Enhanced cellulose degradation by targeted integration of a cohesin-fused β-glucosidase into the Clostridium thermocellum cellulosome

Abstract

The conversion of recalcitrant plant-derived cellulosic biomass into biofuels is dependent on highly efficient cellulase systems that produce near-quantitative levels of soluble saccharides. Similar to other fungal and bacterial cellulase systems, the multienzyme cellulosome system of the anaerobic, cellulolytic bacterium Clostridium thermocellum is strongly inhibited by the major end product cellobiose. Cellobiose-induced inhibition can be relieved via its cleavage to noninhibitory glucose by the addition of exogenous noncellulosomal enzyme β-glucosidase; however, because the cellulosome is adsorbed to the insoluble substrate only a fraction of β-glucosidase would be available to the cellulosome. Towards this end, we designed a chimeric cohesin-fused β-glucosidase (BglA-CohII) that binds directly to the cellulosome through an unoccupied dockerin module of its major scaffoldin subunit. The β-glucosidase activity is thus focused at the immediate site of cellobiose production by the cellulosomal enzymes. BglA-CohII was shown to retain cellobiase activity and was readily incorporated into the native cellulosome complex. Surprisingly, it was found that the native C. thermocellum cellulosome exists as a homooligomer and the high-affinity interaction of BglA-CohII with the scaffoldin moiety appears to dissociate the oligomeric state of the cellulosome. Complexation of the cellulosome and BglA-CohII resulted in higher overall degradation of microcrystalline cellulose and pretreated switchgrass compared to the native cellulosome alone or in combination with wild-type BglA in solution. These results demonstrate the effect of enzyme targeting and its potential for enhanced degradation of cellulosic biomass.

Toward combined delignification and saccharification of wheat straw by a laccase-containing designer cellulosome

“…The incorporation of nonconventional enzymes into designer cellulosomes, such as β-glucosidases, LPMOs, and expansins, has proved an efficient strategy for enhancement of biomass degradation by glycoside hydrolases (22,(60)(61)(62). These accessory enzymes act in concert with glycoside hydrolases, either by releasing product inhibition or by acting directly on the substrate.…”

Immobilization of β-glucosidase onto Magnetic Nanoparticles and Evaluation of the Enzymatic Properties

“…Compared to these chemical methods, enzymatic hydrolysis methods involve lower energy consumption, milder reaction conditions, higher yields, and substrate specificity (Maeda et al 2012;MartináAlonso 2013). β-glucosidase (EC 3.2.1.21) can hydrolyze various glycosides, and multiple potential applications have been developed in cellulose degradation (Gefen et al 2012). However, free β-glucosidase has some limitations for industrial applications, such as a tedious recycle process and poor stability.…”

Evaluation of Cellulase Production by Zymomonas mobilis

“…This is the first report on the level of native cellulolytic activity of Z. mobilis against various cellulosic substrates. The level of cellulolytic activities has been dramatically reported for recombinant Escherichia coli, Saccharomyces cerevisiae and Kluyveromyces lactis through the engineered cellulase obtaining from native Bacillus sp., Clostridium thermocellum and fungal sources (Garvey et al 2013;Gefen et al 2012). The recombinant strains elaborated significant enzyme activity level against CMC and microcrystalline cellulose.…”