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Journal of Industrial Microbiology & Biotechnology

, Volume 44, Issue 12, pp 1643–1651 | Cite as

Heterologous expression of a β-d-glucosidase in Caldicellulosiruptor bescii has a surprisingly modest effect on the activity of the exoproteome and growth on crystalline cellulose

  • Sun-Ki Kim
  • Daehwan Chung
  • Michael E. Himmel
  • Yannick J. Bomble
  • Janet WestphelingEmail author
Genetics and Molecular Biology of Industrial Organisms - Original Paper

Abstract

Members of the genus Caldicellulosiruptor are the most thermophilic cellulolytic bacteria so far described and are capable of efficiently utilizing complex lignocellulosic biomass without conventional pretreatment. Previous studies have shown that accumulation of high concentrations of cellobiose and, to a lesser extent, cellotriose, inhibits cellulase activity both in vivo and in vitro and high concentrations of cellobiose are present in C. bescii fermentations after 90 h of incubation. For some cellulolytic microorganisms, β-d-glucosidase is essential for the efficient utilization of cellobiose as a carbon source and is an essential enzyme in commercial preparations for efficient deconstruction of plant biomass. In spite of its ability to grow efficiently on crystalline cellulose, no extracellular β-d-glucosidase or its GH1 catalytic domain could be identified in the C. bescii genome. To investigate whether the addition of a secreted β-d-glucosidase would improve growth and cellulose utilization by C. bescii, we cloned and expressed a thermostable β-d-glucosidase from Acidothermus cellulolyticus (Acel_0133) in C. bescii using the CelA signal sequence for protein export. The effect of this addition was modest, suggesting that β-d-glucosidase is not rate limiting for cellulose deconstruction and utilization by C. bescii.

Keywords

Consolidated bioprocessing Biomass deconstruction β-d-Glucosidase Caldicellulosiruptor 

Notes

Acknowledgements

We thank Shreena Patel for technical assistance and Joseph Groom for critical review of the manuscript. This work was supported by the BioEnergy Science Center, US DOE Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the US DOE under contract DE-AC05-00OR22725. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Supplementary material

10295_2017_1982_MOESM1_ESM.docx (430 kb)
Supplementary material 1 (DOCX 428 kb)

References

  1. 1.
    Antelmann H, van Dijl JM, Bron S, Hecker M (2005) Proteomic survey through secretome of Bacillus subtilis. In: microbial proteomics. Wiley, New York, pp 179–208. doi: 10.1002/0471973165.ch12
  2. 2.
    Baker JO, Adney WS, Nieves RA, Thomas SR, Wilson DB, Himmel ME (1994) A new thermostable endoglucanase, Acidothermus cellulolyticus E1—synergism with Trichoderma reesei CBH-I and comparison to Thermomonospora fusca E5. Appl Biochem Biotechnol 45:245–256. doi: 10.1007/Bf02941803 CrossRefGoogle Scholar
  3. 3.
    Barabote RD, Parales JV, Guo YY, Labavitch JM, Parales RE, Berry AM (2010) Xyn10A, a thermostable endoxylanase from Acidothermus cellulolyticus 11B. Appl Environ Microbiol 76:7363–7366. doi: 10.1128/Aem.01326-10 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Brunecky R, Alahuhta M, Xu Q, Donohoe BS, Crowley MF, Kataeva IA, Yang SJ, Resch MG, Adams MWW, Lunin VV, Himmel ME, Bomble YJ (2013) Revealing nature’s cellulase diversity: the digestion mechanism of Caldicellulosiruptor bescii CelA. Science 342:1513–1516. doi: 10.1126/science.1244273 CrossRefPubMedGoogle Scholar
  5. 5.
    Chung D, Cha M, Farkas J, Westpheling J (2013) Construction of a stable replicating shuttle vector for Caldicellulosiruptor species: use for extending genetic methodologies to other members of this genus. PLoS One 8:e62881. doi: 10.1371/journal.pone.0062881 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Chung D, Cha M, Guss AM, Westpheling J (2014) Direct conversion of plant biomass to ethanol by engineered Caldicellulosiruptor bescii. Proc Natl Acad Sci USA 111:8931–8936. doi: 10.1073/pnas.1402210111 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Chung D, Cha M, Snyder EN, Elkins JG, Guss AM, Westpheling J (2015) Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii. Biotechnol Biofuels. doi: 10.1186/S13068-015-0346-4 Google Scholar
  8. 8.
    Chung D, Young J, Bomble YJ, Vander Wall TA, Groom J, Himmel ME, Westpheling J (2015) Homologous expression of the Caldicellulosiruptor bescii CelA reveals that the extracellular protein is glycosylated. PLoS One. doi: 10.1371/journal.pone.0119508 Google Scholar
  9. 9.
    Chung D, Young J, Cha M, Brunecky R, Bomble YJ, Himmel ME, Westpheling J (2015) Expression of the Acidothermus cellulolyticus E1 endoglucanase in Caldicellulosiruptor bescii enhances its ability to deconstruct crystalline cellulose. Biotechnol Biofuels. doi: 10.1186/S13068-015-0296-X Google Scholar
  10. 10.
    Chung D, Huddleston JR, Farkas J, Westpheling J (2011) Identification and characterization of CbeI, a novel thermostable restriction enzyme from Caldicellulosiruptor bescii DSM 6725 and a member of a new subfamily of HaeIII-like enzymes. J Ind Microbiol Biotechnol 38:1867–1877. doi: 10.1007/s10295-011-0976-x CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Chung D, Farkas J, Westpheling J (2013) Overcoming restriction as a barrier to DNA transformation in Caldicellulosiruptor species results in efficient marker replacement. Biotechnol Biofuels. doi: 10.1186/1754-6834-6-82 Google Scholar
  12. 12.
    Dam P, Kataeva I, Yang SJ, Zhou FF, Yin YB, Chou WC, Poole FL, Westpheling J, Hettich R, Giannone R, Lewis DL, Kelly R, Gilbert HJ, Henrissat B, Xu Y, Adams MWW (2011) Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725. Nucleic Acids Res 39:3240–3254. doi: 10.1093/nar/gkq1281 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Dashtban M, Qin WS (2012) Overexpression of an exotic thermotolerant β-glucosidase in Trichoderma reesei and its significant increase in cellulolytic activity and saccharification of barley straw. Microb Cell Fact. doi: 10.1186/1475-2859-11-63 PubMedPubMedCentralGoogle Scholar
  14. 14.
    Durmaz E, Hu Y, Aroian RV, Klaenhammer TR (2016) Intracellular and extracellular expression of Bacillus thuringiensis crystal protein Cry5B in Lactococcus lactis for use as an anthelminthic. Appl Environ Microbiol 82:1286–1294. doi: 10.1128/Aem.02365-15 CrossRefPubMedCentralGoogle Scholar
  15. 15.
    Farkas J, Chung D, Cha M, Copeland J, Grayeski P, Westpheling J (2013) Improved growth media and culture techniques for genetic analysis and assessment of biomass utilization by Caldicellulosiruptor bescii. J Ind Microbiol Biotechnol 40:41–49. doi: 10.1007/s10295-012-1202-1 CrossRefPubMedGoogle Scholar
  16. 16.
    Groom J, Chung D, Young J, Westpheling J (2014) Heterologous complementation of a pyrF deletion in Caldicellulosiruptor hydrothermalis generates a new host for the analysis of biomass deconstruction. Biotechnol Biofuels. doi: 10.1186/S13068-014-0132-8 PubMedPubMedCentralGoogle Scholar
  17. 17.
    Kado Y, Inoue T, Ishikawa K (2011) Structure of hyperthermophilic β-glucosidase from Pyrococcus furiosus. Acta Crystallogr Sect F Struct Biol Cryst Commun 67:1473–1479. doi: 10.1107/S1744309111035238 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kanafusa-Shinkai S, Wakayama J, Tsukamoto K, Hayashi N, Miyazaki Y, Ohmori H, Tajima K, Yokoyama H (2013) Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii. J Biosci Bioeng 115:64–70. doi: 10.1016/j.jbiosc.2012.07.019 CrossRefPubMedGoogle Scholar
  19. 19.
    Kim SK, Chung D, Himmel ME, Bomble YJ, Westpheling J (2016) Heterologous expression of family 10 xylanases from Acidothermus cellulolyticus enhances the exoproteome of Caldicellulosiruptor bescii and growth on xylan substrates. Biotechnol Biofuels. doi: 10.1186/s13068-016-0588-9 Google Scholar
  20. 20.
    Lochner A, Giannone RJ, Rodriguez M Jr, Shah MB, Mielenz JR, Keller M, Antranikian G, Graham DE, Hettich RL (2011) Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis. Appl Environ Microbiol 77:4042–4054. doi: 10.1128/AEM.02811-10 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lou J, Dawson KA, Strobel HJ (1996) Role of phosphorolytic cleavage in cellobiose and cellodextrin metabolism by the ruminal bacterium Prevotella ruminicola. Appl Environ Microbiol 62:1770–1773PubMedPubMedCentralGoogle Scholar
  22. 22.
    Maki ML, Armstrong L, Leung KT, Qin WS (2013) Increased expression of β-glucosidase A in Clostridium thermocellum 27405 significantly increases cellulase activity. Bioengineered 4:15–20. doi: 10.4161/bioe.21951 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Mohagheghi A, Grohmann K, Himmel M, Leighton L, Updegraff DM (1986) Isolation and characterization of Acidothermus cellulolyticus gen. nov., sp. nov., a new genus of thermophilic, acidophilic, cellulolytic bacteria. Int J Syst Bacteriol 36:435–443. doi: 10.1099/00207713-36-3-435 CrossRefGoogle Scholar
  24. 24.
    Rosenberg HF (1998) Isolation of recombinant secretory proteins by limited induction and quantitative harvest. Biotechniques 24:188–190PubMedGoogle Scholar
  25. 25.
    Teugjas H, Valjamae P (2013) Product inhibition of cellulases studied with 14C-labeled cellulose substrates. Biotechnol Biofuels. doi: 10.1186/1754-6834-6-104 Google Scholar
  26. 26.
    Tjalsma H, Bolhuis A, Jongbloed JDH, Bron S, van Dijl JM (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol Mol Biol Rev 64:515–547. doi: 10.1128/Mmbr.64.3.515-547.2000 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Yang SJ, Kataeva I, Hamilton-Brehm SD, Engle NL, Tschaplinski TJ, Doeppke C, Davis M, Westpheling J, Adams MW (2009) Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe “Anaerocellum thermophilum” DSM 6725. Appl Environ Microbiol 75:4762–4769. doi: 10.1128/AEM.00236-09 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Yi Z, Su X, Revindran V, Mackie RI, Cann I (2013) Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose. PLoS One 8:e84172. doi: 10.1371/journal.pone.0084172 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Young J, Chung D, Bomble YJ, Himmel ME, Westpheling J (2014) Deletion of Caldicellulosiruptor bescii CelA reveals its crucial role in the deconstruction of lignocellulosic biomass. Biotechnol Biofuels. doi: 10.1186/s13068-014-0142-6 Google Scholar
  30. 30.
    Zhang YHP, Lynd LR (2005) Cellulose utilization by Clostridium thermocellum: bioenergetics and hydrolysis product assimilation. Proc Natl Acad Sci USA 102:7321–7325. doi: 10.1073/pnas.0408734102 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2017

Authors and Affiliations

  • Sun-Ki Kim
    • 1
    • 3
  • Daehwan Chung
    • 2
    • 3
  • Michael E. Himmel
    • 2
    • 3
  • Yannick J. Bomble
    • 2
    • 3
  • Janet Westpheling
    • 1
    • 3
    Email author
  1. 1.Department of GeneticsUniversity of GeorgiaAthensUSA
  2. 2.Biosciences Center, National Renewable Energy LaboratoryGoldenUSA
  3. 3.The BioEnergy Science Center, Oak Ridge National LaboratoryOak RidgeUSA

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