Artificial metalloenzyme assembly in cellular compartments for enhanced catalysis.

Area of Science:

• Biocatalysis
• Synthetic Biology
• Chemical Engineering

Background:

• Artificial metalloenzymes (ArMs) offer catalytic potential but suffer from metal center instability in whole-cell systems.
• This instability leads to reduced activity and limited turnover rates, hindering practical applications.

Purpose of the Study:

• To enhance the stability and performance of ArMs within whole-cell catalysts.
• To develop a protective compartment for ArM assembly and function in Escherichia coli.
• To demonstrate the in vivo applicability of ArM-based whole-cell catalysts.

Main Methods:

• Induction of in cellulo liquid-liquid phase separation using a HaloTag-SNAPTag self-labeling fusion protein.
• Creation of membraneless, isolated liquid condensates within E. coli to compartmentalize ArMs.
• Localization and stabilization of ArMs within these phase-separated regions.

Main Results:

• Achieved high ArM loading and stabilization within the protective compartments.
• Demonstrated a significant improvement in ArM-based whole-cell catalyst performance, with turnover numbers up to 7.1 × 10^9 for olefin metathesis.
• Successfully applied the system in live mice for in vivo catalysis and in a colorectal cancer model for prodrug activation.

Conclusions:

• The developed strategy effectively protects ArMs within bacterial cells, overcoming previous limitations.
• This compartmentalization approach enhances catalytic efficiency and opens new avenues for in vivo biocatalysis.
• The technology shows promise for therapeutic applications, including targeted drug delivery and cancer treatment.