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

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.

Awakening a latent carbon fixation cycle inEscherichia coli

“…We sequenced the genome of the mutated strain and identified a single mutation (compared to the parental strain): the mobile element IS5 42 was inserted 104 base pairs upstream of the pntAB operon (Supplementary Data), which encodes for the membrane-bound transhydrogenase that plays a key role in supplying the cell with NADPH 43,44 . Insertion of the IS5 mobile element is well-known to occur in adaptive evolution experiments, increasing the expression levels of the downstream genes 45,46 .…”

“…We wondered whether other metabolic manipulations that target NADPH homeostasis could also enable the growth of the ΔtktAB +pGED strain. We tested the deletion of the gene encoding for the soluble transhydrogenase (Δsth), as this enzyme is known to provide a strong sink for NADPH 43,44 . Yet, the ΔtktAB Δsth +pGED strain did not grow on xylose even at an elevated CO2 concentration (less than two doublings, brown lines in Fig.…”

In vivoselection for formate dehydrogenases with high efficiency and specificity towards NADP+

“…, with the exception of 6-phosphogluconate dehydrogenase 20 . This strain is auxotrophic for NADPH and can grow on a minimal medium only when gluconate is added as a source of NADPH (doubling time of 2.1 hours, Figure 4a).…”

“…This strain is auxotrophic for NADPH and can grow on a minimal medium only when gluconate is added as a source of NADPH (doubling time of 2.1 hours, Figure 4a). Without gluconate, this strain can be used as a biosensor for the ability of different enzymes to support the in vivo regeneration of this cofactor 20 . We showed that expression of an FDH variant from Mycobacterium vaccae N10 (C145S/A198G/D221Q/C255V, MvaFDH 4M ) -an engineered enzyme with one of the highest reported activities towards NADPH, k cat /K M = 21 s -1 mM -1 12can rescue the growth of this strain upon addition of 75 mM formate (red line in Figure 4a, doubling time of 4.1 hours) 20 .…”

“…Without gluconate, this strain can be used as a biosensor for the ability of different enzymes to support the in vivo regeneration of this cofactor 20 . We showed that expression of an FDH variant from Mycobacterium vaccae N10 (C145S/A198G/D221Q/C255V, MvaFDH 4M ) -an engineered enzyme with one of the highest reported activities towards NADPH, k cat /K M = 21 s -1 mM -1 12can rescue the growth of this strain upon addition of 75 mM formate (red line in Figure 4a, doubling time of 4.1 hours) 20 . As the NAD-dependent malic enzyme, encoded by maeA, is also known to exhibit some NADP + reduction activity 33 , we decided to delete it as well.…”