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Journal Articles ACS Synthetic Biology Year : 2018

NADPH-Auxotrophic E-coil: A Sensor Strain for Testing in Vivo Regeneration of NADPH

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Abstract

Insufficient rate of NADPH regeneration often limits the activity of biosynthetic pathways. Expression of NADPH-regenerating enzymes is commonly used to address this problem and increase cofactor availability. Here, we construct an Escherichia coli NADPH-auxotroph strain, which is deleted in all reactions that produce NADPH with the exception of 6-phosphogluconate dehydrogenase. This strain grows on a minimal medium only if gluconate is added as NADPH source. When gluconate is omitted, the strain serves as a "biosensor" for the capability of enzymes to regenerate NADPH in vivo. We show that the NADPH-auxotroph strain can be used to quantitatively assess different NADPH-regenerating enzymes and provide essential information on expression levels and concentrations of reduced substrates required to support optimal NADPH production rate. The NADPH-auxotroph strain thus serves as an effective metabolic platform for evaluating NADPH regeneration within the cellular context.

Dates and versions

cea-02291351 , version 1 (18-09-2019)

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Steffen N. Lindner, Liliana Calzadiaz Ramirez, Jan L. Kruesemann, Oren Yishai, Sophia Belkhelfa, et al.. NADPH-Auxotrophic E-coil: A Sensor Strain for Testing in Vivo Regeneration of NADPH. ACS Synthetic Biology, 2018, 7 (12), pp.2742-2749. ⟨10.1021/acssynbio.8b00313⟩. ⟨cea-02291351⟩
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