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

Liliana Calzadiaz Ramirez, Carla Calvó-Tusell, Gabriele M. M. Stoffel, View ORCID ProfileSteffen N. Lindner, View ORCID ProfileSílvia Osuna, View ORCID ProfileTobias J. Erb, View ORCID ProfileMarc Garcia-Borràs, View ORCID ProfileArren Bar-Even, View ORCID ProfileCarlos G. Acevedo-Rocha
doi: https://doi.org/10.1101/2020.04.02.022350
Liliana Calzadiaz Ramirez
1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany
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Carla Calvó-Tusell
2Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, Girona 17003, Catalonia, Spain
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Gabriele M. M. Stoffel
3Max Planck Institute of Terrestrial Microbiology, Karl-von-Frisch-Str. 10, D-35043 Marburg, Germany
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Steffen N. Lindner
1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany
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Sílvia Osuna
2Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, Girona 17003, Catalonia, Spain
4ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain
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Tobias J. Erb
3Max Planck Institute of Terrestrial Microbiology, Karl-von-Frisch-Str. 10, D-35043 Marburg, Germany
5LOEWE Research Center for Synthetic Microbiology (SYNMIKRO), Karl-von-Frisch-Str. 16, D-35043 Marburg, Germany
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Marc Garcia-Borràs
2Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, Carrer Maria Aurèlia Capmany 69, Girona 17003, Catalonia, Spain
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  • For correspondence: marcgbq@gmail.com Bar-Even@mpimp-golm.mpg.de car@biosyntia.com
Arren Bar-Even
1Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany
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  • For correspondence: marcgbq@gmail.com Bar-Even@mpimp-golm.mpg.de car@biosyntia.com
Carlos G. Acevedo-Rocha
6Biosyntia ApS, 2100 Copenhagen, Denmark
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  • For correspondence: marcgbq@gmail.com Bar-Even@mpimp-golm.mpg.de car@biosyntia.com
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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 (CO2). 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 >106 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.

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Posted April 03, 2020.
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In vivo selection 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
bioRxiv 2020.04.02.022350; doi: https://doi.org/10.1101/2020.04.02.022350
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In vivo selection 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
bioRxiv 2020.04.02.022350; doi: https://doi.org/10.1101/2020.04.02.022350

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