UNRAVELING THE UNIQUE CELLULOSOMAL COMPONENTS OF THE LARGEST KNOWN CELLULOSOME SYSTEM IN (PSEUDO) BACTEROIDES CELLULOSOLVENS

Olga Nissan-Zhivin ¹ Bareket Dassa ¹ Morais Sarah ^1,2 Sagar Utturkar ³ Steven Brown ³ Bernard Henrissat ⁴ Rafael Lamed ⁵ Ed Bayer ¹

¹Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel
²Faculty of Natural Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
³Graduate School of Genome Science and Technology, University of Tennessee, Knoxville, USA
⁴Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille University and CNRS, Marseille, France
⁵Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Tel Aviv, Israel

(Pseudo)Bacteroides cellulosolvens is an anaerobic, mesophilic, cellulolytic, cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources. Recently, we sequenced the B. cellulosolvens genome, and revealed an unprecedented number of cellulosome-related components, including 79 cohesin modules, scattered among 32 scaffoldins, and more than 200 dockerin-bearing ORFs. The organization of the B. cellulosolvens cellulosome is unique compared to previously described systems. In contrast to all other known cellulosomes, the cohesin types are reversed for all scaffoldins: that is, the type II cohesins are located on the enzyme-integrating primary scaffoldins (instead of the usual type I cohesins), whereas the type I cohesins are located on the anchoring scaffoldins (as opposed to the usual type II cohesins). We revealed the architectural arrangement of cellulosome structure in this bacterium by examining numerous interactions between the various cohesin and dockerin modules. Then we discovered the cellulosomal content by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Proteomic analysis revealed 24 scaffoldin structural units and 166 dockerin-bearing enzymes, in addition to free enzymatic subunits. All these components comprise cell-free and cell-bound cellulosomes for more efficient carbohydrate degradation. Cellulose-derived cellulosomes showed higher expression levels of the cellulosomal components and exhibited the highest degradation activity on five different carbohydrates. The cellulosomal activity of B. cellulosolvens showed very promising results and was compatible with the activity levels exhibited by C. thermocellum purified cellulosomes. The results of this study provide novel insight into the architecture and function of the most intricate and extensive cellulosomal system known today. The robust cellulosome system of B. cellulosolvens, with its unique binding specificities and reversed types of cohesin and dockerin components, has served to amend our views of the cellulosome paradigm. Revealing new cellulosomal interactions and arrangements is critical for designing high-efficiency artificial cellulosomes for conversion of plant-derived cellulosic biomass towards improved production of biofuels.