Protein engineering and bioprospecting for selective hydroxyacid production in engineered Escherichia coli
| Autor: | Tarasova, Yekaterina |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: | |
| Druh dokumentu: | Diplomová práce |
| Popis: | Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2016. Cataloged from PDF version of thesis. Includes bibliographical references (pages 103-110). Engineering of microbes can allow for the sustainable production of a variety of useful chemical compounds upon which we rely in our everyday lives. The many advantages of metabolic engineering include the use of renewable resources, mild reaction conditions, such as ambient pressure, pH and temperature, as well as the avoidance of toxic chemicals in the conversion process. The 3-hydroxyacid (3HA) pathway, also known as coenzyme-A (CoA) dependent chain elongation, can allow for the synthesis of dozens of diverse classes of chemicals. However, this pathway suffers from byproduct formation, which is due to the promiscuous activities of the pathway enzymes. On one hand, this enables the synthesis of such a diverse set of chemicals, but on the other, prohibits commercialization due to high downstream separation costs and low yields of desired longer chain products. The goal of this thesis was to improve this pathway though engineering and bioprospecting of more selective pathway enzymes. Engineering efforts were focused on the thiolase enzyme, which catalyzes the first step of the 3HA pathway, while bioprospecting efforts were focused on the enzyme immediately downstream of the thiolase - the 3-ketoacyl-CoA reductase. Our computationally guided protein engineering efforts identified a thiolase point mutant which allowed for more selective synthesis of longer chain 3HAs. Bioinformatics approaches were used to identify a set of ten diverse 3-ketoacyl-CoA reductases which were then screened and characterized within the context of the 3HA pathway. Two of the reductases led to increased selectivity for longer chain products. Combinations of the most selective thiolase and reductase enzyme pairs almost eliminated C4 byproduct formation by the 3HA pathway. In all, the thiolase and 3-ketoacyl-CoA reductase enzyme variants identified in this work should be applicable to other heterologous pathways where formation of short chain products is undesired, bringing such pathways closer to commercialization. by Yekaterina Tarasova. Ph. D. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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