Substantial Improvement of an Epimerase for the Synthesis of D‐Allulose by Biosensor‐Based High‐Throughput Microdroplet Screening

Abstract

Biosynthesis of D‐allulose has been achieved using ketose 3‐epimerases (KEases), but its application is limited by poor catalytic performance. In this study, we redesigned a genetically encoded biosensor based on a D‐allulose‐responsive transcriptional regulator for real‐time monitoring of D‐allulose. An ultrahigh‐throughput droplet‐based microfluidic screening platform was further constructed by coupling with this D‐allulose‐detecting biosensor for the directed evolution of the KEases. Structural analysis of Sinorhizobium fredii D‐allulose 3‐epimerase (SfDAE) revealed that a highly flexible helix/loop region exposes or occludes the catalytic center as an essential lid conformation regulating substrate recognition. We reprogrammed SfDAE using structure‐guided rational design and directed evolution, in which a mutant M3‐2 was identified with 17‐fold enhanced catalytic efficiency. Our research offers a paradigm for the design and optimization of a biosensor‐based microdroplet screening platform.

Sequence- and Structure-Based Mining of Thermostable D-Allulose 3-Epimerase and Computer-Guided Protein Engineering To Improve Enzyme Activity

“…Once this was completed, the reaction was halted through boiling for a duration of 10 min, after which the mixture was subjected to centrifugation. Measurement of D -allulose, which was formed from D -fructose found in the supernatant, was conducted using the CSA that we previously designed to measure the activity of the mutants. , For further analysis, the mutants selected were those having a greater absorbance at 340 nm (A 340 ) compared to the wild type (WT).…”

“…Measurement of D-allulose, which was formed from D-fructose found in the supernatant, was conducted using the CSA that we previously designed to measure the activity of the mutants. 19,20 For further analysis, the mutants selected were those having a greater absorbance at 340 nm (A 340 ) compared to the wild type (WT).…”

Strategies to increase the robustness of microbial cell factories

“…For example, the DREM CELL platform allows for the screening of target strains at a picoliter scale (Meng et al 2022). Depending on the fluorescence output, a biosensor based on transcription factors or riboswitches can significantly increase the efficiency of a screening process (Li et al 2023). In addition, biosensors with appropriate sensitivity and dynamic range can be used to dynamically regulate the biosynthesis of many compounds (Hossain et al 2020).…”