Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Heyland, Jan; Blank, Lars M.; Schmid, Andreas

doi:10.1016/j.jbiotec.2011.06.016

Cited by 63 publications

(52 citation statements)

References 49 publications

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“…Heyland et al 49 experimentally proved the limited resources of adenine nucleotides in a cell, even if internal control circuits are constantly changing ATP/ADP/AMP ratios, trying to compensate the metabolic burden by enhancing the production of ACCOA and acetate allowing NADH and ATP synthesis via TCA cycle, concomitantly with the conversion of NADPH excess in NADH (through oxidative phosphorylation, not included in the TRM). Such a complex inter-dependence between energy resources and free-energy dissipation was considered by Termonia and Ross 30,31 in a simple way, by including only two reactions (Table 2, last rows) by which ATP is converted to ADP, while ADP quickly reaches its equilibrium state vs. ATP and AMP, the sum of adenine nucleotide concentrations [A(MDT) P] remaining all times constant (see the mass balances of Table 3).…”

Section: Kinetic Model Parameters Reactionmentioning

confidence: 99%

unIn silico Derivation of a Reduced Kinetic Model for Stationary or Oscillating Glycolysis in Escherichia coli Bacterium

Maria

2015

Chem.Biochem.Eng.Q.

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Section: Kinetic Model Parameters Reactionmentioning

confidence: 99%

unIn silico Derivation of a Reduced Kinetic Model for Stationary or Oscillating Glycolysis in Escherichia coli Bacterium

Maria

2015

Chem.Biochem.Eng.Q.

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“…During redox biocatalysis in living recombinant microorganisms, host metabolism may be influenced by several factors, including recombinant protein synthesis, the activity of the recombinant enzyme (e.g., cofactor/cosubstrate withdrawal, uncoupling), as well as the substrate supply and product formation (toxicity, cometabolism). Heyland et al (44) studied the effect of various dmpA expression levels in recombinant E. coli. In contrast to P4H, the catalytic activity of the ␤-aminopeptidase DmpA is not directly connected to host metabolism.…”

Section: Discussionmentioning

confidence: 99%

“…After centrifugation for 10 min at 17,000 ϫ g and 4°C, the cells were washed once with sterile water, and the pellets were stored at Ϫ20°C. The pellets were treated, and proteogenic amino acids were analyzed as described in the literature (44). Hydrolyzed biomass was resuspended in 30 l acetonitrile, to which 30 l N-methyl-N-tert-butyldimethylsilyl-trifluoracetamide (MBDSTFA) was added.…”

Section: Strains and Constructsmentioning

confidence: 99%

“…The mobile phase was composed of a gradient of 3 different eluents, described in File S3 of the supplemental material. Glucose and acetate were quantified via similar equipment, as described in the literature (44). Glucose measurements were routinely corroborated by spectrophotometric analysis with the Enzytec D-glucose kit (Scil Diagnostics GmbH, Martinsried, Germany).…”

Section: Strains and Constructsmentioning

confidence: 99%

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Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

Falcioni

Blank

Frick

et al. 2013

Appl Environ Microbiol

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Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio-and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors. I n redox biocatalysis, especially when activation of molecular oxygen is involved, the whole cell still remains the preferred catalyst. Besides the capability to continuously regenerate redox cofactors via central carbon metabolism, the whole-cell approach provides advantages with respect to operational catalyst stability, e.g., via continuous (multicomponent) enzyme synthesis, assembly, and stabilization, as well as degradation of reactive oxygen species (1-3).Oxygenases are of special interest for industrial applications, since they catalyze the highly specific oxyfunctionalization of unactivated C-H bonds under mild conditions, with molecular oxygen as the oxygen donor (4). The efficiency of an oxygenase-containing whole-cell biocatalyst depends on both enzyme and host cell performance. Hence, biocatalyst engineering must encompass both factors and be integrated with biochemical process engineering (5, 6). The technological progress in enzyme engineering of the recent decades now allows fast generation of enzyme variants with improved properties (7). For in vivo applications, intracellular conditions have to be considered, and host cell engineering often is necessary to allow the exploitation of the catalytic capability of (engineered) enzymes. The benefits of metabolic engineering have especially become evident in fermentation processes, as ex...

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“…The requirement to express two proteins for MS2-based imaging also creates a metabolic burden for the cells that could have unintended impacts on cellular activities and might also affect the transcriptional response that is monitored. 20 Although technologies for imaging mRNA synthesis are likely to always have a footprint on the cell, it is important that this should be minimal. Aptamer tags that bind small molecules as opposed to proteins are likely to have a smaller impact on cell metabolism and behavior.…”

Section: Discussionmentioning

confidence: 99%

Imaging gene expression in real-time using aptamers

Shin

2012

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Quantification of metabolic limitations during recombinant protein production in Escherichia coli

Cited by 63 publications

References 49 publications

unIn silico Derivation of a Reduced Kinetic Model for Stationary or Oscillating Glycolysis in Escherichia coli Bacterium

unIn silico Derivation of a Reduced Kinetic Model for Stationary or Oscillating Glycolysis in Escherichia coli Bacterium

Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli

Imaging gene expression in real-time using aptamers

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