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

Nature Catalysis volume 2, pages 809–819 (2019)Cite this article

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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 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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Fig. 1: Schematic of mSEAs.
Fig. 2: Characterizing aScaf–pScaf assembly on yeast-cell surface.
Fig. 3: Modelling aScaf distribution and aScaf–pScaf assembly on yeast-cell surface.
Fig. 4: Quantitative flow cytometric analysis of yeast cells assembling mSEAs.
Fig. 5: Modelling theoretical mSEA assembly.
Fig. 6: Structure–performance relationship of multi-enzyme assemblies.
Fig. 7: Time course of ethanol production from PASC by aScaf3–mSEA.

Data availability

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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Acknowledgements

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.

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  1. These authors contributed equally: Mason R. Smith, Hui Gao

Authors and Affiliations

  1. Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA

    Mason R. Smith, Hui Gao, Ponnandy Prabhu, Luke F. Bugada, Cori Roth, Deepika Mutukuri, Christine M. Yee, Lester Lee, Robert M. Ziff & Fei Wen

  2. Department of Chemical Engineering, Konkuk University, Seoul, Republic of Korea

    Hui Gao & Jung-Kul Lee

  3. Catalysis Science and Technology Institute, University of Michigan, Ann Arbor, MI, USA

    Luke F. Bugada & Fei Wen

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Contributions

F.W. conceived the idea behind this work. F.W. and J.-K.L. supervised the project. F.W., M.R.S., H.G. and C.R. designed the experiments. M.R.S., H.G., P.P., C.R., D.M., L.L., C.M.Y. and L.F.B. carried out the experiments. M.R.S., R.M.Z. and F.W. carried out the modelling work. M.R.S. and F.W. analysed the data and wrote the paper with H.G.’s assistance. All authors discussed and commented on the manuscript. All authors have given approval for the final version of the manuscript.

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Correspondence to Jung-Kul Lee or Fei Wen.

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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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