(341b) 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.