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Insights into the functionality and stability of designer cellulosomes at elevated temperatures

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Abstract

Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50 °C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme-scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6 h at 60 °C. Finally, within this time frame and conditions, the full complex appeared over 50 % more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.

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Acknowledgments

This research has been co-financed by: (i) The European Union (European Social Fund—ESF) and Greek National funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF) - Research Funding Program “Excellence II (Aristeia II)”, Grant No. 4641 “Engineering advanced biocatalysts for the conversion of lignocellulosic biomass into fermentable sugars (ABCAT)”. (ii) The European Union, Area NMP.2013.1.1-2: Self-assembly of naturally occurring nanosystems: CellulosomePlus Project number: 604530. (iii) Grant (No. 1349/13) from the Israel Science Foundation (ISF), Jerusalem, Israel. (iv) Grant from the US-Israel Binational Science Foundation (BSF). E.A.B. is the incumbent of The Maynard I. and Elaine Wishner Chair of Bio-organic Chemistry.

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  1. Faculty of Biology, Microbiology Group, National and Kapodistrian University of Athens, Zografou Campus, 15784, Zografou, Attica, Greece

    Anastasia P. Galanopoulou, Anastasios Georgoulis & Dimitris G. Hatzinikolaou

  2. Department of Biomolecular Sciences, The Weizmann Institute of Science, 76100, Rehovot, Israel

    Sarah Moraïs, Ely Morag & Edward A. Bayer

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  1. Anastasia P. Galanopoulou
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  2. Sarah Moraïs
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  3. Anastasios Georgoulis
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  4. Ely Morag
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  5. Edward A. Bayer
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  6. Dimitris G. Hatzinikolaou
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Correspondence to Dimitris G. Hatzinikolaou.

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Galanopoulou, A.P., Moraïs, S., Georgoulis, A. et al. Insights into the functionality and stability of designer cellulosomes at elevated temperatures. Appl Microbiol Biotechnol 100, 8731–8743 (2016). https://doi.org/10.1007/s00253-016-7594-5

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