Engineering Embden-Meyerhof-Parnas Glycolysis to Generate Noncanonical Reducing Power

Noncanonical cofactors such as nicotinamide mononucleotide (NMN⁺) supplant the electron-transfer functionality of the natural cofactors, NAD(P)⁺, at a lower cost in cell-free biomanufacturing and enable orthogonal electron delivery in whole-cell metabolic engineering. Here, we redesign the high-flux Embden-Meyerhof-Parnas (EMP) glycolytic pathway to generate NMN⁺-based reducing power, by engineering Streptococcus mutans glyceraldehyde-3-phosphate dehydrogenase (Sm GapN) to utilize NMN⁺. Through iterative rounds of rational design, we discover the variant GapN Penta (P179K-F153S-S330R-I234E-G210Q) with high NMN⁺-dependent activity and GapN Ortho (P179K-F153S-S330R-I234E-G214E) with ~3.4 × 10⁶-fold switch in cofactor specificity from its native cofactor NADP⁺ to NMN⁺. GapN Ortho is further demonstrated to function in Escherichia coli only in the presence of NMN⁺, enabling orthogonal control of glucose utilization. Molecular dynamics simulation and residue network connectivity analysis indicate that mutations altering cofactor specificity must be coordinated to maintain the appropriate degree of backbone flexibility to position the catalytic cysteine. These results provide a strategy to guide future designs of NMN⁺-dependent enzymes and establish the initial steps toward an orthogonal EMP pathway with biomanufacturing potential.