Enzyme Assembly Guided via Modified Signal Peptide for Enhanced Cascade Biocatalysis

Area of Science:

• Biocatalysis
• Protein Engineering
• Synthetic Biology

Background:

• Enzyme aggregation is crucial for enhancing multienzyme cascade reactions.
• A universal and tunable strategy for enzyme aggregation is currently lacking.
• Existing methods face limitations in efficiency and applicability.

Purpose of the Study:

• To develop a rational design-based strategy for inducing enzyme aggregation.
• To engineer a peptide tag that imparts defined aggregation properties.
• To enhance the efficiency of biocatalytic processes through enzyme clustering.

Main Methods:

• Reprogramming native signal peptides from α-helical to β-sheet conformation.
• Engineering a peptide tag for enzyme clustering and fusion protein construction.
• Optimizing linker length to control inclusion body (IB) formation.
• Validating the tag's function across multiple bacterial strains.

Main Results:

• The engineered peptide tag successfully induced enzyme clustering while maintaining protein solubility and activity.
• Tunable inclusion body formation was achieved by optimizing the linker.
• Cellular systems showed enhanced sequential production of EGT (173%) and 6-HHA (111%).
• The tag demonstrated broad compatibility and flexibility across different bacterial strains.

Conclusions:

• A simple, genetically encodable strategy for designing signal peptides to create self-assembling biocatalysts was established.
• This approach significantly improves upon current methods for enzyme aggregation.
• The developed strategy offers a versatile tool for constructing highly efficient biocatalysts.