Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.
Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
FEBS J. 2020 Oct;287(20):4370-4388. doi: 10.1111/febs.15251. Epub 2020 Mar 10.
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.
生物量解构对于实现规模化第二代生物燃料生产至关重要。然而，实现生物质显著溶解所必需的几个步骤，特别是苛刻的预处理条件，给商业化带来了经济障碍。通过使用超耐热纤维素酶机制，可以提高生物量解构的效率，从而采用更温和的预处理条件，最终降低生产成本。嗜热菌 Caldicellulosiruptor bescii 产生极其活跃的超耐热纤维素酶，包括超活性多功能纤维素酶 CbCel9A/Cel48A。重组 CbCel9A/Cel48A 组件以前曾在大肠杆菌中生产，并整合到合成超耐热设计纤维素酶复合物中。此后，糖基化已被证明对 CbCel9A/Cel48A 的高活性和稳定性至关重要。在这里，我们研究了糖基化对超耐热设计纤维素酶系统的影响，其中两个纤维素酶组件，即支架蛋白和 CbCel9A/Cel48A 的 GH9 结构域，由于使用 Ca. bescii 作为表达宿主而发生糖基化。包含糖基化组件产生了一种活性纤维素酶系统，在 75°C 下具有长期稳定性。所得糖基化设计纤维素酶显示出与单独酶组件相比显著更高的协同活性，以及比类似的非糖基化设计纤维素酶更高的热稳定性。这些结果表明，糖基化可用作改善设计纤维素酶特性的基本工程工具。此外，Ca. bescii 被证明是生产糖基化设计纤维素酶组件的有吸引力的候选者，这可能进一步促进该细菌作为纤维素酶表达宿主以及作为潜在的综合生物加工平台生物的生存能力。
