Glycosylation of Hyperthermostable Designer Cellulosome Components Yields Enhanced Stability and Cellulose Hydrolysis

Yannick Bomble, Neal Hengge, Michael Himmel, Daehwan Chung, Amaranta Kahn, Sarah Morais, Audrey Kahn, Edward Bayer, Nicholas Sarai

Research output: Contribution to journalArticlepeer-review

12 Scopus Citations

Abstract

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.

Original languageAmerican English
Pages (from-to)4370-4388
Number of pages19
JournalFEBS Journal
Volume287
Issue number20
DOIs
StatePublished - 1 Oct 2020

Bibliographical note

Publisher Copyright:
© 2020 Federation of European Biochemical Societies

NREL Publication Number

  • NREL/JA-2700-76211

Keywords

  • Caldicellulosiruptor bescii
  • cellulosome
  • expression host
  • glycosylation
  • thermostability

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