Abstract
Background
Designer cellulosomes (DCs) are engineered multi-enzyme complexes that exhibit high potential to be applied in the biomass-to-liquid process of biorefineries. Their architecture allows to bring a high load of enzymes with various catalytic activities in close proximity to each other and to the substrate, which may result in enhanced depolymerization efficiencies compared to freely acting enzymes. As such, DCs have often been proposed as an elegant alternative to (hemi)-cellulolytic enzyme cocktails. However, the practical use of DCs in industrial contexts remains elusive. This can partially be attributed to lacking knowledge about the reaction conditions wherein DCs may offer an added benefit. Therefore, we customized a DC for the efficient saccharification of relevant industrial substrates. These substrates are in great contrast with the conventional pure substrates frequently used in lab environments and are envisioned to provide us with a better understanding of DC behavior on complex lignocellulosic material.
Results
In this work, a DC possessing endoglucanase, cellobiohydrolase, β-glucosidase and endoxylanase activity was developed. For this, multiple enzymes originating from Cellvibrio japonicus, known as a true carbohydrate-bioconversion specialist, were converted to docking enzymes. After a three-faceted selection process of optimal docking enzyme configurations, selected variants were colocalized on a common scaffoldin to finally construct a tetravalent DC. This DC successfully released glucose from agro-industrial wheat fibers and genome-edited low-lignin poplar biomass, representing two main feedstock types of a circular bioeconomy. Comparing saccharification efficiencies of the enzyme systems in their free and complexed form allowed us to outline reaction conditions wherein enzyme colocalization may boost synergism. In fact, the evaluation of DC behavior on recalcitrant lignocellulosic substrates revealed that the effect of colocalization depends on the specific substrate used and is most outspoken under conditions that mimic the natural environment of biomass degradation, deviating from typical industrial settings.
Conclusion
DCs were explored for their industrial applicability, in order to map possible processes where they could offer an added benefit. We anticipate this work to contribute to the broader insights we need to investigate the economic viability of DCs.