Synthetic fungal multifunctional cellulases for enhanced biomass conversion

Significant effort has been expended toward the discovery and/or engineering of improved cellulases. An alternative to this approach is utilizing multifunctional enzymes; however, essentially most if not all relevant bacterial enzymes of this type do not express well in fungi. Therefore, developing a systematic understanding of how to construct multifunctional enzymes that are expressible in commercial fungal hosts is crucial to developing next generation enzymes for biomass deconstruction. Multifunctional cellulolytic enzymes, such as CelA from Caldicellulosiruptor bescii , show extremely high cellulolytic activity; however, a systematic understanding of its mechanism of action does not exist and it is not readily expressed in current industrial hosts. CelA is comprised of GH 9 and GH 48 catalytic domains connected by three type III cellulose-binding modules (CBMs). We have engineered several multifunctional enzymes designed to mimic CelA, and successfully expressed them in T. reesei . We then assessed their biophysical and kinetic performance parameters. The CBM3b-containing construct demonstrated increased initial binding rate to cellulose, enhanced digestion of biomass, and was able to increase the activity of a commercial cellulase formulation acting on pretreated biomass. The same construct containing CBM1 also demonstrated enhancement of a commercial cellulase formulation in the digestion of pretreated biomass; however, it had lower initial binding rates and did not demonstrate improved activity on its own. Interestingly, a CBM-less construct decreased the activity of the commercial cellulase formulation slightly. Examination of the mechanism of the CBM3b-containing construct revealed a novel biomass deconstruction behavior similar to, but yet distinct from that of native CelA. Synthetic multifunctional enzymes exhibit superior performance to their non-multifunctional enzyme components and exhibit novel cellulose deconstruction mechanisms.