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Investigating Metabolic Limitations to Cellulosic Ethanol Production in Thermophilic Bacteria
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Investigating Metabolic Limitations to Cellulosic Ethanol Production in Thermophilic Bacteria. |
| 개인저자 | Cui, Jingxuan. |
| 단체저자명 | Dartmouth College. Biology. |
| 발행사항 | [S.l.]: Dartmouth College., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 238 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-06B. Dissertation Abstract International |
| ISBN | 9781392727232 |
| 학위논문주기 | Thesis (Ph.D.)--Dartmouth College, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-06, Section: B.
Advisor: Lynd, Lee R. |
| 이용제한사항 | This item must not be sold to any third party vendors. |
| 요약 | The thermophilic anaerobic bacteria Thermoanaerobacterium saccharolyticum and Clostridium thermocellum are good candidates for lignocellulosic ethanol production via consolidated bioprocessing (CBP). T. saccharolyticum has been successfully engineered to produce ethanol at high titer (70 g/L). The maximum ethanol titer of engineered strains of C. thermocellum is only 25 g/L. For commercialization, the final ethanol titer needs to be further improved to > 40 g/L. In this thesis, we used several strategies to study metabolic bottlenecks that potentially limit ethanol production in C. thermocellum. First, we focused on the enzymes responsible for the part of the ethanol production pathway from pyruvate to ethanol. In T. saccharolyticum, we replaced all of the genes encoding proteins in this pathway with their homologs from C. thermocellum and examined what combination of gene replacements restricted high ethanol titer. We found that a pathway consisting of Ct_nfnAB, Ct_fd, Ct_adhE and Ts_pforA was sufficient to support ethanol titer greater than 50 g/L, however replacement of Ts_pforA by Ct_pfor1 dramatically decreased the maximum ethanol titer to 14 g/L. We then demonstrated that the reason for reduced ethanol production is that the Ct_pfor1 is inhibited by accumulation of ethanol and NADH, while Ts_pforA is not. We next expanded our studies to the entire cellobiose to ethanol pathway by developing a cell-free extract reaction (CFER) system that recapitulates in vivo ethanol production. We demonstrated that the CFER was able to produce ethanol to a similar yield compared to whole cells but with a much slower rate. Metabolomics analysis revealed potential rate limiting enzymes downstream of F6P. Through supplementing the CFER with exogenous enzymes, we identified the phosphoenolpyruvate to pyruvate and acetaldehyde to ethanol reactions as bottlenecks. These results revealed metabolic bottlenecks that limit ethanol production in C. thermocellum and provide insights on future engineering targets to create a strain of C. thermocellum capable of producing ethanol at a titer higher than 40 g/L. |
| 일반주제명 | Microbiology. Molecular biology. Bioengineering. |
| 언어 | 영어 |
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