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

Abstract

Cooperative enzyme catalysis in nature has long inspired the application of engineered multi-enzyme assemblies for industrial biocatalysis. Despite considerable interest, efforts to harness the activity of cell-surface displayed multi-enzyme assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. In this study, we have developed a quantitative approach to whole-cell biocatalyst characterization, enabling a comprehensive study of how yeast-surface displayed multi-enzyme assemblies form. Here we show that the multi-enzyme assembly efficiency is limited by molecular crowding on the yeast-cell surface, and that maximizing enzyme density is the most important parameter for enhancing cellulose hydrolytic performance. Interestingly, we also observed that proximity effects are only synergistic when the average inter-enzyme distance is greater than ~130\u2009nm. The findings and the quantitative approach developed in this work should help to advance the field of biocatalyst engineering from trial and error to rational design. Efforts to harness the cellulolytic activity of enzyme assemblies have been mainly empirical due to the lack of quantification tools. Now this work reports experimental and theoretical approaches to quantify enzyme assemblies, revealing the parameters that are important for cellulolytic activity.