Minimalistic cellulosome of the butanologenic bacterium clostridium saccharoperbutylacetonicum

Bosmat Levi Hevroni, Sarah Moraïs, Yonit Ben-David, Ely Morag, Edward A. Bayer

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome con-tains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two co-hesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and puri-fied. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellu-losic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a man-nanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylace-tonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports sub-strate-dependent in vivo secretion of cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellu-losic biomass. IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellu-lose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective “first responders.” The mas-sive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and buta-nol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

Original languageEnglish
Article numbere00443-20
JournalmBio
Volume11
Issue number2
DOIs
StatePublished - 1 Mar 2020

Keywords

  • CBM
  • Cohesin specificity
  • Cohesin-dockerin specificity
  • Enzymatic profiling
  • Glycoside hydrolases
  • Scaffoldin

ASJC Scopus subject areas

  • Microbiology
  • Virology

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