Expanding the product portfolio of Yarrowia lipolytica through metabolic engineering and synthetic biology tool development

Yarrowia lipolytica is a potent microbial cell factory for metabolic engineering that will enable the sustainable production of chemicals beyond first generation biofuels and polymer precursors. As an alternative host, there are many limitations yet to be resolved that require much more intensive research. Yet, this oleaginous yeast has already proven superior to its conventional yeast counterpart Saccharomyces cerevisiae for the production of a variety of different products most notably including lipids. The work presented here offers two metabolic engineering stories demonstrating the potential of Y. lipolytica to produce value-added chemicals at high titers. Additionally, seeking to alleviate bottlenecks in the engineering process, this work presents details of an optimized and streamlined protocol for transformation as well as a strategy for tuning gene expression via synthetic terminators. First, the focus was set on engineering a high-lipid strain for cyclopropane fatty acid production. This work leveraged a strain of Y. lipolytica that had been previously metabolically engineered to produce high levels of fatty acids. Cyclopropane fatty acids (a non-native molecule to Y. lipolytica) serve as a simple way to improve biodiesel properties and are additionally useful in lubricants. Through engineering, 3 g/L production was enabled. In a second study, the inherent flux towards lipids was hijacked and redirected towards polyketides. Specifically, extensive metabolic engineering led to production of 36 g/L of triacetic acid lactone, an important biorenewable precursor molecule. Third, the development and optimization of an electroporation transformation protocol alleviated a critical bottleneck in the engineering process, allowing for highly efficient incorporation of exogenous DNA into Y. lipolytica. This protocol streamlines transformations and does not require the addition of carrier DNA. Fourth, synthetic terminators originally developed for S. cerevisiae were tested in Y. lipolytica enabling improved tuning of expression. Incorporation of these synthetic terminators allowed for a 70% increase in gene expression over a commonly used terminator. Collectively, these chapters demonstrate the potential of Y. lipolytica as a host for metabolic engineering through study of tools and investigation of native pathways that can be redirected towards non-native productsChemical Engineerin