Programming Integrative Multienzyme Systems and Ionic Strength For Recyclable Synthesis of Glutathione

Area of Science:

• Biocatalysis
• Synthetic Biology
• Enzyme Engineering

Background:

• Enzymatic cascade catalysis faces challenges in creating stable, reusable enzyme catalysts.
• Artificial multienzyme systems (MESs) offer a promising approach to enhance enzyme catalysis performance.

Purpose of the Study:

• To construct a stable and reusable artificial multienzyme system (MES) for glutathione synthesis.
• To develop a method for assembling MESs into nanoaggregates for improved biocatalysis.

Main Methods:

• Assembled polyphosphate kinase (PPK) and glutathione synthetase (GshF) using the SpyCatcher/SpyTag system.
• Formed nanoaggregates from MESs by adjusting ionic strength.
• Optimized in vivo MES production via gene duplication and coexpression.

Main Results:

• Achieved robust and reusable synthesis of glutathione (GSH) using MES-based nanoaggregates.
• Optimized production of GSH and oxidized glutathione (GSSG) reached 102.6 and 6.7 mM within 2 hours.
• Demonstrated high stability, with only a 9.4% yield decrease after five biocatalysis rounds.

Conclusions:

• The developed MES nanoaggregate system provides a stable and reusable platform for efficient glutathione synthesis.
• The system shows significant potential for industrial applications in biocatalysis and enzyme engineering.