Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium

Levine, Steven M.; Ardeshir, Feroza; Ames, Giovanna Ferro‐Luzzi

doi:10.1128/jb.143.2.1081-1085.1980

Cited by 69 publications

(29 citation statements)

References 15 publications

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“…It seems more sensible, therefore, to consider these primarily as a safety valve for acetate excretion. Be that as it may, it is relatively easy to raise mutants deficient in the acetate excretion enzymes by enriching those insensitive to fluoroacetate [18][19][20]. This mutant was derived from E. coli.…”

Section: Cont;ersion Of Accoa To Acetatementioning

confidence: 99%

Flux analysis and control of the central metabolic pathways in Escherichia coli

Holms¹

1996

FEMS Microbiology Reviews

111

149

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The growth of the bacterial cell involves the co-ordination of the fluxes of carbon into a considerable diversity of products that are the components of the cell. Fortunately the monomers from which the cell's polymers are made are themselves synthesised from a relatively small group of precursors that are the products of the central metabolic pathways. This simplification renders cell metabolism accessible to flux analysis, a method for handling experimental data to derive metabolic fluxes. Through such analysis of the growth of Escherichia coli ML308 on 11 single carbon sources in batch, turbidostat or chemostat culture general patterns are discernible. Most significant among these are that growth on different carbon sources is achieved without any obvious enzyme acting as a regulator of metabolic flux, except when acetate is the sole source of carbon. In this case a junction is created at which iso citrate dehydrogenase (ICDH) and isocitrate lyase (ICL) compete for their common substrate and this competition is resolved by partial inactivation of ICDH to match flux through ICL and this balance limits growth rate. In this sense, flux through ICDH and ICL is 'rate-limiting'. Uptake of six of the remaining carbon inputs exceeds the capacity of the central metabolic pathways (CMPs) to sustain flux to the precursors required for growth and the CMPs are balanced by excretion of acetate. Restriction of carbon uptake by chemostat progressively diminishes growth rate and acetate excretion until acetate excretion is prevented. For the four remaining carbon sources, uptake is apparently restricted and the products are biomass, carbon dioxide and water. Carbon sources feeding the phosphorylated parts of the CMPs flux relatively more carbon to precursors (Pre-C) than CO2 when compared with carbon sources which feed into the non-phosphorylated pathways. Pre-C/CO2 ratios for the former are 1.73-3.91 and for the latter are 0.46-0.78. Flux analysis of all 11 carbon sources shows that there is an overabundant supply of 'energy' (ATP + [2H]), generated by the CMPs, in all phenotypes and conditions down to a glucose chemostat at mu of 0.72. This excess energy is a thermodynamic inefficiency which must be dissipated as heat. E. coli ML308 probably evolved in circumstances of 'feast' and 'famine'. The two strategies selected (excretion of surplus carbon and restriction of mu) would appear to be defences against 'feast'. Presumably there are defences against 'famine'. These are not made obvious by flux analysis but allosteric control of irreversible enzymes would protect pools of essential nutrients from rapid depletion on the sudden onset of 'famine'.

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Section: Cont;ersion Of Accoa To Acetatementioning

confidence: 99%

Flux analysis and control of the central metabolic pathways in Escherichia coli

Holms¹

1996

FEMS Microbiology Reviews

111

149

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“…pGTlOO was transformed into strain TA3476 (see ref. 16) mutated in the ack and ptu genes. In a first experiment, TA3476/pGT100 was grown to late exponential phase on LB medium with 0.2% glucose; then, the tuc promoter was induced by the addition of various concentrations of IPTG, and the time trajectories of glycogen accumulation were determined.…”

Section: -mentioning

confidence: 99%

Overproduction of glycogen in Escherichia coli blocked in the acetate pathway improves cell growth

Dedhia

Hottiger

Bailey

1994

Biotech & Bioengineering

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Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations.

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“…Mutations affecting phosphotransacetylase (pta) and acetate kinase (ackA) map at 50 mitautes [85,87]. The pta and ackA genes form an operon and evidence from Mud and TnlO insertions in Salmonella indicates that ackA is promoter proximal [88,89].…”

Section: Ch3co-scoa + Pi ~ Ch3co-p + Coashmentioning

confidence: 99%

The fermentation pathways of Escherichia coli

Clark¹

1989

FEMS Microbiology Reviews

351

240

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Under anaerobic conditions and in the absence of alternative electron acceptors Escherichia coli converts sugars to a mixture of products by fermentation. The major soluble products are acetate, ethanol, acetate and formate with smaller amounts of succinate. In addition the gaseous products hydrogen and carbon dioxide are produced in substantial amounts. The pathway generating fermentation products is branched and the flow down each branch is varied in response both to the pH of the culture medium and the nature of the fermentation substrate. In particular, the ratio of the various fermentation products is manipulated in order to balance the number of reducing equivalents generated during glycolytic breakdown of the substrate. The enzymes and corresponding genes involved in these fermentation pathways are described. The regulatory responses of these genes and enzymes are known but the details of the underlying regulatory mechanisms are still obscure.

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Isolation and Characterization of acetate kinase and phosphotransacetylase mutants of Escherichia coli and Salmonella typhimurium

Cited by 69 publications

References 15 publications

Flux analysis and control of the central metabolic pathways in Escherichia coli

Flux analysis and control of the central metabolic pathways in Escherichia coli

Overproduction of glycogen in Escherichia coli blocked in the acetate pathway improves cell growth

The fermentation pathways of Escherichia coli

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