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

Abstract

CGAR) 2 A b s t r a c t 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.3 I n t r o d u c t i o n Co-factor regeneration is vital for the continuous operation of biocatalytic processes taking place either within a living cell or in a cell free system 1 . A considerable amount of research has therefore been invested in developing and optimizing enzymatic systems for the in situ regeneration of key cofactors such as ATP, NADH, and NADPH 2,3 . Formate has been long considered as a suitable reducing agent for the regeneration of NADH both in vivo and in vitro 2,4 . This is due to several properties: (i) abundance of formate dehydrogenases (FDHs) that can efficiently transfer reducing power from formate to NAD + 5 ; (ii) formate oxidation is practically irreversible, increasing the efficiency of NADH regeneration; (iii) formate, a small molecule, can easily cross membranes, thus being accessible within cellular compartments; and (iv) the byproduct of formate oxidation, CO 2 , is non-toxic and can be easily expelled from the system.Many biocatalytic processes rely on NADPH rather than NADH 6, 7 . Therefore, in the last 20 years multiple studies aimed at identifying FDHs that can naturally accept NADP + or engineering NAD-dependent FDHs to accept the phosphorylated cofactor [8][9][10][11][12][13][14][15][16] . Whi...

Switching the Cofactor Preference of Formate Dehydrogenase to Develop an NADPH-Dependent Biocatalytic System for Synthesizing Chiral Amino Acids

“…26 One unit (U) of enzyme activity was defined as the amount of enzyme catalyzing the conversion of 1 μmol of substrate per minute under standard conditions. According to previous reports, 27,28 the kinetic constants were measured by varying the ammonium formate from 5 to 250 mM, with the NADP + (5−10) were used to determine the optimum pH for ApFDH and its variants. Citric acid−sodium citrate buffer (100 mM) was prepared for pH 5.0−6.0, phosphate buffer (100 mM) was prepared for pH 6.0−8.0, Tris-HCl buffer (100 mM) was prepared for pH 8.0−9.0, and glycine−NaOH buffer (100 mM) was prepared for pH 9.0−10.0.…”

Emergence of NADP+-reducing enzymes inEscherichia colicentral metabolism via adaptive evolution

“…Several studies also constructed mutant libraries of specific oxidoreductases and harnessed natural selection to identify variants with altered cofactor specificity [5][6][7]. Yet, until now, no study has attempted to systematically explore the overall evolvability of central metabolism oxidoreductases towards the use of a different cofactor.…”