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Leveraging Extreme Thermoacidophily for Archaeal Metabolic Engineering
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Leveraging Extreme Thermoacidophily for Archaeal Metabolic Engineering. |
| 개인저자 | Zeldes, Benjamin. |
| 단체저자명 | North Carolina State University. |
| 발행사항 | [S.l.]: North Carolina State University., 2018. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2018. |
| 형태사항 | 161 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-03B. Dissertation Abstract International |
| ISBN | 9781085644365 |
| 학위논문주기 | Thesis (Ph.D.)--North Carolina State University, 2018. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-03, Section: B.
Advisor: Kelly, Robert |
| 이용제한사항 | This item must not be sold to any third party vendors. |
| 요약 | Recent improvements in molecular genetics tools for extreme thermophiles mean that microbial metabolic engineering is now possible at temperatures in excess of 70째C. Thermophilic organisms have had a dramatic impact in both science and industry based on the utility of their thermostable, thermoactive enzymes. Extreme thermophile metabolic engineering means that more complex bio-transformations involving multi-enzyme pathways are now possible. Among the many promising microorganisms for industrial biotechnology are members of the thermoacidophilic (Topt > 75째C, pHopt < 3) archaeal order Sulfolobales, many of which are chemolithoautotrophs. As such, they contain pathways for acquiring energy from inorganic chemical sources, such as metal ores and elemental sulfur, and a carbon fixation cycle for taking up CO2. Portions of the carbon fixation cycle expressed in another extreme thermophile, Pyrococcus furiosus, have produced the bioplastic precursor 3-hydroxypropionate (3HP), where one-third of the carbon in the final product is derived from CO2. Expression of chemical production pathways within a chemolithoautotrophic species of Sulfolobales would allow for production of carbon chemicals entirely from carbon dioxide, using inorganic chemical energy sources which are plentiful and inexpensive.Metabolic engineering also has the potential to provide insights into aspects of thermophilic metabolism that remain poorly understood. Co-expression of additional enzymes alongside those for carbon fixation in P. furiosus determined that carbonic anhydrase plays an important role in CO2 uptake in the Sulfolobales, and a biotinylating maturation enzyme dramatically improved function of the first enzyme in the cycle. Similar insights into the process of sulfur oxidation in Sulfolobales were obtained by cloning two sulfur oxidation enzymes into Sulfolobus acidocaldarius, a species in which lithoautotrophic sulfur oxidation has been lost. While the sulfur oxygenase reductase (SOR) and thiosulfate quinone oxidoreductase (TQO) had been characterized individually, their co-expression revealed cooperative effects as a full sulfur oxidation pathway. Sulfur was toxic to the strain expressing SOR alone, but adding TQO led to robust growth in the presence of sulfur and significant sulfur oxidation. Transcriptomic analysis revealed that S. acidocaldarius retains mechanisms to detect and respond to the presence of sulfur, but exhibits minimal response to CO2. Future work will focus on a carbon-fixation associated regulatory system, and the new model species for sulfur oxidation, Acidianus brierleyi, for which a genome sequence has just become available, and sulfur-transcriptome data is pending.Continued progress in extreme thermophile metabolic engineering will depend on fully realizing the unique advantages found at high temperatures. One of the most promising is the potential for facilitated purification and continuous removal of a volatile chemical as it is produced by a thermophilic host, termed "bio-reactive distillation" (BRD). Acetone has both the requisite volatility, and value as a chemical product. Moderately thermophilic acetone production has been demonstrated, but BRD requires temperatures in excess of 70째C. Despite a dearth of thermophilic native acetone producers, enzyme candidates were identified, including the first thermophilic acetoacetyl-CoA-transferases to be characterized, and an unusually thermostable enzyme from the mesophile Clostridium acetobutylicum. The isolated subunits of CoA transferase exhibit dramatically different thermostabilities, but in complex alpha protects the more labile beta. Together with a previously characterized thermophilic thiolase, these enzymes function as an in vitro synthetic pathway to produce acetone from acetyl-CoA at 70째C.The work reported here provides improved understanding of chemolithoautotrophic energy and carbon fixation pathways in the Sulfolobales, as well as thermostable enzymatic routes for production of 3HP and acetone. Together with rapidly improving molecular genetics techniques, these results constitute the first steps towards creation of a metabolically engineered Sulfolobus strain for production of volatile bio-based chemicals from inorganic carbon and energy sources. |
| 일반주제명 | Chemical engineering. Microbiology. Genetics. |
| 언어 | 영어 |
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