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Creation of a functional hyperthermostable designer cellulosome

Kahn, Amaranta and Moraïs, Sarah and Galanopoulou, Anastasia P. and Chung, Daehwan and Sarai, Nicholas S. and Hengge, Neal and Hatzinikolaou, Dimitris G. and Himmel, Michael E. and Bomble, Yannick J. and Bayer, Edward A. (2019) Creation of a functional hyperthermostable designer cellulosome. Biotechnology for Biofuels, 12 . Art. No. 44. ISSN 1754-6834. PMCID PMC6394049. doi:10.1186/s13068-019-1386-y.

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Background: Renewable energy has become a field of high interest over the past decade, and production of biofuels from cellulosic substrates has a particularly high potential as an alternative source of energy. Industrial deconstruction of biomass, however, is an onerous, exothermic process, the cost of which could be decreased significantly by use of hyperthermophilic enzymes. An efficient way of breaking down cellulosic substrates can also be achieved by highly efficient enzymatic complexes called cellulosomes. The modular architecture of these multi-enzyme complexes results in substrate targeting and proximity-based synergy among the resident enzymes. However, cellulosomes have not been observed in hyperthermophilic bacteria. Results: Here, we report the design and function of a novel hyperthermostable “designer cellulosome” system, which is stable and active at 75 °C. Enzymes from Caldicellulosiruptor bescii, a highly cellulolytic hyperthermophilic anaerobic bacterium, were selected and successfully converted to the cellulosomal mode by grafting onto them divergent dockerin modules that can be inserted in a precise manner into a thermostable chimaeric scaffoldin by virtue of their matching cohesins. Three pairs of cohesins and dockerins, selected from thermophilic microbes, were examined for their stability at extreme temperatures and were determined stable at 75 °C for at least 72 h. The resultant hyperthermostable cellulosome complex exhibited the highest levels of enzymatic activity on microcrystalline cellulose at 75 °C, compared to those of previously reported designer cellulosome systems and the native cellulosome from Clostridium thermocellum. Conclusion: The functional hyperthermophilic platform fulfills the appropriate physico-chemical properties required for exothermic processes. This system can thus be adapted for other types of thermostable enzyme systems and could serve as a basis for a variety of cellulolytic and non-cellulolytic industrial objectives at high temperatures.

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URLURL TypeDescription CentralArticle
Kahn, Amaranta0000-0001-8516-7296
Sarai, Nicholas S.0000-0002-4655-0038
Bomble, Yannick J.0000-0001-7624-8000
Additional Information:© The Author(s) 2019. Open Access - This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated. Received: 1 November 2018. Accepted: 20 February 2019. Published online 28 February 2019. A.K. greatly appreciates scholarships received from the Ministry of Immigrant Absorption, Jerusalem, Israel and from the ministry of Foreign Affairs, Paris, France. A.K. is a Sustainability and Energy Weizmann Fellow. E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry. This research was supported by the United States—Israel Binational Science Foundation (BSF Grant No. 2013284), Jerusalem, Israel; the Israel Science Foundation (ISF Grant No. 1349/13); the European Union NMP.2013.1.1-2: CellulosomePlus Project number 604530. Funding was also provided by the BioEnergy Science Center (BESC) and the Center for Bioenergy Innovation (CBI), from the U.S. Department of Energy Bioenergy Research Centers supported by the Office of Biological and Environmental Research in the DOE Office of Science. 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. The U.S. Government and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this work, or allow others to do so, for U.S. Government purposes. Authors’ contributions: AK designed the research, performed the experiments and wrote the manuscript. AK, SM and DC designed and performed the experiments for the cloning and selection of the best-performing chimaeric glycoside hydrolases. AK and APG designed the methodology for thermostability assay of the cellulosomal components. AK and NSS produced and purified the proteins. AK, NH and YJB designed and conducted the activity assay. AK, SM, APG, DGH, DC, NSS, NH, YJB, MEH and EAB analyzed the results. AK, SM and EAB wrote the manuscript. All authors read and approved the final manuscript. Availability of data and materials: Not applicable. Consent for publication: Not applicable. Ethics approval and consent to participate: Not applicable. The authors declare that they have no competing interests.
Funding AgencyGrant Number
Ministry of Immigrant Absorption (Israel)UNSPECIFIED
Ministry of Foreign Affairs (France)UNSPECIFIED
Weizmann Institute of ScienceUNSPECIFIED
Binational Science Foundation (USA-Israel)2013284
Israel Science Foundation1349/13
European Research Council (ERC)604530
Department of Energy (DOE)DE-AC36-08GO28308
Subject Keywords:Multi-enzyme complex, Cellulases, Thermostability, Caldicellulosiruptor bescii, Scaffoldin, Dockerin, Cohesin
PubMed Central ID:PMC6394049
Record Number:CaltechAUTHORS:20190228-085142512
Persistent URL:
Official Citation:Kahn, A., Moraïs, S., Galanopoulou, A.P. et al. Biotechnol Biofuels (2019) 12: 44.
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:93330
Deposited By: George Porter
Deposited On:28 Feb 2019 20:42
Last Modified:16 Nov 2021 16:56

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