Over millions of years of evolution, highly efficient cooperative enzyme assemblies have developed in nature that allow cells to perform life-sustaining complex chemical transformations using a variety of natural substrates. One of the most elegant enzyme assemblies to emerge in nature is the cellulosome – a modular, multi-protein complex of synergistic enzymes that catalyzes cellulose hydrolysis. Despite considerable interest in assembling cellulosome-inspired multi-enzyme complexes on microbial surfaces, efforts to harness the activity of these assemblies have been based on trial and error rather than rational design due to a lack of quantitative tools. This empirical approach has created a gap in our understanding of how multi-enzyme complexes assemble, and what parameters affect assembly efficiency and overall activity. In this study, we aimed to address these challenges by developing and validating a quantitative approach to whole-cell biocatalyst characterization. Using this approach, we characterized a series of yeast whole-cell biocatalysts displaying tetrafunctional multi-enzyme assemblies of varying complexity and at varying display levels. Combined with a mathematical model, we revealed that the assembly efficiency is limited by molecular crowding on the yeast cell surface, and the significance of this crowding phenomenon is dependent on the surface anchor protein size. In addition, we found that the multi-enzyme assembly complexity (i.e. enzyme proximity) does not enhance the overall performance of yeast whole-cell biocatalysts. We further revealed that synergistic enzyme proximity effects are only appreciable on a length scale where the average inter-enzyme distance is &gt; ~130 nm. Therefore, enzyme density on a whole-cell biocatalyst surface is the most important parameter for cellulose hydrolysis. We believe the quantitative approach underlying these observations is broadly applicable and may serve as a model for benchmarking whole-cell biocatalyst performance in the future.

Structure-performance relationships in cooperative  biocatalysis by multi-enzyme assemblies displayed on cell surface

2019 Fall National Meeting

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