Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Alper, H. & Stephanopoulos, G. Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat. Rev. Microbiol. 7, 715–723 (2009).

    Article  CAS  Google Scholar 

  2. Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309 (2016).

    Article  CAS  Google Scholar 

  3. Avalos, J. L., Fink, G. R. & Stephanopoulos, G. Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nat. Biotechnol. 31, 335–341 (2013).

    Article  CAS  Google Scholar 

  4. Dodds, D. R. & Gross, R. A. Chemicals from biomass. Science 318, 1250–1251 (2007).

    Article  CAS  Google Scholar 

  5. Gupta, N. K., Fukuoka, A. & Nakajima, K. Amorphous Nb2O5 as a selective and reusable catalyst for furfural production from xylose in biphasic water and toluene. ACS Catal. 7, 2430–2436 (2017).

    Article  CAS  Google Scholar 

  6. Zinoviev, S. et al. Next-generation biofuels: survey of emerging technologies and sustainability issues. ChemSusChem 3, 1106–1133 (2010).

    Article  CAS  Google Scholar 

  7. Huber, G. W., Iborra, S. & Corma, A. Synthesis of transportation fuels from biomass: chemistry, catalysts and engineering. Chem. Rev. 106, 4044–4098 (2006).

    Article  CAS  Google Scholar 

  8. Isikgor, F. H. & Becer, C. R. Lignocellulosic biomass: a sustainable platform for the production of bio-based chemicals and polymers. Polym. Chem. 6, 4497–4559 (2015).

    Article  CAS  Google Scholar 

  9. Caes, B. R., Teixeira, R. E., Knapp, K. G. & Raines, R. T. Biomass to furanics: renewable routes to chemicals and fuels. ACS Sustain. Chem. Eng. 3, 2591–2605 (2015).

    Article  CAS  Google Scholar 

  10. Himmel, M. E. & Bayer, E. A. Lignocellulose conversion to biofuels: current challenges, global perspectives. Curr. Opin. Biotechnol. 20, 316–317 (2009).

    Article  CAS  Google Scholar 

  11. Yeh, T. M. et al. Hydrothermal catalytic production of fuels and chemicals from aquatic biomass. J. Chem. Technol. Biotechnol. 88, 13–24 (2013).

    Article  CAS  Google Scholar 

  12. Wen, F., Nair, N. U. & Zhao, H. Protein engineering in designing tailored enzymes and microorganisms for biofuels production. Curr. Opin. Biotechnol. 20, 412–419 (2009).

    Article  CAS  Google Scholar 

  13. Horn, S. J., Vaaje-Kolstad, G., Westereng, B. & Eijsink, V. G. Novel enzymes for the degradation of cellulose. Biotechnol. Biofuels 5, 45 (2012).

    Article  CAS  Google Scholar 

  14. Percival Zhang, Y.-H., Himmel, M. E. & Mielenz, J. R. Outlook for cellulase improvement: screening and selection strategies. Biotechnol. Adv. 24, 452–481 (2006).

    Article  CAS  Google Scholar 

  15. Schwarz, W. H. The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol. Biotechnol. 56, 634–649 (2001).

    Article  CAS  Google Scholar 

  16. Lynd, L. R., Weimer, P. J., van Zyl, W. H. & Pretorius, I. S. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66, 739–739 (2002).

    Article  Google Scholar 

  17. Shang, B. Z. & Chu, J. W. Kinetic modeling at single-molecule resolution elucidates the mechanisms of cellulase synergy. ACS Catal. 4, 2216–2225 (2014).

    Article  CAS  Google Scholar 

  18. Percival Zhang, Y. H. et al. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 7, 644–648 (2006).

    Article  Google Scholar 

  19. Artzi, L., Bayer, E. A. & Moraïs, S. Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides. Nat. Rev. Microbiol. 15, 83–95 (2016).

    Article  Google Scholar 

  20. Fierobe, H.-P. et al. Action of designer cellulosomes on homogeneous versus complex substrates: controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin. J. Biol. Chem. 280, 16325–16334 (2005).

    Article  CAS  Google Scholar 

  21. Moraïs, S. et al. Cellulase–xylanase synergy in designer cellulosomes for enhanced degradation of a complex cellulosic substrate. MBio 1, e00285–10 (2010).

    Article  Google Scholar 

  22. Vazana, Y., Moraïs, S., Barak, Y., Lamed, R. & Bayer, E. A. Interplay between Clostridium thermocellum family 48 and family 9 cellulases in cellulosomal versus noncellulosomal states. Appl. Environ. Microbiol. 76, 3236–3243 (2010).

    Article  CAS  Google Scholar 

  23. Fierobe, H. P. et al. Degradation of cellulose substrates by cellulosome chimeras: substrate targeting versus proximity of enzyme components. J. Biol. Chem. 277, 49621–49630 (2002).

    Article  CAS  Google Scholar 

  24. Sun, J., Wen, F., Si, T., Xu, J. H. & Zhao, H. Direct conversion of xylan to ethanol by recombinant Saccharomyces cerevisiae strains displaying an engineered minihemicellulosome. Appl. Environ. Microbiol. 78, 3837–3845 (2012).

    Article  CAS  Google Scholar 

  25. Smith, M. R., Khera, E. & Wen, F. Engineering novel and improved biocatalysts by cell surface display. Ind. Eng. Chem. Res. 54, 4021–4032 (2015).

    Article  CAS  Google Scholar 

  26. You, C., Zhang, X.-Z., Sathitsuksanoh, N., Lynd, L. R. & Zhang, Y.-H. P. Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome–microbe complex. Appl. Environ. Microbiol. 78, 1437–1444 (2012).

    Article  CAS  Google Scholar 

  27. Wieczorek, A. S. & Martin, V. J. J. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis. Microb. Cell Fact. 9, 69 (2010).

    Article  Google Scholar 

  28. Lu, Y., Zhang, Y.-H. P. & Lynd, L. R. Enzyme–microbe synergy during cellulose hydrolysis by Clostridium thermocellum. Proc. Natl Acad. Sci. USA 103, 16165–16169 (2006).

    Article  CAS  Google Scholar 

  29. Wen, F., Sun, J. & Zhao, H. Yeast surface display of trifunctional minicellulosomes for simultaneous saccharification and fermentation of cellulose to ethanol. Appl. Environ. Microbiol. 76, 1251–1260 (2010).

    Article  CAS  Google Scholar 

  30. Liang, Y., Si, T., Ang, E. L. & Zhao, H. An engineered pentafunctional minicellulosome for simultaneous saccharification and ethanol fermentation in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 80, 6677–6684 (2014).

    Article  Google Scholar 

  31. Tsai, S.-L., Oh, J., Singh, S., Chen, R. & Chen, W. Functional assembly of minicellulosomes on the Saccharomyces cerevisiae cell surface for cellulose hydrolysis and ethanol production. Appl. Environ. Microbiol. 75, 6087–6093 (2009).

    Article  CAS  Google Scholar 

  32. Bugada, L. F., Smith, M. R. & Wen, F. Engineering spatially organized multienzyme assemblies for complex chemical transformation. ACS Catal. 8, 7898–7906 (2018).

    Article  CAS  Google Scholar 

  33. Tsai, S.-L. L., DaSilva, N. A. & Chen, W. Functional display of complex cellulosomes on the yeast surface via adaptive assembly. ACS Synth. Biol. 2, 14–21 (2013).

    Article  CAS  Google Scholar 

  34. Fan, L.-H., Zhang, Z.-J., Yu, X.-Y., Xue, Y.-X. & Tan, T.-W. Self-surface assembly of cellulosomes with two miniscaffoldins on Saccharomyces cerevisiae for cellulosic ethanol production. Proc. Natl Acad. Sci. USA 109, 13260–13265 (2012).

    Article  CAS  Google Scholar 

  35. Matano, Y., Hasunuma, T. & Kondo, A. Display of cellulases on the cell surface of Saccharomyces cerevisiae for high yield ethanol production from high-solid lignocellulosic biomass. Bioresour. Technol. 108, 128–133 (2012).

    Article  CAS  Google Scholar 

  36. Meyer, A. et al. Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors. Nat. Chem. 7, 673–678 (2015).

    Article  CAS  Google Scholar 

  37. Bligaard, T. et al. Toward benchmarking in catalysis science: best practices, challenges and opportunities. ACS Catal. 6, 2590–2602 (2016).

    Article  CAS  Google Scholar 

  38. Boder, E. T. & Wittrup, K. D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

    Article  CAS  Google Scholar 

  39. Bayer, E. A., Shimon, L. J. W., Shoham, Y. & Lamed, R. Cellulosomes—structure and ultrastructure. J. Struct. Biol. 124, 221–234 (1998).

    Article  CAS  Google Scholar 

  40. Kataeva, I., Li, X. L., Chen, H., Choi, S. K. & Ljungdahl, L. G. Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum: evidence for gene duplication and recombination. J. Bacteriol. 181, 5288–5295 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Béguin, P., Cornet, P. & Aubert, J. P. Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum. J. Bacteriol. 162, 102–105 (1985).

    PubMed  PubMed Central  Google Scholar 

  42. Reverbel-Leroy, C., Pages, S., Belaich, A., Belaich, J. P. & Tardif, C. The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form. J. Bacteriol. 179, 46–52 (1997).

    Article  CAS  Google Scholar 

  43. Jeng, W.-Y. et al. Structural and functional analysis of three β-glucosidases from bacterium Clostridium cellulovorans, fungus Trichoderma reesei and termite Neotermes koshunensis. J. Struct. Biol. 173, 46–56 (2011).

    Article  CAS  Google Scholar 

  44. Bayer, E. A., Belaich, J.-P., Shoham, Y. & Lamed, R. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. Annu. Rev. Microbiol. 58, 521–554 (2004).

    Article  CAS  Google Scholar 

  45. Oliveira, C., Carvalho, V., Domingues, L. & Gama, F. M. Recombinant CBM-fusion technology—applications overview. Biotechnol. Adv. 33, 358–369 (2015).

    Article  CAS  Google Scholar 

  46. Davis, K. A., Abrams, B., Iyer, S. B., Hoffman, R. A. & Bishop, J. E. Determination of CD4 antigen density on cells: role of antibody valency, avidity, clones and conjugation. Cytometry 33, 197–205 (1998).

    Article  CAS  Google Scholar 

  47. Gratama, J. W. et al. Flow cytometric quantitation of immunofluorescence intensity: problems and perspectives. European working group on clinical cell analysis. Cytometry 33, 166–178 (1998).

    Article  CAS  Google Scholar 

  48. Smith, M. R., Tolbert, S. V. & Wen, F. Protein-scaffold directed nanoscale assembly of T cell ligands: artificial antigen presentation with defined valency, density and ratio. ACS Synth. Biol. 7, 1629–1639 (2018).

    Article  CAS  Google Scholar 

  49. Ripley, B. D. Modelling spatial patterns. J. R. Stat. Soc. Series B 39, 172–192 (1977).

    Google Scholar 

  50. Wang, X. X. & Shusta, E. V. The use of scFv-displaying yeast in mammalian cell surface selections. J. Immunol. Methods 304, 30–42 (2005).

    Article  CAS  Google Scholar 

  51. Jindou, S. et al. Cohesin–dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity. J. Biol. Chem. 279, 9867–9874 (2004).

    Article  CAS  Google Scholar 

  52. Bae, J., Kuroda, K. & Ueda, M. Proximity effect among cellulose-degrading enzymes displayed on the Saccharomyces cerevisiae cell surface. Appl. Environ. Microbiol. 81, 59–66 (2015).

    Article  Google Scholar 

  53. Erickson, H. P. Size and shape of protein molecules at the nanometer level determined by sedimentation, gel filtration and electron microscopy. Biol. Proced. Online 11, 32–51 (2009).

    Article  CAS  Google Scholar 

  54. Chen, L., Mulchandani, A. & Ge, X. Spore-displayed enzyme cascade with tunable stoichiometry. Biotechnol. Prog. 33, 383–389 (2017).

    Article  CAS  Google Scholar 

  55. Chen, L., Holmes, M., Schaefer, E., Mulchandani, A. & Ge, X. Highly active spore biocatalyst by self-assembly of co-expressed anchoring scaffoldin and multimeric enzyme. Biotechnol. Bioeng. 115, 557–564 (2018).

    Article  CAS  Google Scholar 

  56. Lin, J. L., Zhu, J. & Wheeldon, I. Synthetic protein scaffolds for biosynthetic pathway colocalization on lipid droplet membranes. ACS Synth. Biol. 6, 1534–1544 (2017).

    Article  CAS  Google Scholar 

  57. Fu, J. et al. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nat. Nanotechnol. 9, 531–536 (2014).

    Article  CAS  Google Scholar 

  58. García-Alvarez, B. et al. Molecular architecture and structural transitions of a Clostridium thermocellum mini-cellulosome. J. Mol. Biol. 407, 571–580 (2011).

    Article  Google Scholar 

  59. Wood, T. M. & Bhat, K. M. Methods for measuring cellulase activities. Methods Enzymol. 160, 87–112 (1988).

    Article  CAS  Google Scholar 

  60. Liu, Z. et al. Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production. Sci. Rep. 6, 24550 (2016).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  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

Authors
  1. Mason R. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hui Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ponnandy Prabhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Luke F. Bugada
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cori Roth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Deepika Mutukuri
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Christine M. Yee
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lester Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Robert M. Ziff
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jung-Kul Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Fei Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Jung-Kul Lee or Fei Wen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary methods, Supplementary Figs. 1–12, Supplementary Tables 1–3, Supplementary references

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-019-0321-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research