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

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.

Metagenomic Discovery and Characterization of Multi-Functional and Monomodular Processive Endoglucanases as Biocatalysts

“…Xu et al (2013) [101] used domain engineering to exploit the advantage of intramolecular and intermolecular proximity synergies by designing mini-cellulosomes to improve cellulase specific activity as well as to enhance their biomass-penetrating feature. Other strategies such as glycosylation by overexpression designer cellulosomes in other microbial platforms could further improve designer cellulosomes' thermostability and activity [102].…”

Clostridium thermocellum as a Promising Source of Genetic Material for Designer Cellulosomes: An Overview

“…The lack of a proper glycosylation system in E. coli seems to prevent C. bescii enzymes from fully expressing their capability [17]. Therefore, to improve the cellulolytic activity of the DCs through glycosylation, in a recently published paper, Kahn and coworkers [61] After 72 h of incubation at optimal temperature 60 • C for C. thermocellum enzymes, with the synergy of Cel48S and Cel9R [60], the C. thermocellum-based DC (i.e., ScafTBA:Cel5G-t/b-Cel48S/a-Cel9R) [31] produced higher reducing sugar release than the ScafGTV:Cel5Dg/Cel48A-lk-t/Cel9A-lk-v, indicating a predominant efficiency of the C. thermocellum enzymes at mild thermophilic conditions. However, after 72 h of incubation at 75 • C, an opposite situation was observed as the complexation of ScafGTV:Cel5D-g/Cel48A-lkt/Cel9A-lk-v showed a far higher saccharolytic activity than that of the C. thermocellumbased DC and even higher than that of the native C. thermocellum cellulosome whose cellulose-degrading capacity dramatically lost at such a higher temperature (Figure 7C,D).…”

“…Synergy between enzymes, however, was not observed in the C. bescii-based DCs. In fact, the catalytic efficiencies of the individual bound Cel48A-lk-t and Cel48A-sk-t and their unbound forms were dramatically low on Avicel due to the lack of an appropriate glycosylation system in the E. coli XL1 cells [61].…”

“…The lack of a proper glycosylation system in E. coli seems to prevent C. bescii enzymes from fully expressing their capability [17]. Therefore, to improve the cellulolytic activity of the DCs through glycosylation, in a recently published paper, Kahn and coworkers [61] used C. bescii as an expression host to produce glycosylated CbCel9A-lk-v, CbCel48A-lk-t, and chimeric CbScaf-GTV. The DocI-bearing Cel5D failed to express in C. bescii and the CbCel48A-lk-t could not bind to Scaf-T, thus the non-glycosylated DocI-bearing versions of Cel48A and Cel5D, with short linker, were expressed in E. coli to produce EcCel48Ask-t and EcCel5D-g, respectively (Figure 8A).…”

Trichoderma harzianum cerato‐platanin enhances hydrolysis of lignocellulosic materials

“…In this direction, some hurdles still exist, such as physico‐chemical, structural and compositional factors hindering the hydrolysis of cellulose present in biomasses, beyond other economic aspects (Kahn et al ., 2020). A necessary step is the removal or reduction of any obstacle to hydrolysis, in order to maximize the yields of fermentable sugars from cellulose or hemicellulose (Mosier et al ., 2005).…”