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

Chao Li1, Xin Gao1, Huimin Li1

  • 1Key Laboratory of Industrial Fermentation Microbiology of the Ministry of Education, Tianjin Key Laboratory of Industrial Microbiology, National Engineering Laboratory for Industrial Enzymes, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, P. R. China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|February 2, 2024
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel biosensor screening platform to improve ketose 3-epimerases (KEases) for D-allulose synthesis. This method enhances enzyme activity and stability, paving the way for efficient biocatalyst engineering.

Keywords:
D‐allulose 3‐epimerasebiosensorscrystal structure analysisgrowth‐coupled selection platformprotein engineering

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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.