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

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 bacteriumCaldicellulosiruptor besciiproduces extremely active hyperthermostable cellulases, including the hyperactive multifunctional cellulaseCbCel9A/Cel48A. RecombinantCbCel9A/Cel48A components have been previously produced inEscherichia coliand integrated into synthetic hyperthermophilic designer cellulosome complexes. Since then, glycosylation has been shown to be vital for the high activity and stability ofCbCel9A/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 ofCbCel9A/Cel48A, were glycosylated as a consequence of employingCa. besciias an expression host.Inclusion of the glycosylated components yielded an active cellulosome system that exhibited long-term stability at 75 degrees 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. besciiwas 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.