In Vitro Reconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>Artificial cellulase complexes active on crystalline cellulose were reconstituted<jats:italic>in vitro</jats:italic>from a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of a<jats:italic>C. thermocellum cipA</jats:italic>mutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. The<jats:italic>in vitro</jats:italic>-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities for<jats:italic>in vitro</jats:italic>complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.</jats:p>

Proximity Effect among Cellulose-Degrading Enzymes Displayed on the Saccharomyces cerevisiae Cell Surface

“…Krauss et al (24) reported that the addition of scaffoldin to the scaffoldin-disrupted C. thermocellum culture supernatant increased the cellulase activity only when Avicel was used as a substrate, whereas it did not differ greatly when PASC or soluble cellulose was used, which suggests an exclusive proximity effect on Avicel. From this report, we expected that the arming yeast would show an even greater proximity effect on Avicel and we measured the Avicel-degrading activity of MIX-yeast and ALLyeast (see Fig.…”

“…The cell pellet was resuspended using 500 l of a 100 mM citrate buffer and incubated in a heat block at 50°C for 1 h, after which 500 l of 2% PASC was added to start the reaction (which was performed at 50°C). Reaction samples were withdrawn at 6, 12, 24, and 48 h of incubation in the case of OD10 yeast cells and at 24,48,84, and 120 h of incubation in the case of OD0.1 yeast cells, and then the supernatants were mixed with 3,5-dinitrosalicylic acid (DNS) solution (NaOH, 16 g/liter; potassium sodium tartrate, 300 g/liter; DNS, 5 g/liter) in a 1:2 ratio to quantify the reduced ends of the degraded products. The mixed solutions were incubated at 100°C for 5 min, and after cooling them to room temperature, 200 l of each solution was used for measuring the A 530 using the Fluoroskan Ascent fluorometer and 96-well black plates.…”

Stoichiometric Assembly of the Cellulosome Generates Maximum Synergy for the Degradation of Crystalline Cellulose, as Revealed by In Vitro Reconstitution of the Clostridium thermocellum Cellulosome

“…These deletion variants display two or three enzyme molecules (8,(11)(12)(13), whereas the number of cohesin modules in native cellulosomal scaffoldin proteins is typically between 6 and 11 (1). These minicellulosomes usually show significantly lower activity toward crystalline cellulose than do native cellulosomes (8,12,14). Thus, elucidating the mechanism responsible for the high activity for crystalline cellulose exhibited by native cellulosomes requires in vitro reconstitution of the cellulosome complexes from their large scaffoldin proteins.…”

“…The cellulolytic activity of the in vitro-reconstituted cellulosome, comprising full-length CipA produced by recombinant E. coli cells and the protein mixture secreted from cipA-deficient C. thermocellum cells, has recently been investigated (14). The reconstituted cellulosome was assembled by assuming that the secreted protein mixture has an average molecular mass of 80 kDa.…”

Bioprospecting thermostable cellulosomes for efficient biofuel production from lignocellulosic biomass

“…Localization of broad substrate range enzymes and their close proximity for efficient formation of enzyme-substrate complex are the significant factors for increasing the efficiency of the cellulosomes. Cellulosomes are known to have better enzymatic activity as compared to free enzymes because of the close proximity of various cellulases which act synergistically (Blanchette et al 2012;Krauss et al 2012;You et al 2012a). However, the mechanism of protein assembly and organization of various catalytic enzymes in the cellulosomes is partially known (Wilson 2011).…”