Engineered repeat proteins as scaffolds to assemble multi-enzyme systems for efficient cell-free biosynthesis

Area of Science:

• Biochemistry
• Synthetic Biology
• Biocatalysis

Background:

• Multi-enzymatic cascades require precise enzyme organization for efficient substrate channeling and cofactor reuse.
• Designing protein scaffolds for nanometric enzyme arrangement remains a significant challenge in biocatalysis.

Purpose of the Study:

• To develop a nanometrically organized multi-enzymatic system using engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolds.
• To enhance catalytic efficiency and cofactor recycling in cell-free biosynthetic pathways.

Main Methods:

• Genetically fusing TRAP domains to create selective and orthogonal enzyme binding sites.
• Programming TRAP scaffolds to sequester reaction intermediates like cofactors via electrostatic interactions.
• Demonstrating the system for amino acid and amine biosynthesis using up to three enzymes.

Main Results:

• Scaffolded multi-enzyme systems achieved up to a 5-fold increase in specific productivity compared to non-scaffolded systems.
• NADH cofactor channeling between assembled enzymes enhanced cascade throughput and product yield.
• Immobilization of the scaffold created reusable heterogeneous biocatalysts for multiple operational cycles.

Conclusions:

• TRAP-scaffolding systems offer a powerful tool for spatial organization of enzymes in cell-free biosynthesis.
• This approach significantly improves the efficiency and reusability of multi-enzymatic biocatalysts.
• The developed system holds potential for industrial applications in chemical synthesis.