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A parts list for fungal cellulosomes revealed by comparative genomics

Abstract

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis1. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds1. The versatile protein assembly mechanism conferred by the bacterial cohesin–dockerin interaction is now a standard design principle for synthetic biology2,3. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs)4. However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown5,6. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure—an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.

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Figure 1: Overview of gut fungal cellulosome components.
Figure 2: Sequence analysis of repeating cohesin motifs.
Figure 3: Dockerins self-assemble onto scaffoldin fragments.
Figure 4: CAZyme domains of fungal cellulosomes probably originated from bacteria by HGT.

References

  1. 1

    Fontes, C. M. G. A. & Gilbert, H. J. Cellulosomes: highly efficient nanomachines designed to deconstruct plant cell wall complex carbohydrates. Annu. Rev. Biochem. 79, 655–681 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2

    You, C., Myung, S. & Zhang, Y.-H. P. Facilitated substrate channeling in a self-assembled trifunctional enzyme complex. Angew. Chem. Int. Ed. 51, 8787–8790 (2012).

    CAS  Article  Google Scholar 

  3. 3

    Liu, F., Banta, S. & Chen, W. Functional assembly of a multi-enzyme methanol oxidation cascade on a surface-displayed trifunctional scaffold for enhanced NADH production. Chem. Commun. 49, 3766–3768 (2013).

    CAS  Article  Google Scholar 

  4. 4

    Fanutti, C. C., Ponyi, T. T., Black, G. W. G., Hazlewood, G. P. G. & Gilbert, H. J. H. The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain. J. Biol. Chem. 270, 29314–29322 (1995).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5

    Nagy, T. T. et al. Characterization of a double dockerin from the cellulosome of the anaerobic fungus Piromyces equi. J. Mol. Biol. 373, 612–622 (2007).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. 6

    Haitjema, C. H., Solomon, K. V., Henske, J. K., Theodorou, M. K. & O'Malley, M. A. Anaerobic gut fungi: advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production. Biotechnol. Bioeng. 111, 1471–1482 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Solomon, K. V. et al. Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes. Science 351, 1192–1195 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. 8

    Anderson, T. D. et al. Assembly of minicellulosomes on the surface of Bacillus subtilis. Appl. Environ. Microb. 77, 4849–4858 (2011).

    CAS  Article  Google Scholar 

  9. 9

    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. Microb. 75, 6087–6093 (2009).

    CAS  Article  Google Scholar 

  10. 10

    Tsai, S.-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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11

    Wilson, C. A. & Wood, T. M. The anaerobic fungus Neocallimastix frontalis—isolation and properties of a cellulosome-type enzyme fraction with the capacity to solubilize hydrogen-bond-ordered cellulose. Appl. Microbiol. Biotechnol. 37, 125–129 (1992).

    CAS  Article  Google Scholar 

  12. 12

    Nguyen, K. B. et al. Phosphorylation of spore coat proteins by a family of atypical protein kinases. Proc. Natl Acad. Sci. USA 113, E3482–E3491 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13

    Steenbakkers, P. J. M. et al. beta-Glucosidase in cellulosome of the anaerobic fungus Piromyces sp. strain E2 is a family 3 glycoside hydrolase. Biochem. J. 370, 963–970 (2003).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. 14

    Youssef, N. H. et al. The genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader. Appl. Environ. Microb. 79, 4620–4634 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Shah, D. S., Joucla, G., Remaud-Simeon, M. & Russell, R. R. B. Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides. J. Bacteriol. 186, 8301–8308 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. 16

    Raghothama, S. et al. Characterization of a cellulosome dockerin domain from the anaerobic fungus Piromyces equi. Nat. Struct. Biol. 8, 775–778 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. 17

    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).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18

    Garcia-Vallve, S., Romeu, A. & Palau, J. Horizontal gene transfer of glycosyl hydrolases of the rumen fungi. Mol. Biol. Evol. 17, 352–361 (2000).

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Grigoriev, I. V. et al. Mycocosm portal: gearing up for 1000 fungal genomes. Nucleic Acids Res. 42, D699–D704 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. 20

    Markowitz, V. M. et al. IMG/m 4 version of the integrated metagenome comparative analysis system. Nucleic Acids Res. 42, D568–D573 (2014).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21

    Goodstein, D. M. et al. Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res. 40, D1178–D1186 (2012).

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Steenbakkers, P. J. P. et al. Noncatalytic docking domains of cellulosomes of anaerobic fungi. J. Bacteriol. 183, 5325–5333 (2001).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. 23

    Gilmore, S. P., Henske, J. K. & O'Malley, M. A. Driving biomass breakdown through engineered cellulosomes. Bioengineered 6, 204–208 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24

    Martin, J. et al. Rnnotator: an automated de novo transcriptome assembly pipeline from stranded RNA-Seq reads. BMC Genomics 11, 663 (2010).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25

    Solomon, K. V., Henske, J. K., Theodorou, M. K. & O'Malley, M. A. Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi. Anaerobe 38, 39–46 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26

    Lam, K. K., LaButti, K., Khalak, A. & Tse, D. FinisherSC: a repeat-aware tool for upgrading de novo assembly using long reads. Bioinformatics 31, 3207–3209 (2015).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. 27

    Chin, C. S. et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat. Methods 10, 563–569 (2013).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. 28

    Margulies, M. et al. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437, 376–380 (2005); corrigendum 441, 120 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29

    Zerbino, D. R. & Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18, 821–829 (2008).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30

    Trong, S. et al. Gap resolution: a software package for improving newbler genome assemblies. in Proceedings of the 4th Annual Meeting on Sequencing Finishing, Analysis in the Future 35 (2009).

  31. 31

    Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. 32

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    CAS  Article  Google Scholar 

  34. 34

    Eddy, S. R. Multiple alignment using hidden Markov models. Proc. Int. Conf. Intell. Syst. Mol. Biol. 3, 114–120 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. 36

    Capella-Gutierrez, S., Silla-Martinez, J. M. & Gabaldon, T. Trimal: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37

    Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22, 2688–2690 (2006).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. 38

    Ali, B. R. S. et al. Cellulases and hemicellulases of the anaerobic fungus Piromyces constitute a multiprotein cellulose-binding complex and are encoded by multigene families. FEMS Microbiol. Lett. 125, 15–21 (1995).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. 39

    Xiao, Z., Storms, R. & Tsang, A. Microplate-based carboxymethylcellulose assay for endoglucanase activity. Anal. Biochem. 342, 176–178 (2005).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  40. 40

    Vizcaino, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016).

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors acknowledge funding support from the Office of Science (BER), US Department of Energy (DE-SC0010352), the US Department of Agriculture (award no. 2011-67017-20459), the National Science Foundation (DGE 1144085) and the Institute for Collaborative Biotechnologies through grant no. W911NF-09-0001 from the US Army Research Office. A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) exploratory effort and used resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under contract nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). The authors acknowledge support from the California NanoSystems Institute (CNSI), supported by the University of California, Santa Barbara, and the University of California, Office of the President. SPR data were generated in the UCSB and UCOP-supported Biological Nanostructures Laboratory within the California NanoSystems Institute. The authors thank P.J. Weimer (US Dairy Forage Research Center) for lignocellulosic substrates. B.H. acknowledges IDEX Aix-Marseille (Grant Microbio-E) and Agence Nationale de la Recherche (grant no. ANR-14-CE06-0020) for funding.

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Authors

Contributions

C.H.H., S.P.G. and M.A.O. planned the experiments. C.H.H. and R.D. performed ELISA and S.P.G. performed SPR experiments. C.H.H., S.P.G., A.K. and M.A.O. wrote the manuscript. H.M.B., S.O.P. and A.T.W. performed proteomic analyses. K.V.S. and J.K.H. prepared and analysed genomic samples for N. californiae, P. finnis and A. robustus. B.B., T.v.A. and J.H.P.H. prepared and analysed genomic samples for Piromyces sp. E2. Z.Z. and J.C. sequenced, K.L. assembled, and A.K., S.J.M. and A.A.S. annotated and analysed genomes. B.H. and M.H. analysed and classified carbohydrate-active enzymes. M.A.O., S.E.B., K.B. and I.V.G. coordinated genome projects at JGI.

Corresponding author

Correspondence to Michelle A. O'Malley.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Description of Supplementary Datasets, Supplementary Tables 1–7 and Supplementary Figures 1–9 (PDF 2587 kb)

Supplementary Datasets

The supplementary datasets contain 3 types of files related to each of the 10 Pfam domains for which there is evidence of DDP HGT between Neocallimastigomycota and Bacteria. (ZIP 333 kb)

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Haitjema, C., Gilmore, S., Henske, J. et al. A parts list for fungal cellulosomes revealed by comparative genomics. Nat Microbiol 2, 17087 (2017). https://doi.org/10.1038/nmicrobiol.2017.87

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