Thermostability Engineering of Ribitol Dehydrogenase and Its Application in Whole-Cell Catalytic Production of Allitol

Allitol is a six-carbon rare sugar alcohol with physiological activities such as antihypertensive and anti-obesity effects, holding application value in the functional food and pharmaceutical industries. Currently, whole-cell catalysis for allitol production is still limited by relatively low reaction temperatures. Ribitol dehydrogenase (PaRDH), which catalyzes the reduction of psicose to allitol, typically functions optimally under low to moderate temperature conditions, which not only reduces conversion efficiency but also increases process risks such as microbial contamination. To enhance the application performance of PaRDH, multiple strategies including PROSS web-based prediction, free energy analysis, and conserved site analysis were employed to systematically modify the thermostability of PaRDH. In the first round of mutagenesis, 11 mutants with improved performance were obtained. After another round of combinatorial mutagenesis, 10 mutants with significantly enhanced thermostability were generated. Among them, the initial enzyme activity of mutant PaRDH^VM-N7G-I93V increased by 30.68% compared to the template PaRDH^VM, and its half-life was extended from 7.66 h to 16.26 h. This mutant was then introduced into a three-enzyme cascade system, and the resulting engineered strain was designated as Ec/DRF-Therm-VMGV. Using a cell dosage of 10 OD₆₀₀ at 45 ℃ for 24 h, the allitol conversion rate reached 79.43%, which was 4.10% higher than that of the control strain harboring PaRDH^VM. Subsequently, the concentration of sodium formate in the reaction system was optimized and increased from 0.75 mol/L to 1.00 mol/L. Finally, using the engineered strain Ec/DRF-Therm-VMGV as the catalyst at a cell concentration of 15 OD₆₀₀, the allitol conversion rates reached 83.10% and 90.45% after 12 h and 24 h of reaction, respectively, representing the highest levels reported to date. This study aimed to provide a theoretical reference for improving the production efficiency of allitol and promoting its industrial application.