Deciphering domain structures of Aspergillus and Streptomyces GH3-ß-Glucosidases: a screening system for enzyme engineering and biotechnological applications.

ABSTRACT

The glycoside hydrolase family 3 (GH3) ß-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 ß-glucosidases. Two distinct configurations were identified in fungal GH3 ß-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in ß-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger ß-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric ß-glucosidases enzymes for industrial application.