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

• Published in: ACS synthetic biology Vol. 7; no. 12; pp. 2742 - 2749
• Main Authors: Lindner, Steffen N., Ramirez, Liliana Calzadiaz, Kruesemann, Jan L., Yishai, Oren, Belkhelfa, Sophia, He, Hai, Bouzon, Madeleine, Doring, Volker, Bar-Even, Arren
• Format: Journal Article
• Language: English
• Published: American Chemical Society 01.12.2018
• Subjects: Life Sciences; enzyme screening; NADPH regeneration; dihydrolipoamide dehydrogenase; formate dehydrogenase; cinnamyl alcohol; selection strain; auxotroph strain; glyceraldehyde 3-phosphate dehydrogenase
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.8b00313

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.