Enhancing L-Arabinose Assimilation by Saccharomyces cerevisiae Using Combinatorial Pathways and Regulon Engineering
Hart, Taylor K.
2019
- Extending the substrate range of Saccharomyces cerevisiae has been an area of interest since the conception of metabolic engineering due to the ease with which this yeast can be genetically manipulated. Although its preferred substrate is glucose, extensive work has been previously carried out in order to allow S. cerevisiae to process lactose, L-malate, melibiose, D-xylose, cellobiose, and ... read moreL-arabinose. The general strategy involves isolating the metabolic pathways for utilization these carbon sources from organisms that can naturally metabolize them and expressing them in S. cerevisiae. Such efforts have yielded strains that are able to utilize non-native substrates for growth and fermentation to ethanol. For the combinatorial pathway engineering, we built six permutations of three metabolic genes (araA, araB, araD) and three native yeast galactose-responsive promoters and assembled them into expression vectors. We then built three of the constructs via Gibson Assembly or yeast assembly. These constructs, along with a template construct supplied and created by Dr. Vikas Trivedi in the Nair laboratory were ligated into a parent plasmid pRS416 (Appendix D) and transformed into S. cerevisiae. A growth study was performed on these transformed yeast cells using media with arabinose as the sole carbon source. The results of this study were used to determine the optimal among the six permutations for growth of yeast on this non-native substrate. When analyzed, the results indicate that strain TH4, containing the template plasmid supplied by the lab, has the most optimized construction, with a growth rate of 0.20 h-1 and final OD of 4.16. The next best strain, TH3, was assembled in this study and was contained in the engineered plasmid pTH-3, with a growth rate of 0.18 h-1 and a final OD600 of 3.02.read less
- ID:
- 2801pv36r
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- TARC Citation Guide EndNote
