Tools, strategies, and applications of synthetic biology in Saccharomyces cerevisiae

Synthetic biology is founded on the idea that cells are living machines that execute genetically encoded programs, and that we as engineers can reprogram them to perform new functions. Unlike man-made machines that are designed from the ground up, cells have been shaped and molded by evolution, and this makes them much more difficult to engineer. As synthetic biology has grown and matured, the field has shifted from focusing primarily on simpler bacterial chassis to engineering more complex and more powerful eukaryotic hosts, especially the budding yeast, Saccharomyces cerevisiae. Here we present technologies and strategies for efficiently engineering yeast, with an emphasis on metabolic engineering. First, we describe a practical framework for designing and constructing DNA for expression in yeast. This framework standardizes--and as a consequence, accelerates--the process of building new strains, enabling more rapid iteration and experimentation. We then develop a strategy for optimizing metabolic pathways by assembling combinatorial libraries that simultaneously titrate the expression of many genes. We identify strains with improved pathway flux for violacein biosynthesis and xylose utilization using mathematical modeling and selection, respectively. Finally, we attempt to alter the specificity of a native hexose transporter to exclusively import xylose without inhibition by glucose. In summary, this work is a combination of developing fundamental tools for engineering yeast and the application of those tools for lignocellulosic biomass fermentation