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 nm. 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.
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Biodegradation of highly crystallized poly(ethylene terephthalate) through cell surface codisplay of bacterial PETase and hydrophobin
Nature Communications Open Access 21 November 2022
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The data that support the plots within this paper and other findings of this study are available from corresponding author F.W. upon reasonable request.
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This work was partly supported by the National Science Foundation (NSF) under grants nos. 1511720 and 1645229 and CAREER award 1653611, by the National Institutes of Health under grants nos. CA191952 and OD020053, and by MCubed at the University of Michigan. H.G. and J.-K.L. were partly supported by the Basic Science Research Program through the National Research Foundation of Korea (2017R1A2B3011676 and 2013M3A6A8073184) and by a WTU joint research grant from Konkuk University. The authors thank L. Zhang and B.D. Hill for assistance with the confocal microscopy experiments, and C. Jackman for help with setting up the anaerobic chamber for fermentation.
The authors declare no competing interests.
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Smith, M.R., Gao, H., Prabhu, P. et al. Elucidating structure–performance relationships in whole-cell cooperative enzyme catalysis. Nat Catal 2, 809–819 (2019). https://doi.org/10.1038/s41929-019-0321-8
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