Bootstrapped Biocatalysis: Biofilm-Derived Materials as Reversibly Functionalizable Multienzyme Surfaces

Area of Science:

• Biotechnology
• Biocatalysis
• Materials Science

Background:

• Cell-free biocatalysis offers advantages for chemical manufacturing but faces challenges.
• High costs of enzyme purification, modification, and immobilization limit widespread application.
• Existing substrate materials add significant expense to biocatalytic processes.

Purpose of the Study:

• To develop a cost-effective and efficient biocatalysis substrate.
• To enable simultaneous enzyme purification and immobilization directly from crude cell lysates.
• To demonstrate a reusable and versatile biocatalyst system.

Main Methods:

• Engineered bacterial biofilm matrix proteins to self-assemble into a nanofibrous mesh.
• Utilized genetically programmed, orthogonal conjugation domains for site-specific enzyme attachment.
• Demonstrated enzyme purification and immobilization directly from crude cell lysates.
• Implemented a bienzymatic reaction coupled with cofactor recycling.

Main Results:

• Successfully produced a
• bootstrapped
• biocatalysis substrate material directly in bacterial culture.
• Achieved simultaneous purification and site-specific immobilization of multiple enzymes from crude lysates.
• Demonstrated efficient bienzymatic stereoselective reduction with cofactor recycling.
• Showcased selective enzyme removal and replacement for catalyst regeneration.

Conclusions:

• The novel biofilm-derived substrate significantly reduces costs associated with enzyme preparation and immobilization.
• This approach offers a versatile platform for developing robust and reusable cell-free biocatalysis systems.
• The technique facilitates efficient multi-enzyme catalysis and catalyst regeneration for sustainable chemical manufacturing.