(4an) Engineering the Yeast Saccharomyces Cerevisiae for Drug Discovery and Bioenergy Applications

Molecular engineering has enabled the economical production
of several valuable products with the aid of microbial systems, from vaccines and
therapeutics to biofuels.  Yeast systems, such as S. cerevisiae, are
ideal for protein production and other industrial applications because they are
easy to grow and scale-up, inexpensive to work with, and straightforward to
genetically manipulate.  Since yeast have a eukaryotic secretory pathway, they
also are equipped with the cellular machinery to facilitate protein folding and
critical post-translational processing steps that often are necessary for the
production of authentic proteins.    

My doctoral research involved the expression, purification,
and biophysical characterization of human G-protein coupled receptors (GPCRs)
from S. cerevisiae.  GPCRs are membrane proteins that mediate responses
to extracellular stimuli, represent over 50% of all drug targets, and have been
directly linked to several diseases including HIV infection, cancer, and
diabetes.  However, the development of improved, structure-based therapeutics
to target these proteins requires milligram amounts of properly folded,
purified receptors, which are generally not achievable from native tissues.  To
structurally characterize this important class of proteins, GPCRs were over-expressed
in S. cerevisae to circumvent the problem of low natural abundance, and their
trafficking, folding, and activity were evaluated.  Although all GPCRs were successfully
expressed at the mg/L scale, we discovered that most receptors trigger cellular
stress responses when expressed in yeast and exhibit compromised activity.  These
observations were closely linked to improper processing of the N-terminal
leader sequence, which suggests that translocation is a critical bottleneck for
receptor over-expression in yeast.  Of all the receptors analyzed, only the
human adenosine A₂a receptor (hA₂aR) was found in large
quantities at the plasma membrane, where it was able to bind to adenosine
analogs.  The hA₂aR was purified to homogeneity in milligram amounts
and reconstituted with mixed surfactant micelles, which allowed for extensive
structural characterization using biophysical techniques.

Building on my expertise with S. cerevisiae, my
postdoctoral research is focused on engineering S. cerevisiae as a tool
for biofuel production from diverse cellulose-rich feedstocks.  Our approach
involves the characterization of fungal cellulosomes, or large multisubunit
complexes of carbohydrate hydrolyzing enzymes and cellulose binding domains,
from anaerobic fungi that are native to the rumen of large herbivorous animals. 
The multicomponent cellulosome complexes produced by these fungi are superior
to similar complexes that have been widely studied from bacteria since they are
much more efficient at degrading cellulose and have novel cellulolytic
properties.  As the components of the fungal cellulosome have not been
completely identified, we are using powerful genomic tools to identify genes
within the anaerobic fungi Piromyces equi that are upregulated when
grown in the presence of cellulose-rich feedstocks.  We are currently working
towards expressing and reconstituting components of the Piromyces
cellulosome within S. cerevisiae to facilitate consolidated
bioprocessing of cellulose to ethanol.  Furthermore, we seek to understand the structural
basis for efficient cellulose degredation, and optimize the cellulosome through
genetic engineering to maximize S. cerevisiae's ability to degrade
complex forms of cellulose.