Minimalistic Cellulosome of the Butanologenic Bacterium Clostridium saccharoperbutylacetonicum

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 contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. 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 cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, 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. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-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 cellulosic 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 cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective “first responders.” The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. 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.

Cell‐surface binding domains from Clostridium cellulovorans can be used for surface display of cellulosomal scaffoldins in Lactococcus lactis

“…[ 25 ] X2 modules have been described to bind to different polysaccharides, such as cellulose and chitin, [ 18,19 ] and those found in bacterial cell walls, [ 25 ] but also to possibly stabilize the structure of the neighboring Coh domains. [ 26 ] More in detail, the binding affinity of CbpA X2 domains for C. cellulovorans cell wall fragments is lower than that for cellulose or chitin. [ 24 ] Surface‐display of C. cellulovorans cellulosomes is probably mediated by multiple proteins.…”

A miniature cellulosome with novel interaction modes

“…We have demonstrated that in the genome of C. lentocellum , a functional comparatively miniature cellulosome complex is encoded. Interestingly, recently, a small cellulosome was also discovered in Ruminoclostridium saccharoperbutylacetonicum 26 . The genome of this organism encodes two cohesin domains and eight dockerin-bearing enzymes.…”

Milling byproducts are an economically viable substrate for butanol production using clostridial ABE fermentation

“…Recently, putative cellulosomal genes were identified in three mesophilic Clostridium species, including Clostridium acetobutylicum and Clostridium saccharoperbutylacetonicum (Dassa et al 2017). However, only the cellulosome of C. saccharoperbutylacetonicum N1-4 (HMT) appears to be actively expressed, although the organism was still not able to utilize purified cellulose as substrate (Levi Hevroni et al 2020). Levi Hevroni et al hypothesize that cellulosome expression in C. saccharoperbutylacetonicum might be dependent on specific inducers which can only be found in complex substrates, such as plant biomasses (Montoya et al 2001;Levi Hevroni et al 2020).…”

“…However, only the cellulosome of C. saccharoperbutylacetonicum N1-4 (HMT) appears to be actively expressed, although the organism was still not able to utilize purified cellulose as substrate (Levi Hevroni et al 2020). Levi Hevroni et al hypothesize that cellulosome expression in C. saccharoperbutylacetonicum might be dependent on specific inducers which can only be found in complex substrates, such as plant biomasses (Montoya et al 2001;Levi Hevroni et al 2020). While we did not examine the cellulose utilization of our strains in this study, we would like to speculate that at least some of them might be able to use a small part of the cellulose content of our complex substrates as a carbon source.…”