Towards Consolidated Bio-Processing: Engineering Saccharomyces Cerevisiae for the Expression and Evaluation of Novel Anaerobic Fungal Cellulases

The most promising approach for harnessing the energy contained within cellulosic biomass is to break down the cellulose polymer into simple sugars that can be converted via fermentation into energy-rich chemicals such as ethanol.  At present time, no single microorganism has been identified that can perform both cellulolytic and fermentation processes with suitable efficiency.  Thus, industrial approaches to biomass conversion typically rely on co-culturing two different organisms or pre-treating cellulose-rich biomass with expensive chemicals to reduce lignin content prior to incubation with purified cellulolytic enzymes.  More recently, advances in the identification of the genes responsible for cellulose breakdown in native cellulolytic organisms as well as advances in our ability to genetically modify a wide variety of microbial species has opened the way to genetic engineering of a single microbe that can efficiently carry out both cellulose breakdown and fermentation – a strategy referred to as Consolidated Bio-processing (CBP). 

In order to facilitate CBP, we are developing the yeast Saccharomyces cerevisae as a robust expression platform for the production of novel cellulases derived from anerobic fungi that naturally inhabit the rumen of large herbivorous animals.  Despite the powerful cellulolytic properties of rumen fungi, remarkably few enzymes have been characterized or cloned from these organisms due to difficulties associated with the isolation and culture of these microbes.  Towards introducing these enzymes into yeast, we have developed unique methods to culture the anaerobic fungi Piromyces equi, which produces a collection of catabolically-regulated cellulases.  Among these enzymes, most exist in multi-enzyme complexes termed cellulosomes, which tether cellulases together for the targeted, synergistic hydrolysis of cellulose.  We will discuss the properties of these enzymes as they are expressed in S. cerevisiae, including their enhanced specific activities, thermostability, resistance to inhibitors, and their ability to cleave novel chemical linkages in cell wall material.  Reconstitution, assembly, and catalytic activity of fungal cellulosomes produced in S. cerevisiae will also be discussed.