An "energy‐auxotroph" Escherichia coli provides an in vivo platform for assessing NADH regeneration systems. Issue 11 (22nd July 2020)

• Record Type: Journal Article
• Authors: Wenk, Sebastian; Schann, Karin; He, Hai; Rainaldi, Vittorio; Kim, Seohyoung; Lindner, Steffen N.; Bar‐Even, Arren
• Is Part Of: Biotechnology and bioengineering. Volume 117:Issue 11(2020)
• Page Start: 3422
• Page End: 3434
• Publication Date: 2020-07-22
• Subjects: auxotrophy -- biosensor -- energy metabolism -- in vivo selection -- methylotrophy; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27490
• Languages: English
• ISSNs: 0006-3592

Abstract

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. Abstract : Wenk, Schann and coworkers have constructed an Escherichia coli strain deleted in dihydrolipoyl dehydrogenase, which participates in the pyruvate and 2‐ketoglutarate dehydrogenase complexes. When cultivated on acetate, this strain is auxotrophic to NADH and ATP, and thus provides a useful platform for comparing and optimizing enzymatic systems for cofactor regeneration under physiological conditions. Several NAD‐dependent formate dehydrogenases and methanol dehydrogenases were compared according to their ability to regenerate NADH, and optimal feedstock concentrations that maximize NADH biosynthesis were identified.