A High-Throughput Method for Directed Evolution of NAD(P)⁺-Dependent Dehydrogenases for the Reduction of Biomimetic...

Engineering flavin-free NAD(P)⁺-dependent dehydrogenases to reduce biomimetic nicotinamide analogues (mNAD⁺s) is of importance for eliminating the need for costly NAD(P)⁺ in coenzyme regeneration systems. Current redox dye-based screening methods for engineering the mNAD⁺ specificity of dehydrogenases are frequently encumbered by a background signal from endogenous NAD(P) and intracellular reducing compounds, making the detection of low mNAD⁺-based activities a limiting factor for directed evolution. Here, we develop a high-throughput screening method, NAD(P)-eliminated solid-phase assay (NESPA), which can reliably identify mNAD⁺-active mutants of dehydrogenases with a minimal background signal. This method involves (1) heat lysis of colonies to permeabilize the cell membrane, (2) colony transfer onto filter paper, (3) washing to remove endogenous NAD(P) and reducing compounds, (4) enzyme-coupled assay for mNADH-dependent color production, and (5) digital imaging of colonies to identify mNAD⁺-active mutants. This method was used to improve the activity of 6-phosphogluconate dehydrogenase on nicotinamide mononucleotide (NMN⁺). The best mutant obtained after six rounds of directed evolution exhibits a 50-fold enhancement in catalytic efficiency (k _cat/K _M) and a specific activity of 17.7 U/mg on NMN⁺, which is comparable to the wild-type enzyme on its natural coenzyme, NADP⁺. The engineered dehydrogenase was then used to construct an NMNH regeneration system to drive an ene-reductase catalysis. A comparable level of turnover frequency and product yield was observed using the engineered system relative to NADPH regeneration by using the wild-type dehydrogenase. NESPA provides a simple and accurate readout of mNAD⁺-based activities and the screening at high-throughput levels (approximately tens of thousands per round), thus opening up an avenue for the evolution of dehydrogenases with specific activities on mNAD⁺s similar to the levels of natural enzyme/coenzyme pairs.