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Abstract Background Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. Results In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. Conclusion This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.

Abstract Background In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive. Results Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library. Conclusions The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.