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

Abstract

Efficient formate dehydrogenase (FDH)-based cofactor regeneration systems are widely used for biocatalytic processes due to their ready availability, low reduction potential, and production of only benign byproducts. However, FDHs are usually specific to NAD + , and NADPH regeneration with formate is challenging. Herein, an FDH with a preference for NAD + from Azospirillum palustre (ApFDH) was selected owing to its high activity. By static and dynamic structural analyses, a beneficial substitution, D222Q, was identified for cofactor-preference switching. However, its total activity was substantially decreased by 90% owing to the activity−specificity trade-off. Subsequently, a semirational library was designed and screened, which yielded a variant ApFDH D222Q+A199G+H380S with satisfactory activity and NADP + specificity. Our analysis of dynamical cross-correlations revealed a substitution combination that brought balance to the dynamical correlation network. This combination successfully overcame the activity−specificity−stability trade-off and resulted in a beneficial outcome. The substitution combination (D222Q-A199G/H380S-C256A/C146S) enabled the simultaneous improvement of activity, specificity, and stability and was successfully applied to other 17 FDHs. Finally, by employing engineered ApFDH, an NADPH regeneration system was developed, optimized, and utilized for the asymmetric biosynthesis of L-phosphinothricin.

Hierarchical Engineering of meso-Diaminopimelate Dehydrogenase for Efficient Synthesis of Bulky d-Amino Acids

“…Specifically, variant M3 even exhibited slightly better thermostability than the WT. These results indicate that introducing mutations (W129T, D134C, F154V, S177A, and H235I) into the active pocket of Bt DAPDH not only significantly improved the catalytic efficiency toward model substrate benzoylformic acid but also retained the thermostability well, and the well-known activity-stability trade-off was not observed in this case. Therefore, the engineered Bt DAPDH mutants might serve as promising biocatalysts for the asymmetric synthesis of structurally bulky d -amino acids.…”

Engineering Redox Cofactor Balance for Improved 5-Methyltetrahydrofolate Production in Escherichia coli

“…This study focuses on FDH derived from Candida boidinii (CbFDH) and a mutant variant of FDH from Candida dubliniensis (CdFDH), identified as 8J3P, as reported in the literature . CbFDH displays a strict preference for NAD + , whereas the CdFDH mutant, created through structure-guided rational design, has shifted from a high dependence on NAD + to a more relaxed NAD + preference, exhibiting effective utilization of NADP + . We integrated these two enzymes into the motA site of the genome for tandem expression using the P J23119 inducible promoter and two ribosome binding sites (RBS) (Figure A).…”

“…36 CbFDH displays a strict preference for NAD + , whereas the CdFDH mutant, created through structure-guided rational design, has shifted from a high dependence on NAD + to a more relaxed NAD + preference, exhibiting effective utilization of NADP + . 37 We integrated these two enzymes into the motA site of the genome for tandem expression using the P J23119 inducible promoter and two ribosome binding sites (RBS) (Figure 3A). The use of an IPTG-inducible promoter was to avoid imposing excessive metabolic burden on bacterial growth in the early stage, affecting growth or causing death.…”

Correlation Analysis of Key Residue Sites between Computational-Aided Design Thermostability d-Amino Acid Oxidase and Ancestral Enzymes

“…One of the main rational strategies involves enhancing the folding stability of natural proteins, which includes improving hydrophobic surfaces, substituting electrostatic surfaces, and flexible regions. ,, Computer algorithms have aided in accelerating the exploration of potential stabilizing substitutions within the vast sequence space, increasing the success rate of these strategies . Semirational strategies based on consensus and ancestral sequence reconstruction (ASR) exploiting the greater contribution of consensus amino acids to protein stability without structural requirements are widely used. − It is worth noting that ASR is generated within a related group, taking branch lengths into consideration, and is distinct from consensus proteins. , Nevertheless, the practical implementation of ASR still faces some uncertain limitations, such as low solubility during expression and a lack of comprehensive quantitative characterization . Therefore, exploring the relationship between ASR and the original enzyme is crucial as potential beneficial residues identified through ASR could contribute to enhancing the thermostability of this specific enzyme class.…”