Development of a robust cellulase expression system in Trichoderma reesei

Improving efficiency in enzymatic hydrolysis of biomass remains paramount to producing economically viable cellulosic biofuels, yet the full mechanistic understanding and fundamental knowledge of fungal cellulases remains incomplete. Given the vast differences in enzymatic activity of the same enzymes produced in varied heterologous hosts, it is imperative to study these enzymes in their native and industrially relevant host strains.  Accordingly, we have generated a robust expression system using the filamentous fungal host Trichoderma reesei.  This system allows us to focus on high level expression of a single enzyme with no background cellulases, easing purification and limiting contaminating or synergistic activities.  We have developed a production pipeline from DNA cloning and transformation through enzyme purification and characterization that generates multitudes of phylogenetically diverse enzymes and mutants designed to better understand the fundamental mechanistic actions of cellulases. While we currently use this system for producing various classes of enzymes, here we will focus on Cel7 proteins. We will discuss aspects of strain development, protein expression, purification, and glycosylation effects on activity.  Furthermore, we will discuss how we have streamlined our approach towards enzyme production and enzyme screening.  In essence, we will detail the progressive history of the development of our current expression system including pitfalls and breakthroughs, and where we envision taking this system in the future.