Functional and structural study of a cellulosomal cellulase from anaerobic fungus and its dockerin-scaffoldin interaction

Abstract

The lignocellulosic biomass is the most abundant renewable carbon source on the planet, with several biotechnological applications, including the conversion of cellulose into ethanol. The cellulosome is a modular multi-enzymatic complex with high molecular mass present in anaerobic microorganisms and which presents a high efficiency in the degradation of cellulose. As a comparison, the cellulosome of the anaerobic bacterium Clostridium thermocellum presents one of the highest degradation rates with efficiency dozens of times higher in the degradation of crystalline cellulose when compared to the cellulolytic system of the fungus of the genus Trichoderma. Similarly to the bacterium C. thermocellum, the enzymes of anaerobic fungi that degrade the plant cell wall are organized into cellulosomes with high molecular mass. In contrast to bacterial cellulosomes, fungal cellulosomes are still much less studied and characterized. Even after all the research developed, currently an effective commercial process using cellulosomes is still not feasible. Therefore, more studies need to be carried out to better understand the mechanism of assembly and action of cellulosomes, especially the cellulosomes of anaerobic fungi. To this end, in this research project, we propose to study a cellulosomal cellulase identified in the genome of the anaerobic fungus Piromyces finnis and its interaction with scaffoldins of the same fungus. The sequence of a cellulase from the GH5 family from P. finnis e two scaffoldins were cloned into bacterial expression vectors. P. finnis cellulase is composed of a GH5 catalytic domain connected to a CBM1 domain through a dockerin module, and showed promising expression in E. coli cells. Efforts will be directed at the initiative to build mini-cellulosomes of P. finnis, based on the recombinant expression of two different scaffoldins and evaluation of their interaction with the modular cellulase (dockerin-scaffoldin interaction). The structure of the modular cellulase and the formation of complexes with scaffoldins will be analyzed by physical techniques such as circular dichroism spectroscopy, dynamic light scattering, small angle X-rays scattering, protein crystallography and cryogenic electron microscopy. The results from this research can provide original and innovative information on the structure, organization and functioning of cellulosome complexes of anaerobic fungi. (AU)