(5bq) Engineering the Yeast Saccharomyces Cerevisiae for Protein Production 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 with Anne Robinson (Dept. of Chemical Engineering, University of Delaware) 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, we over-expressed GPCRs in S. cerevisae to circumvent the problem of low natural abundance, and systematically evaluated their trafficking, folding, and activity. 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. We found that 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 within Chris Kaiser's lab (Dept. of Biology, MIT) is focused on engineering S. cerevisiae as a tool for ethanol production from diverse cellulose-rich feedstocks. Our approach involves the characterization of fungal cellulosomes, 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. As the components of the fungal cellulosome have not been completely identified, we are using powerful genomic tools to identify genes within anaerobic fungi that are upregulated when grown in the presence of different cellulosic feedstocks. We are currently working towards expressing and reconstituting components of the anaerobic fungal 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.