Growth-Coupled Evolutionary Pressure Improving Epimerases for D-Allulose Biosynthesis Using a Biosensor-Assisted In...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Metabolic Engineering

Background:

• Ketose 3-epimerases (KEases) exhibit poor catalytic performance, limiting efficient D-allulose synthesis.
• High-throughput screening is essential for identifying improved enzyme variants from large libraries.

Purpose of the Study:

• To develop a high-throughput, growth-coupled in vivo screening platform for engineering D-allulose-producing enzymes.
• To enhance the catalytic activity and stability of ketose 3-epimerases (KEases) for D-allulose production.

Main Methods:

• Designed a D-allulose-dependent biosensor system with optimized genetic elements for improved dynamic range.
• Employed structure-guided rational design and directed evolution on Agrobacterium sp. SUL3 D-allulose 3-epimerase (ADAE).
• Utilized a growth-coupled evolutionary pressure strategy integrated with structural analysis.

Main Results:

• Achieved a ≈30-fold facilitated growth optical density and a high signal-to-noise ratio for the biosensor.
• Identified a mutant ADAE (M42) with a 6.28-fold increase in catalytic activity.
• Enhanced mutant ADAE thermostability, showing a 2.5-fold increase in half-life at 60 °C.

Conclusions:

• The developed biosensor-assisted screening platform enables efficient engineering of KEases.
• Combining evolutionary pressure with rational design offers a universal strategy for enzyme improvement.
• This work facilitates the biocatalytic synthesis of D-allulose through enhanced enzyme performance.