Protein Engineering of an Inositol-1-phosphate Synthase Based on the Active-Site Redesign Facilitates the Biomanufacturing of myo-Inositol from Starch via In Vitro Synthetic Enzymatic Biosystem

myo-Inositol, a water-soluble B vitamin compound, has broad applications in the food, pharmaceutical, and feed industries. Sustainable production of myo-inositol from starch can be achieved using an in vitro synthetic enzymatic biosystem (ivSEB) comprising four key enzymes. The NAD⁺/NADH self-recycling hyperthermophilic inositol 1-phosphate synthase (AfIPS) from Archaeoglobus fulgidus catalyzes the rate-limiting reaction. Utilizing a combinatorial active-site saturation test and iterative saturation mutagenesis (CAST/ISM), an optimized AfIPS mutant (I11C/I334V) was obtained. This mutant retained thermal stability comparable to the wild-type enzyme and exhibited a 2-fold increase in the specific activity (1.80 to 3.83 U/mg at 70 °C), and a 3-fold improvement in catalytic efficiency (k_cat/K_m: 7.46 to 22.1 mM^–1 min^–1). Molecular dynamics (MD) simulations revealed a novel hydrogen bond between the C11 side chain and the NAD⁺ pyrophosphate, enhancing cofactor binding and stabilizing the active conformation. This stabilization promotes optimal substrate alignment and improved hydride transfer, reducing total enzyme loading in the ivSEB by approximately 40% at varying substrate levels. These findings highlight the potential of semirational engineering of rate-limiting enzymes to enhance process efficiency and reduce costs, thus advancing the feasibility of scalable and economically sustainable myo-inositol production.