Growth‐Coupled Evolutionary Pressure Improving Epimerases for D‐Allulose Biosynthesis Using a Biosensor‐Assisted In Vivo Selection Platform

Abstract

Abstract Fast screening strategies that enable high‐throughput evaluation and identification of desired variants from diversified enzyme libraries are crucial to tailoring biocatalysts for the synthesis of D‐allulose, which is currently limited by the poor catalytic performance of ketose 3‐epimerases (KEases). Here, the study designs a minimally equipment‐dependent, high‐throughput, and growth‐coupled in vivo screening platform founded on a redesigned D‐allulose‐dependent biosensor system. The genetic elements modulating regulator PsiR expression levels undergo systematic optimization to improve the growth‐responsive dynamic range of the biosensor, which presents ≈30‐fold facilitated growth optical density with a high signal‐to‐noise ratio (1.52 to 0.05) toward D‐allulose concentrations from 0 to 100 mm. Structural analysis and evolutionary conservation analysis of Agrobacterium sp. SUL3 D‐allulose 3‐epimerase (ADAE) reveal a highly conserved catalytic active site and variable hydrophobic pocket, which together regulate substrate recognition. Structure‐guided rational design and directed evolution are implemented using the growth‐coupled in vivo screening platform to reprogram ADAE, in which a mutant M42 (P38N/V102A/Y201L/S207N/I251R) is identified with a 6.28‐fold enhancement of catalytic activity and significantly improved thermostability with a 2.5‐fold increase of the half‐life at 60 °C. The research demonstrates that biosensor‐assisted growth‐coupled evolutionary pressure combined with structure‐guided rational design provides a universal route for engineering KEases.

Keywords

• biosensors
• crystal structure analysis
• D‐allulose 3‐epimerase
• growth‐coupled selection platform
• protein engineering