Elucidating structure-performance relationships in whole-cell cooperative enzyme catalysis

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Biology
• Cell Surface Display Technology

Background:

• Nature utilizes cooperative enzyme catalysis, inspiring engineered multi-enzyme assemblies for industrial biocatalysis.
• Current cell-surface displayed multi-enzyme assembly efforts rely on trial-and-error due to a lack of quantitative tools.

Purpose of the Study:

• To develop a quantitative approach for whole-cell biocatalyst characterization.
• To comprehensively study the formation of yeast-surface displayed multi-enzyme assemblies.
• To identify key parameters for enhancing biocatalytic performance.

Main Methods:

• Development of a quantitative whole-cell biocatalyst characterization method.
• Analysis of multi-enzyme assembly formation on the yeast cell surface.
• Investigation of molecular crowding and enzyme density effects.

Main Results:

• Multi-enzyme assembly efficiency is limited by molecular crowding on the yeast cell surface.
• Maximizing enzyme density is crucial for enhancing cellulose hydrolytic performance.
• Synergistic proximity effects occur only when inter-enzyme distance exceeds approximately 130 nm.

Conclusions:

• The developed quantitative approach enables rational design of biocatalysts.
• Understanding molecular crowding and enzyme density is key for optimizing multi-enzyme assemblies.
• This work transitions biocatalyst engineering from empirical methods to rational design.