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  An “energy-auxotroph” E. coli provides an in vivo platform for assessing NADH regeneration systems

Wenk, S., Schann, K., He, H., Rainaldi, V., Kim, S., Lindner, S. N., et al. (2020). An “energy-auxotroph” E. coli provides an in vivo platform for assessing NADH regeneration systems. Biotechnology and Bioengineering, 117(11), 3422-3434. doi:10.1002/bit.27490.

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Wenk, S.1, Autor           
Schann, K.1, Autor           
He, H.1, Autor           
Rainaldi, V.1, Autor           
Kim, S.1, Autor           
Lindner, S. N.1, Autor           
Bar-Even, A.1, Autor           
Affiliations:
1Systems and Synthetic Metabolism, Max Planck Research Groups, Max Planck Institute of Molecular Plant Physiology, Max Planck Society, ou_2035297              

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Schlagwörter: auxotrophy, biosensor, energy metabolism, in vivo selection, methylotrophy
 Zusammenfassung: Abstract An efficient in vivo regeneration of the primary cellular resources NADH and ATP is vital for optimizing the production of value-added chemicals and enabling the activity of synthetic pathways. Currently, such regeneration routes are tested and characterized mainly in vitro before being introduced into the cell. However, in vitro measurements could be misleading as they do not reflect enzyme activity under physiological conditions. Here, we construct an in vivo platform to test and compare NADH regeneration systems. By deleting dihydrolipoyl dehydrogenase in Escherichia coli, we abolish the activity of pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase. When cultivated on acetate, the resulting strain is auxotrophic to NADH and ATP: acetate can be assimilated via the glyoxylate shunt but cannot be oxidized to provide the cell with reducing power and energy. This strain can therefore serve to select for and test different NADH regeneration routes. We exemplify this by comparing several NAD-dependent formate dehydrogenases and methanol dehydrogenases. We identify the most efficient enzyme variants under in vivo conditions and pinpoint optimal feedstock concentrations that maximize NADH biosynthesis while avoiding cellular toxicity. Our strain thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions. This article is protected by copyright. All rights reserved.

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Sprache(n): eng - English
 Datum: 2020
 Publikationsstatus: Erschienen
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 Ort, Verlag, Ausgabe: -
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 Art der Begutachtung: -
 Identifikatoren: DOI: 10.1002/bit.27490
BibTex Citekey: doi:10.1002/bit.27490
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Titel: Biotechnology and Bioengineering
  Andere : Biotechnol. Bioeng.
Genre der Quelle: Zeitschrift
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Ort, Verlag, Ausgabe: New York : Wiley [etc.]
Seiten: - Band / Heft: 117 (11) Artikelnummer: - Start- / Endseite: 3422 - 3434 Identifikator: ISSN: 0006-3592
CoNE: https://pure.mpg.de/cone/journals/resource/111088195273104