A Toxin-Antitoxin-Driven Biosensor Platform for the Directed Evolution of d-Allulose 3-Epimerase

Area of Science:

• Biotechnology
• Enzyme Engineering
• Synthetic Biology

Background:

• Biosensor-assisted screening is crucial for enzyme directed evolution.
• Existing antibiotic resistance-based methods face declining selection pressure.
• A robust, sustainable selection pressure is needed for high-throughput screening.

Purpose of the Study:

• To develop a novel toxin-antitoxin-based high-throughput screening platform (TASP).
• To engineer d-allulose 3-epimerase (DAE) using the TASP system.
• To enhance enzyme catalytic activity and stability through directed evolution.

Main Methods:

• Developed a TASP incorporating the toxin-antitoxin system (AtaT-AtaR) for cell-autonomous selection pressure.
• Engineered a d-allulose-responsive biosensor (DB) with an enhanced pPsiA promoter for a wider dynamic range and improved signal-to-noise ratio.
• Utilized mCherry as a reporter gene to link DAE activity to cell growth phenotypes for visual and quantitative analysis.

Main Results:

• The engineered pPsiA promoter achieved a 150 mM d-allulose dynamic range and a 14.2-fold signal-to-noise enhancement.
• The TASP platform successfully linked DAE catalytic activity to cell growth.
• Identified a DAE mutant (M4-2) with a 5.2-fold increase in catalytic activity and a 3.5-fold longer half-life at 60 °C.

Conclusions:

• Established a reliable TASP for high-throughput screening of DAE variants.
• Advanced the development of growth-coupled selection strategies for enzyme engineering.
• The TASP platform offers a sustainable and effective approach for directed enzyme evolution.