Metabolic engineering has enabled the advancement of biotechnology over the past few decades through the use of cells as biochemical factories. Cellular factories have now provided many new safe and sustainable routes to fuels, pharmaceuticals, polymers, and specialty chemicals. Many of these successes have been achieved using the yeast Saccharomyces cerevisiae, which has been used and shaped by humans for millennia for the production of food and drink. Consequently, S. cerevisiae is one of the most studied eukaryotic organisms in existence, and has established genetic tools for engineering efforts.
However, despite the many achievements in metabolic engineering of S. cerevisiae, it is still significantly more difficult to engineer than its prokaryotic counterpart, Escherichia coli. As a result, there is an unmet need to further develop genetic tools in yeast and to do so in the context of metabolic pathway engineering. The work presented here addresses this need through the study and engineering of heterologous gene expression. First, a new biosynthetic pathway is engineered for the production of muconic acid in yeast. Muconic acid is a dicarboxylic acid that can serve as a platform chemical for the production of several bio-polymers. The final muconic acid producing strain was developed through significant metabolic engineering efforts and reached a titer of nearly 141 mg/L muconic acid, a value nearly 24-fold higher than the initial strain. Second, a new method of engineering promoters is presented that allows for increased expression of native promoters and the de novo design of synthetic promoters. The highest expression synthetic promoter is within the top 6th percentile of native yeast promoters. Third, a study of native and synthetic terminators for heterologous gene expression is completed for the first time. This study demonstrates that terminators can tune heterologous expression by as much as an order of magnitude. Fourth, a comparative study of plasmid components dissects the different contributions to plasmid burden, copy number, and gene expression level. Collectively, this work represents a significant step forward in the metabolic engineering of yeast through the establishment of a new pathway and the study and engineering of new tools for heterologous gene expression. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/30469 |
| Date | 28 August 2015 |
| Creators | Curran, Kathleen Anne |
| Contributors | Alper, Hal S. |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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