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-alluloseresponsive 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.

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“…Therefore, if the high-throughput screening platform of droplet microfluidics can be used for the screening of key enzymes or strains in SA biosynthesis, it will undoubtedly accelerate the development of SA biosynthesis. Based on this, the combination of ultra-high-throughput droplet microfluidic screening platforms and spectroscopic techniques also provides for high-throughput screening of strains, such as Raman-activated cell sorting (RACS) ( Nitta et al, 2020 ) and fluorescence-activated cell sorting (FACS) ( Li C. et al, 2023 ). Combining a droplet-captured microfluidic cell-sorting device with a spectral analysis device allows individual cells to be analyzed by Raman microspectroscopy or fluorescence detection, and cells can be sorted as needed.…”

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“…3,4 However, high-throughput biosensor assays for indicating target secretory cells remain limited. 5,6 Many current assays face challenges in sensitively and flexibly measuring valuable chemicals like naringenins for industrial applications.…”

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

“…[ 9 ] Furthermore, single‐cell evaluation techniques have also been developed employing fluorescence‐activated cell sorting or droplet‐based microfluidic assays, allowing high‐throughput screening of KEase mutants with superior catalytic activity. [ 10 ] However, these existing methods are either limited by their low throughput (chromatography‐reliant assays and colorimetric assays in microtiter plates) or require extensive expertise and expensive equipment (e.g., robotic platforms or microfluidic devices). These limitations have prevented a more widespread implementation of KEase enzymes in biotransformation.…”

“…[ 19 ] Recent expansions in the D‐ allulose‐responsive biosensor founded on the transcription factor PsiR and its cognate promoter pPsiA highlighted the initial proof‐of‐concept that the expression of the green fluorescent protein gene can be linked to KEase activity in Escherichia coli , which together provide insightful guidelines to associate growth phenotype with KEase activity by regulating the expression of antibiotic resistance genes using the genetic biosensor. [ 10b ]…”