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Co-cultivation of the anaerobic fungus Anaeromyces robustus with Methanobacterium bryantii enhances transcription of carbohydrate active enzymes

  • Genetics and Molecular Biology of Industrial Organisms - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Anaerobic gut fungi are biomass degraders that form syntrophic associations with other microbes in their native rumen environment. Here, RNA-Seq was used to track and quantify carbohydrate active enzyme (CAZyme) transcription in a synthetic consortium composed of the anaerobic fungus Anaeromyces robustus with methanogen Methanobacterium bryantii. Approximately 5% of total A. robustus genes were differentially regulated in co-culture with M. bryantii relative to cultivation of A. robustus alone. We found that 105 CAZymes (12% of the total predicted CAZymes of A. robustus) were upregulated while 29 were downregulated. Upregulated genes encode putative proteins with a wide array of cellulolytic, xylanolytic, and carbohydrate transport activities; 75% were fused to fungal dockerin domains, associated with a carbohydrate binding module, or both. Collectively, this analysis suggests that co-culture of A. robustus with M. bryantii remodels the transcriptional landscape of CAZymes and associated metabolic pathways in the fungus to aid in lignocellulose breakdown.

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References

  1. Akin DE, Borneman WS, Lyon CE (1990) Degradation of leaf blades and stems by monocentric and polycentric isolates of ruminal fungi. Anim Feed Sci Technol 31:205–221. https://doi.org/10.1016/0377-8401(90)90125-R

    Article  Google Scholar 

  2. Akin DE, Rigsby LL (1987) Mixed fungal populations and lignocellulosic tissue degradation in the bovine rumen. Appl Environ Microbiol 53:1987–1995. https://doi.org/10.1128/AEM.70.7.4402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Balch WE, Fox GE, Magrum LJ, Woese CR, Wolfe ARS (1979) Methanogens: reevaluation of a unique biological group. Microbiol Rev 43:260–296

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Bauchop T, Mountfort DO (1981) Cellulose fermentation by a rumen anaerobic fungus in both the absence and the presence of rumen methanogens. Appl Environ Microbiol 42:1103–1110

    CAS  PubMed  PubMed Central  Google Scholar 

  5. ter Beek J, Guskov A, Slotboom DJ (2014) Structural diversity of ABC transporters. J Gen Physiol 143:419–435. https://doi.org/10.1085/jgp.201411164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bernard T, Cantarel BL, Henrissat B, Lombard V, Coutinho PM, Rancurel C (2008) The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res 37:D233–D238. https://doi.org/10.1093/nar/gkn663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Gilmore SP, Henske JK, Sexton JA, Solomon KV, Seppälä S, Yoo JI, Huyett LM, Pressman A, Cogan JZ, Kivenson V, Peng X, Tan YP, Valentine DL, O’Malley MA (2017) Genomic analysis of methanogenic archaea reveals a shift towards energy conservation. BMC Genom 18:1–14. https://doi.org/10.1186/s12864-017-4036-4

    Article  CAS  Google Scholar 

  8. Grigoriev IV, Nikitin R, Haridas S, Kuo A, Ohm R, Otillar R, Riley R, Salamov A, Zhao X, Korzeniewski F, Smirnova T, Nordberg H, Dubchak I, Shabalov I (2014) MycoCosm portal: gearing up for 1000 fungal genomes. Nucleic Acids Res 42:699–704. https://doi.org/10.1093/nar/gkt1183

    Article  CAS  Google Scholar 

  9. Haitjema CH, Gilmore SP, Henske JK, Solomon KV, de Groot R, Kuo A, Mondo SJ, Salamov AA, LaButti K, Zhao Z, Chiniquy J, Barry K, Brewer HM, Purvine SO, Wright AT, Hainaut M, Boxma B, van Alen T, Hackstein JHP, Henrissat B, Baker SE, Grigoriev IV, O’Malley MA (2017) A parts list for fungal cellulosomes revealed by comparative genomics. Nat Microbiol 2:17087. https://doi.org/10.1038/nmicrobiol.2017.87

    Article  CAS  PubMed  Google Scholar 

  10. Haitjema CH, Solomon KV, Henske JK, Theodorou MK, O’Malley MA (2014) Anaerobic gut fungi: advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production. Biotechnol Bioeng 111:1471–1482. https://doi.org/10.1002/bit.25264

    Article  CAS  PubMed  Google Scholar 

  11. Henske JK, Gilmore SP, Haitjema CH, Solomon KV, Malley MAO (2018) Biomass-degrading enzymes are catabolite repressed in anaerobic gut fungi. AIChE J. https://doi.org/10.1002/aic.16395

    Article  Google Scholar 

  12. Henske JK, Gilmore SP, Knop D, Cunningham FJ, Sexton JA, Smallwood CR, Shutthanandan V, Evans JE, Theodorou MK, O’Malley MA (2017) Transcriptomic characterization of Caecomyces churrovis: a novel, non-rhizoid-forming lignocellulolytic anaerobic fungus. Biotechnol Biofuels 10:305. https://doi.org/10.1186/s13068-017-0997-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Jin W, Cheng YF, Mao SY, Zhu WY (2011) Isolation of natural cultures of anaerobic fungi and indigenously associated methanogens from herbivores and their bioconversion of lignocellulosic materials to methane. Bioresour Technol 102:7925–7931. https://doi.org/10.1016/j.biortech.2011.06.026

    Article  CAS  PubMed  Google Scholar 

  14. Joblin KN, Naylor GE, Williams AG (1990) Effect of Methanobrevibacter smithii on xylanolytic activity of anaerobic ruminal fungi. Appl Environ Microbiol 56:2287–2295

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Joblin KN, Williams AG (1991) Effect of cocultivation of ruminal chytrid fungi with Methanobrevibacter smithii on lucerne stem degradation and extracellular fungal enzyme activities. Lett Appl Microbiol 12:121–124. https://doi.org/10.1111/j.1472-765X.1991.tb00520.x

    Article  CAS  Google Scholar 

  16. Joblin KN, Campbell GP, Richardson CSS (1989) Fermentation of barley straw by anaerobic rumen bacteria and fungi in axenic culture and in co-culture with methanogens. Lett Appl Microbiol 9:195–197

    Article  Google Scholar 

  17. Kim D, Langmead B, Salzberg SL (2015) HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360. https://doi.org/10.1038/nmeth.3317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Koonin E, Fedorova N, Jackson J, Jacobs A, Krylov D, Makarova K, Mazumder R, Mekhedov S, Nikolskaya A, Rao B, Rogozin I, Smirnov S, Sorokin A, Sverdlov A, Vasudevan S, Wolf Y, Yin J, Natale D (2004) A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes. Genome Biol 5:R7. https://doi.org/10.1186/gb-2004-5-2-r7

    Article  PubMed  PubMed Central  Google Scholar 

  19. Krogh A, Larsson B, von Heijne G, Sonnhammer EL (2001) Predicting transmembrane protein topology with a hidden markov model: application to complete genomes. J Mol Biol 305:567–580. https://doi.org/10.1006/jmbi.2000.4315

    Article  CAS  PubMed  Google Scholar 

  20. Li Y, Jin W, Mu C, Cheng Y, Zhu W (2017) Indigenously associated methanogens intensified the metabolism in hydrogenosomes of anaerobic fungi with xylose as substrate. J Basic Microbiol 57:933–940. https://doi.org/10.1002/jobm.201700132

    Article  CAS  PubMed  Google Scholar 

  21. Liao Y, Smyth GK, Shi W (2014) FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30:923–930. https://doi.org/10.1093/bioinformatics/btt656

    Article  CAS  PubMed  Google Scholar 

  22. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2014) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:490–495. https://doi.org/10.1093/nar/gkt1178

    Article  CAS  Google Scholar 

  23. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550. https://doi.org/10.1186/s13059-014-0550-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Marvin-Sikkema FD, Richardson AJ, Stewart CS, Gottschal JC, Prins RA (1990) Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi. Appl Environ Microbiol 56:3793–3797

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Mi H, Muruganujan A, Casagrande JT, Thomas PD (2013) Large-scale gene function analysis with the PANTHER classification system. Nat Protoc 8:1551–1566. https://doi.org/10.1038/nprot.2013.092

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Mitchell AL, Attwood TK, Babbitt PC, Blum M, Bork P, Bridge A, Brown SD, Chang H-Y, El-Gebali S, Fraser MI, Gough J, Haft DR, Huang H, Letunic I, Lopez R, Luciani A, Madeira F, Marchler-Bauer A, Mi H, Natale DA, Necci M, Nuka G, Orengo C, Pandurangan AP, Paysan-Lafosse T, Pesseat S, Potter SC, Qureshi MA, Rawlings ND, Redaschi N, Richardson LJ, Rivoire C, Salazar GA, Sangrador-Vegas A, Sigrist CJA, Sillitoe I, Sutton GG, Thanki N, Thomas PD, Tosatto SCE, Yong S-Y, Finn RD (2018) InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. https://doi.org/10.1093/nar/gky1100

    Article  PubMed  PubMed Central  Google Scholar 

  27. Mountfort DO, Asher RA, Bauchop T (1982) Fermentation of cellulose to methane and carbon dioxide by a rumen anaerobic fungus in a triculture with Methanobrevibacter sp. strain RA1 and Methanosarcina barkeri. Appl Environ Microbiol 44:128–134

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Nagy T, Tunnicliffe RB, Higgins LD, Walters C, Gilbert HJ, Williamson MP (2007) Characterization of a double dockerin from the cellulosome of the anaerobic fungus Piromyces equi. J Mol Biol 373:612–622. https://doi.org/10.1016/j.jmb.2007.08.007

    Article  CAS  PubMed  Google Scholar 

  29. Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, Kanehisa M (1999) KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res 27:29–34. https://doi.org/10.1093/nar/27.1.29

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Orpin CG (1977) The rumen flagellate Piromonas communis: its life-history and invasion of plant material in the rumen. J Gen Microbiol 99:107–117

    Article  CAS  PubMed  Google Scholar 

  31. Perlin MH, Andrews J, San Toh S (2014) Essential letters in the fungal alphabet: ABC and MFS transporters and their roles in survival and pathogenicity. Adv Genet 85:201–253. https://doi.org/10.1016/B978-0-12-800271-1.00004-4

    Article  CAS  PubMed  Google Scholar 

  32. Schink B (1997) Energetics of syntrophic cooperation in methanogenic degradation. Microbiol Mol Biol Rev 61:262–280

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Seppala S, Solomon KV, Gilmore SP, Henske JK, O’Malley MA (2016) Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters. Microb Cell Fact. https://doi.org/10.1186/s12934-016-0611-7

    Article  PubMed  PubMed Central  Google Scholar 

  34. Solomon KV, Haitjema CH, Henske JK, Gilmore SP, Borges-Rivera D, Lipzen A, Brewer HM, Purvine SO, Wright AT, Theodorou MK, Grigoriev IV, Regev A, Thompson DA, OMalley MA (2016) Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes. Science 351:1192–1195. https://doi.org/10.1126/science.aad1431

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Altschul Stephen F, Gish Warren, Miller Webb, Myers Eugene W, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

    Article  CAS  PubMed  Google Scholar 

  36. Teunissen MJ, Kets EP, Op den Camp HJ, Huis in’t Veld JH, Vogels GD (1992) Effect of coculture of anaerobic fungi isolated from ruminants and non-ruminants with methanogenic bacteria on cellulolytic and xylanolytic enzyme activities. Arch Microbiol 157:176–182

    Article  CAS  PubMed  Google Scholar 

  37. Teunissen MJ, Op den Camp HJM, Orpin CG, Huis in’t Veld JHJ, Vogels GD (1991) Comparison of growth characteristics of anaerobic fungi isolated from ruminant and non-ruminant herbivores during cultivation in a defined medium. J Gen Microbiol 137:1401–1408. https://doi.org/10.1099/00221287-137-6-1401

    Article  CAS  PubMed  Google Scholar 

  38. Theodorou MK, Brookman J, Trinci APJ (2005) Anaerobic fungi. Methods in gut microbial ecology for ruminants. Springer, Berlin, pp 55–66

    Chapter  Google Scholar 

  39. Theodorou MK, Davies DR, Nielsen BB, Lawrence MIG, Trinci APJ (1995) Determination of growth of anaerobic fungi on soluble and cellulosic substrates using a pressure transducer. Microbiology 141:671–678. https://doi.org/10.1099/13500872-141-3-671

    Article  CAS  Google Scholar 

  40. Theodorou MK, Mennim G, Davies DR, Zhu W-Y, Trinci APJ, Brookman JL (1996) Anaerobic fungi in the digestive tract of mammalian herbivores and their potential for exploitation. Proc Nutr Soc 55:913–926. https://doi.org/10.1079/PNS19960088

    Article  CAS  PubMed  Google Scholar 

  41. Wei YQ, Long RJ, Yang H, Yang HJ, Shen XH, Shi RF, Wang ZY, Du JG, Qi XJ, Ye QH (2016) Fiber degradation potential of natural co-cultures of Neocallimastix frontalis and Methanobrevibacter ruminantium isolated from yaks (Bos grunniens) grazing on the Qinghai Tibetan Plateau. Anaerobe 39:158–164. https://doi.org/10.1016/j.anaerobe.2016.03.005

    Article  CAS  PubMed  Google Scholar 

  42. Youssef NH, Couger MB, Struchtemeyer CG, Liggenstoffer AS, Prade RA, Najar FZ, Atiyeh HK, Wilkins MR, Elshahed MS (2013) The genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader. Appl Environ Microbiol 79:4620–4634. https://doi.org/10.1128/AEM.00821-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhang W, Kou Y, Xu J, Cao Y, Zhao G, Shao J, Wang H, Wang Z, Bao X, Chen G, Liu W (2013) Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis. J Biol Chem 288:32861–32872. https://doi.org/10.1074/jbc.M113.505826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors are grateful for funding support from the National Science Foundation (Directorate for Biological Sciences, Grant no. MCB-1553721), the Institute for Collaborative Biotechnologies through Grants W911NF-09-0001 and W911NF-19-D-0001 from the U.S. Army Research Office, and the Camille Dreyfus Teacher-Scholar Awards Program. CLS is also supported by a National Science Foundation Graduate Research Fellowship Program under Grant no. 1650114. We thank Dr. Jennifer Smith, manager of the Biological Nanostructures Laboratory within the California NanoSystems Institute, supported by the University of California, Santa Barbara and the University of California, Office of the President. The authors acknowledge support from the Center for Scientific Computing from the CNSI, MRL: an NSF MRSEC (DMR-1121053) and NSF CNS-0960316.

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  1. Department of Chemical Engineering, University of California Santa Barbara, Rm 3357 Engineering II, Santa Barbara, CA, 93106, USA

    Candice L. Swift, Jennifer L. Brown, Susanna Seppälä & Michelle A. O’Malley

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Swift, C.L., Brown, J.L., Seppälä, S. et al. Co-cultivation of the anaerobic fungus Anaeromyces robustus with Methanobacterium bryantii enhances transcription of carbohydrate active enzymes. J Ind Microbiol Biotechnol 46, 1427–1433 (2019). https://doi.org/10.1007/s10295-019-02188-0

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