Minimal Pathway for the Regeneration of Redox Cofactors

Michele Partipilo, Eleanor J Ewins, Jacopo Frallicciardi, Tom Robinson, Bert Poolman, Dirk Jan Slotboom*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP+ thereby making NAD+ available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism.

Original languageEnglish
Pages (from-to)2280-2293
Number of pages14
JournalJACS Au
Volume1
Issue number12
Early online date12-Nov-2021
DOIs
Publication statusPublished - 27-Dec-2021

Keywords

  • Redox cofactors, metabolic pathways, synthetic biology, liposome confinement, formic acid

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