Metabolic Engineering ofEscherichia colifor Poly(3-hydroxybutyrate) Production under Microaerobic Condition

The alcohol dehydrogenase promoterPadhEand mixed acid fermentation pathway deficient mutants ofEscherichia coliwere employed to produce poly(3-hydroxybutyrate) (P3HB) under microaerobic condition. TheE. colimutant withackA-pta, poxB, ldhA, andadhEdeletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase genepflBdrastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. OverexpressingpflBvia the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineeredE. coliBWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.

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Aerobic Fermentation of d-Glucose by an Evolved Cytochrome Oxidase-Deficient Escherichia coli Strain

Applied and Environmental Microbiology ◽

10.1128/aem.00880-08 ◽

2008 ◽

Vol 74 (24) ◽

pp. 7561-7569 ◽

Cited By ~ 41

Author(s):

Vasiliy A. Portnoy ◽

Markus J. Herrgård ◽

Bernhard Ø. Palsson

Keyword(s):

Escherichia Coli ◽

Oxygen Uptake ◽

Growth Conditions ◽

Aerobic Growth ◽

Acid Fermentation ◽

Mixed Acid Fermentation ◽

E Coli ◽

Knockout Strain ◽

Mixed Acid ◽

Cytochrome Oxidases

ABSTRACT Fermentation of glucose to d-lactic acid under aerobic growth conditions by an evolved Escherichia coli mutant deficient in three terminal oxidases is reported in this work. Cytochrome oxidases (cydAB, cyoABCD, and cbdAB) were removed from the E. coli K12 MG1655 genome, resulting in the ECOM3 (E. coli cytochrome oxidase mutant) strain. Removal of cytochrome oxidases reduced the oxygen uptake rate of the knockout strain by nearly 85%. Moreover, the knockout strain was initially incapable of growing on M9 minimal medium. After the ECOM3 strain was subjected to adaptive evolution on glucose M9 medium for 60 days, a growth rate equivalent to that of anaerobic wild-type E. coli was achieved. Our findings demonstrate that three independently adaptively evolved ECOM3 populations acquired different phenotypes: one produced lactate as a sole fermentation product, while the other two strains exhibited a mixed-acid fermentation under oxic growth conditions with lactate remaining as the major product. The homofermenting strain showed a d-lactate yield of 0.8 g/g from glucose. Gene expression and in silico model-based analyses were employed to identify perturbed pathways and explain phenotypic behavior. Significant upregulation of ygiN and sodAB explains the remaining oxygen uptake that was observed in evolved ECOM3 strains. E. coli strains produced in this study showed the ability to produce lactate as a fermentation product from glucose and to undergo mixed-acid fermentation during aerobic growth.

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Coordination of FocA and Pyruvate Formate-Lyase Synthesis in Escherichia coli Demonstrates Preferential Translocation of Formate over Other Mixed-Acid Fermentation Products

Journal of Bacteriology ◽

10.1128/jb.02166-12 ◽

2013 ◽

Vol 195 (7) ◽

pp. 1428-1435 ◽

Cited By ~ 28

Author(s):

L. Beyer ◽

C. Doberenz ◽

D. Falke ◽

D. Hunger ◽

B. Suppmann ◽

...

Keyword(s):

Escherichia Coli ◽

Acid Fermentation ◽

Pyruvate Formate Lyase ◽

Fermentation Products ◽

Mixed Acid Fermentation ◽

Mixed Acid

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Harnessing Escherichia coli for bio-based production of formate under pressurized H2 and CO2 gases

10.1101/2021.01.06.425572 ◽

2021 ◽

Author(s):

Magali Roger ◽

Tom C. Reed ◽

Frank Sargent

Keyword(s):

Escherichia Coli ◽

Carbon Dioxide ◽

Carbon Capture ◽

Carbon Capture And Storage ◽

Host Strain ◽

Acid Fermentation ◽

E Coli ◽

Mixed Acid ◽

Batch Bioreactor ◽

Growth Deficiency

ABSRACTEscherichia coli is gram-negative bacterium that is a workhorse of the biotechnology industry. The organism has a flexible metabolism and can perform a mixed-acid fermentation under anaerobic conditions. Under these conditions E. coli synthesises a formate hydrogenlyase isoenzyme (FHL-1) that can generate molecular hydrogen and carbon dioxide from formic acid. The reverse reaction is hydrogen-dependent carbon dioxide reduction (HDCR), which has exciting possibilities in bio-based carbon capture and storage if it can be harnessed. In this study, an E. coli host strain was optimised for the production of formate from H2 and CO2 during bacterial growth in a pressurised batch bioreactor. A host strain was engineered that constitutively produced the FHL-1 enzyme and incorporation of tungsten in to the enzyme, in place of molybdenum, helped poise the reaction in the HDCR direction. The engineered E. coli strain showed an ability to grow under fermentative conditions while simultaneously producing formate from gaseous H2 and CO2 supplied in the bioreactor. However, while a sustained pressure of 10 bar N2 had no adverse effect on cell growth, when the culture was placed at or above 4 bar pressure of a H2:CO2 mixture then a clear growth deficiency was observed. Taken together, this work demonstrates that growing cells can be harnessed to hydrogenate carbon dioxide and provides fresh evidence that the FHL-1 enzyme may be intimately linked with bacterial energy metabolism.

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Dynamic metabolomic responses of Escherichia coli to nicotine stress

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0206 ◽

2014 ◽

Vol 60 (8) ◽

pp. 547-556 ◽

Cited By ~ 5

Author(s):

Lijian Ding ◽

Juanjuan Chen ◽

Jianding Zou ◽

Limin Zhang ◽

Yangfang Ye

Keyword(s):

Escherichia Coli ◽

Metabolic Response ◽

Tricarboxylic Acid Cycle ◽

Metabolic Effects ◽

Acid Fermentation ◽

Nucleotide Synthesis ◽

E Coli ◽

Mixed Acid ◽

Degrading Bacterium ◽

Degrading Bacteria

Previously, we reported the metabolic responses of Pseudomonas sp. strain HF-1, a nicotine-degrading bacterium, to nicotine stress. However, the metabolic effects of nicotine on non-nicotine-degrading bacteria that dominate the environment are still unclear. Here, we have used nuclear magnetic resonance based metabolomics in combination with multivariate data analysis methods to comprehensively analyze the metabolic changes in nicotine-treated Escherichia coli. Our results showed that nicotine caused the changes of energy-related metabolism that we believe are due to enhanced glycolysis and mixed acid fermentation as well as inhibited tricarboxylic acid cycle activity. Furthermore, nicotine resulted in the alteration of choline metabolism with a decreased synthesis of betaine but an increased production of dimethylamine. Moreover, nicotine caused a decrease in amino acid concentration and an alteration of nucleotide synthesis. We hypothesize that these changes caused the decrease in bacterial cell density observed in the experiment. These findings provide a comprehensive insight into the metabolic response of E. coli to nicotine stress. Our study highlights the value of metabolomics in elucidating the metabolic mechanisms of nicotine action.

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Formate and its role in hydrogen production in Escherichia coli

Biochemical Society Transactions ◽

10.1042/bst0330042 ◽

2005 ◽

Vol 33 (1) ◽

pp. 42-46 ◽

Cited By ~ 139

Author(s):

R.G. Sawers

Keyword(s):

Escherichia Coli ◽

Hydrogen Production ◽

Enzyme Complex ◽

Acid Fermentation ◽

Mixed Acid Fermentation ◽

E Coli ◽

Mixed Acid ◽

Formate Hydrogenlyase ◽

Absolute Requirement ◽

Formate Metabolism

The production of dihydrogen by Escherichia coli and other members of the Enterobacteriaceae is one of the classic features of mixed-acid fermentation. Synthesis of the multicomponent, membrane-associated FHL (formate hydrogenlyase) enzyme complex, which disproportionates formate into CO2 and H2, has an absolute requirement for formate. Formate, therefore, represents a signature molecule in the fermenting E. coli cell and factors that determine formate metabolism control FHL synthesis and consequently dihydrogen evolution.

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Hydrogen production in an aerobic environment by Escherichia coli K-12

10.1101/2022.01.11.475878 ◽

2022 ◽

Author(s):

George D Metcalfe ◽

Frank Sargent ◽

Michael Hippler

Keyword(s):

Escherichia Coli ◽

Raman Spectroscopy ◽

Formic Acid ◽

H2 Production ◽

Acid Fermentation ◽

E Coli ◽

Mixed Acid ◽

Microaerobic Conditions ◽

K 12 ◽

H2 Metabolism

Escherichia coli (E. coli) is a facultative anaerobe that can grow in a variety of environmental conditions. In the complete absence of O2, E. coli can perform a mixed-acid fermentation that contains within it an elaborate metabolism of formic acid. In this study, we use cavity-enhanced Raman spectroscopy (CERS), FTIR, liquid Raman spectroscopy, isotopic labelling, and molecular genetics to make advances in the understanding of bacterial formate and H2 metabolism. It is shown that, under anaerobic conditions, formic acid is generated endogenously, excreted briefly from the cell, and then taken up again to be disproportionated to H2 and CO2 by formate hydrogenlyase (FHL-1). However, exogenously added D-labelled formate behaves quite differently from the endogenous formate and is taken up immediately, independently, and possibly by a different mechanism, by the cell and converted to H2 and CO2. Our data support an anion-proton symport model for formic acid transport. In addition, when E. coli was grown in a microaerobic environment it was possible to analyse aspects of formate and O2 respiration occurring alongside anaerobic metabolism. While cells growing under microaerobic conditions generated endogenous formic acid, no H2 was produced. However, addition of exogenous formate at the outset of cell growth did induce FHL-1 biosynthesis and resulted in formate-dependent H2 production in the presence of O2.

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Creation of Plasmidless and Markerless Escherichia coli Succinate Producing Strain and Evaluation of its Biosynthetic Potential during Utilization of Lignocellulosic Sugars

Biotekhnologiya ◽

10.21519/0234-2758-2020-36-2-3-11 ◽

2020 ◽

Vol 36 (2) ◽

pp. 3-11

Author(s):

O.A. Zhuravliova ◽

Т.А. Voeikova ◽

A.Yu. Gulevich ◽

V.G. Debabov

Keyword(s):

Escherichia Coli ◽

Succinic Acid ◽

Plant Biomass ◽

Anaerobic Fermentation ◽

Basic Research ◽

Acid Fermentation ◽

Mixed Acid ◽

Gene Coding ◽

Lignocellulosic Sugars ◽

Basic Research Project

The plasmidless and markerless Escherichia coli succinate producing strain SGM2.0Pyc-int has been engineered and characterized. The strain has the inactivated main mixed-acid fermentation pathways due to the deletions of ldhA,poxB, ackA,pta, and adhE genes, constitutively expresses the genes of the aceEF-lpdA operon encoding components of pyravate dehydrogenase complex, and possesses the chromosomally integrated Bacillus subtilis pycA gene coding for pyruvate carboxylase. The capacity of the strain to synthesize succinic acid in course of dual-phase aerobic-anaerobic fermentation with lignocellulosic sugars as substrates was studied. The SGM2.0Pyc-int strain synthesized succinic acid from glucose, xylose, and arabinose with a molar yields of 1.41 mol/mol, 1.18 mol/mol, and 1.18 mol/mol, respectively, during the anaerobic production stage. The constructed strain has great potential for developing efficient processes for the succinic acid production from plant biomass-derived sugars. Escherichia coli, fermentation, arabinose, glucose, xylose, succinic acid. The work was supported by a Grant from the Russian Foundation for Basic Research (Project no. 18-29-14005).

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The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli

Genes ◽

10.3390/genes11040447 ◽

2020 ◽

Vol 11 (4) ◽

pp. 447

Author(s):

Klaudyna Krause ◽

Monika Maciąg-Dorszyńska ◽

Anna Wosinski ◽

Lidia Gaffke ◽

Joanna Morcinek-Orłowska ◽

...

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Protein Interactions ◽

Carbon Metabolism ◽

Molecular Mechanisms ◽

Central Carbon Metabolism ◽

Replication Initiation ◽

Cell Physiology ◽

E Coli ◽

Central Carbon

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

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Heterogeneity of uidA gene in environmental Escherichia coli populations

Journal of Water and Health ◽

10.2166/wh.2005.041 ◽

2005 ◽

Vol 3 (3) ◽

pp. 297-304 ◽

Cited By ~ 15

Author(s):

Clarivel Lasalde ◽

Roberto Rodriguez ◽

Gary A. Toranzos ◽

Henry H. Smith

Keyword(s):

Escherichia Coli ◽

Genetic Heterogeneity ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Dry Weight ◽

Source Tracking ◽

Tropical Rain Forests ◽

Rain Forests ◽

E Coli ◽

Gradient Gel Electrophoresis

Previous studies have shown that Escherichia coli can be isolated from non-polluted rivers and from bromeliad axilae in pristine areas of tropical rain forests. Finding E. coli in pristine environments is unusual because this bacterium is thought to only survive in the gut of warm-blooded animals and thus its presence should indicate recent fecal contamination. The aims of this study were 1) to determine if E. coli is part of the native soil microbiota in tropical rain forests and 2) to determine if genetic heterogeneity exists among E. coli populations. High concentrations of total coliforms (104–105 cells per 10 g of soil dry weight) and low concentrations of thermotolerant coliforms (101–102 cells per 10 g dry soil, the majority of these were found to be E. coli) were detected. PCR using uidA-specific primers was done on DNA purified from E. coli isolates and the resulting amplicons analysed by denaturing-gradient gel electrophoresis (DGGE). Out of several hundred isolates, mixtures of nine different amplicons were consistently observed. The different patterns of DGGE observed indicate that the E. coli populations in these pristine soils are genetically heterogeneous. Fecal and environmental E. coli isolates were also analysed by pulsed-field gel electrophoresis (PFGE) which showed high DNA sequence variation among the E. coli isolates. Because of these differences in the genomes, PFGE did not allow grouping of environmental versus human isolates of E. coli when compared side to side. The apparent genetic polymorphisms, as a result of genetic heterogeneity, observed in isolates from the same pristine site indicate that source tracking may be difficult to carry out using E. coli as the target organism.

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