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Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis

Kahn, Amaranta and Moraïs, Sarah and Chung, Daehwan and Sarai, Nicholas S. and Hengge, Neal N. and Kahn, Audrey and Himmel, Michael E. and Bayer, Edward A. and Bomble, Yannick J. (2020) Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis. FEBS Journal, 287 (20). pp. 4370-4388. ISSN 1742-464X. doi:10.1111/febs.15251.

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Biomass deconstruction remains integral for enabling second‐generation biofuel production at scale. However, several steps necessary to achieve significant solubilization of biomass, notably harsh pretreatment conditions, impose economic barriers to commercialization. By employing hyperthermostable cellulase machinery, biomass deconstruction can be made more efficient, leading to milder pretreatment conditions and ultimately lower production costs. The hyperthermophilic bacterium Caldicellulosiruptor bescii produces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulase CbCel9A/Cel48A. Recombinant CbCel9A/Cel48A components have been previously produced in Escherichia coli and integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability of CbCel9A/Cel48A. Here, we studied the impact of glycosylation on a hyperthermostable designer cellulosome system in which two of the cellulosomal components, the scaffoldin and the GH9 domain of CbCel9A/Cel48A, were glycosylated as a consequence of employing Ca. bescii as an expression host. Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long‐term stability at 75 °C. The resulting glycosylated designer cellulosomes showed significantly greater synergistic activity compared to the enzymatic components alone, as well as higher thermostability than the analogous nonglycosylated designer cellulosomes. These results indicate that glycosylation can be used as an essential engineering tool to improve the properties of designer cellulosomes. Additionally, Ca. bescii was shown to be an attractive candidate for production of glycosylated designer cellulosome components, which may further promote the viability of this bacterium both as a cellulase expression host and as a potential consolidated bioprocessing platform organism.

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URLURL TypeDescription
Kahn, Amaranta0000-0001-8516-7296
Sarai, Nicholas S.0000-0002-4655-0038
Bomble, Yannick J.0000-0001-7624-8000
Additional Information:© 2020 Federation of European Biochemical Societies. Issue Online: 19 October 2020; Version of Record online: 10 March 2020; Accepted manuscript online: 17 February 2020; Manuscript accepted: 14 February 2020; Manuscript revised: 06 January 2020; Manuscript received: 29 August 2019. AmK greatly appreciates scholarships received from the Ministry of Immigrant Absorption, Jerusalem, Israel, and from the ministry of Foreign Affairs, Paris, France. AmK is a Sustainability and Energy Weizmann Fellow. EAB is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio‐organic Chemistry. This work was authored in part by Alliance for Sustainable Energy, LLC, the manager and operator of the National Renewable Energy Laboratory for the U.S. Department of Energy (DOE) under Contract No. DE‐AC36‐08GO28308. The views expressed in the article do not necessarily represent the views of the DOE or the U.S. Government. Funding was also provided by the Center for Bioenergy Innovation (CBI), a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This research was also supported by the United States–Israel Binational Science Foundation (BSF grant No. 2013284), Jerusalem, Israel; the Israel Science Foundation (ISF grant no. 1349/13); and the European Union NMP.2013.1.1‐2: CellulosomePlus Project number 604530. The funding sources were not involved in the design of the study, data interpretation, report writing, or decision in submitting the article for publication. The authors declare no conflict of interest. Author contributions: AmK designed the research, performed the experiments, and wrote the manuscript. AmK, SM, and DC designed and performed the experiments for the cloning and transformation of the plasmid in Ca. bescii. AmK, NH, and NSS produced and purified the proteins. AmK designed and conducted the binding assay. AmK, AuK, and YJB designed and conducted the activity assay. AmK and SM designed and conducted the thermostability assay. All authors analyzed the results. AK, SM, EAB, MEH, and YJB wrote the manuscript. All authors read and approved the manuscript.
Funding AgencyGrant Number
Ministry of Immigrant Absorption (Israel)UNSPECIFIED
Ministry of Foreign Affairs (France)UNSPECIFIED
Weizmann Institute of ScienceUNSPECIFIED
Department of Energy (DOE)DE-AC36-08GO28308
Binational Science Foundation (USA-Israel)2013284
Israel Science Foundation1349/13
European Research Council (ERC)604530
Subject Keywords: Caldicellulosiruptor bescii; cellulosome; expression host; glycosylation; thermostability
Issue or Number:20
Record Number:CaltechAUTHORS:20200224-134411353
Persistent URL:
Official Citation:Kahn, A., Moraïs, S., Chung, D., Sarai, N.S., Hengge, N.N., Kahn, A., Himmel, M.E., Bayer, E.A. and Bomble, Y.J. (2020), Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis. FEBS J., 287: 4370-4388.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:101503
Deposited By: Tony Diaz
Deposited On:24 Feb 2020 22:06
Last Modified:16 Nov 2021 18:03

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