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

-Inositol, a water-soluble B vitamin compound, has broad applications in the food, pharmaceutical, and feed industries. Sustainable production of -inositol from starch can be achieved using an synthetic enzymatic biosystem (ivSEB) comprising four key enzymes. The NAD/NADH self-recycling hyperthermophilic inositol 1-phosphate synthase (AfIPS) from 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 (/: 7.46 to 22.1 mM min). 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 -inositol production.