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD+ ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.

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