Directed evolution of an NAD-preferring shikimate dehydrogenase mutant for enhanced catalytic efficiency

Cofactor imbalance is a pervasive challenge in metabolic engineering, particularly when microbial hosts with native NADH-dominated redox pools express heterologous pathways requiring NADPH. A prominent example is the shikimate pathway, where NADPH-dependent shikimate dehydrogenase (SDH) constitutes a bottleneck. Although rational design has attempted to shift SDH specificity toward NADH, the resulting mutants typically suffer from severely compromised catalytic efficiency (0.5% of wild-type k_cat/K_m). To address this limitation, we implemented a structure-guided saturation mutagenesis strategy targeting the cofactor-binding pocket of SDH, combined with high-throughput screening for NAD utilization. This approach yielded novel SDH mutants with significantly improved NAD-linked catalytic efficiency while maintaining thermal stability. The optimal mutant, SDH_NAD-S193H (S131A/L135A/N149D/V152F/S193H), achieved a 25-fold enhancement in NAD-dependent efficiency (k_cat/K_m) relative to the parental mutant attributed to a substantial reduction in K_m for NAD and exhibited robust activity in both in vitro assays and in vivo complementation of a shikimate-auxotrophic E. coli strain. Our work provides an efficient enzymatic module for shikimate bioproduction and offers mechanistic insights into reprogramming cofactor preference in NADP-dependent enzymes.