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

Liliana Calzadiaz Ramirez, Carla Calvó-Tusell, Gabriele M. M. Stoffel, Steffen N. Lindner, Sílvia Osuna, Tobias J. Erb, Marc Garcia-Borràs, Arren Bar-Even, Carlos G. Acevedo-Rocha
2020 · doi:10.1101/2020.04.02.022350
preprint OA: closed CC-BY-NC-ND-4.0
📄 Open PDF View at publisher

Abstract

Efficient regeneration of cofactors is vital for the establishment of continuous biocatalytic processes. Formate is an ideal electron donor for cofactor regeneration due to its general availability, low reduction potential, and benign byproduct (CO 2 ). However, formate dehydrogenases (FDHs) are usual specific to NAD + , such that NADPH regeneration with formate is challenging. Previous studies reported naturally occurring FDHs or engineered FDHs that accept NADP + , but these enzymes show low kinetic efficiencies and specificities. Here, we harness the power of natural selection to engineer FDH variants to simultaneously optimize three properties: kinetic efficiency with NADP + , specificity towards NADP + , and affinity towards formate. By simultaneously mutating multiple residues of FDH from Pseudomonas sp. 101, which exhibits no initial activity towards NADP + , we generate a library of >10 6 variants. We introduce this library into an E. coli strain that cannot produce NADPH. By selecting for growth with formate as sole NADPH source, we isolate several enzyme variants that support efficient NADPH regeneration. We find that the kinetically superior enzyme variant, harboring five mutations, has 5-fold higher efficiency and 13-fold higher specificity than the best enzyme previously engineered, while retaining high affinity towards formate. By using molecular dynamics simulations, we reveal the contribution of each mutation to the superior kinetics of this variant. We further determine how non-additive epistatic effects improve multiple parameters simultaneously. Our work demonstrates the capacity of in vivo selection to identify superior enzyme variants carrying multiple mutations which would be almost impossible to find using conventional screening methods.

My notes (saved in your browser only)

Citation neighborhood (no data yet)

We don't have any in-corpus citations linked to this paper yet. The paper's references may be in our DB but unresolved to ``paper_id`` (resolution happens at ingest when the cited DOI matches a row we already have). Run the cross-source citation reconcile pass to retry.

References (60)

Source provenance

crossref
last seen: 2026-06-24T06:27:41.342438+00:00
europepmc
last seen: 2026-05-19T01:45:01.086888+00:00
unpaywall
last seen: 2026-05-22T02:00:06.705733+00:00
License: CC-BY-NC-ND-4.0