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Functional Genomics Approach to Identify New Determinants of Butanol Production in Clostridium beijerinckii NCIMB 8052
Functional Genomics Approach to Identify New Determinants of Butanol Production in Clostridium beijerinckii NCIMB 8052
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20260202103655
- ISBN
- 9798314889909
- DDC
- 574
- 서명/저자
- Functional Genomics Approach to Identify New Determinants of Butanol Production in Clostridium beijerinckii NCIMB 8052
- 발행사항
- [Sl] : The Ohio State University, 2023
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2023
- 형태사항
- 501 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 86-11, Section: B.
- 주기사항
- Advisor: Ezeji, Thaddeus C.
- 학위논문주기
- Thesis (Ph.D.)--The Ohio State University, 2023.
- 초록/해제
- 요약The finite nature of natural gas, in addition to environmental and health issues arising from the burning of fossil fuels have propelled increased interest in the development of renewable and clean alternative energy sources. Biofuels, and specifically, biobutanol production through ABE fermentation is a promising means of achieving the goal of replacing fossil fuels with a renewable energy source in the short term. However, low yield and productivity of butanol producing fermentation workhorse, Clostridium beijerinckii, is a major impediment to commercialization of biobutanol production. Extensive metabolic engineering efforts have been made to generate an industrially applicable strain; however, success has been limited. The large genome, complex metabolic and regulatory networks, and the abundance of hypothetical proteins in C. beijerinckii, in addition to the limited success obtained with metabolic engineering efforts, indicate there could be new unidentified butanol production determinants in C. beijerinckii. Therefore, this study explored the use of a ribozyme-based approach as a reverse genetics tool to identify unknown genetic determinants of butanol production in C. beijerinckii. Using Gibson assembly, the Escherichia coli-Clostridium shuttle plasmid carrying the E. coli RNase P (M1 RNA) sequence and synthesized external guide sequences (GS) were assembled, to generate a plasmid library of customized M1-based ribozyme-guide sequence (GS) constructs. The M1GS library was generated to target 31 genes that code for hypothetical proteins, which are among the 100 most expressed genes during the transition from acidogenesis to solventogenesis in C. beijerinckii. Generated customized M1GS plasmid library was used to transform C. beijerinckii to generate individual transformants with targeted mRNA degradation. With selective (antibiotics) medium, high performance liquid chromatography and spectrophotometric assays, transformants with various growth and solvent production profiles were isolated and catalogued. Specifically, 23 transformants exhibited butanol and ABE production profiles of interest, producing concentrations that were ⁓ 4 - 126% of the butanol and ⁓ 7 - 142% of ABE produced by the empty plasmid control were obtained. The second objective of this study was to functionally characterize genes encoding hypothetical proteins identified through the knockdown experiments to be involved in growth and ABE production in C. beijerinckii, using direct knockout strategies. Using CRISPR-Cas9 and allele exchange for homologous recombination, Cbei_1177 gene was deleted and overexpressed in C. beijerinckii. Deletion of Cbei_1177 led to ~ 19%, 23%, and 35% increase in growth, butanol, and ABE production, respectively. Surprisingly, overexpression of Cbei_1177 resulted in ~35%, 22%, and 22% reduction in growth, increase in butanol, and ABE production, respectively. Notably, Cbei_1177 deletion resulted in a ⁓ 97% and 17% increase in volume of hydrogen and total gas produced compared to the wildtype, respectively, while overexpression of Cbei_1177 resulted in 5% increase and 19% reduction in volume of hydrogen and total gas produced, respectively, compared to the wildtype. Furthermore, using CRISPR-Cas9 and allele exchange for homologous recombination, Cbei_4587 deletion strain was generated. Interestingly, deletion of Cbei_4587 in C. beijerinckii led to ~34% increase in growth. However, butanol and ABE production decreased by 51% and 45% respectively. Objective 3 explored the application of lantibiotics immunity genes to confer butanol tolerance in C. beijerinckii and improve butanol production. Using allele exchange for homologous recombination, lantibiotics immunity genes, Cbei_3326 (putative lanI) and Cbei_3325-3324-3325 (lanFEG), were overexpressed in C. beijerinckii. Overexpression of lanFEG in C. beijerinckii led to ~31% and 53% increase in butanol and ABE production, respectively, while growth reduced by 16% compared to the wildtype. However, overexpression of lanI resulted in 13% and 22% increase in growth and ABE production, while butanol production remained unchanged.Taken together, this study uncovered previously unknown genetic determinants affecting growth and butanol production in C. beijerinckii, which can be further explored to improve biobutanol production.
- 일반주제명
- Biochemistry
- 일반주제명
- Alternative energy
- 일반주제명
- Molecular biology
- 일반주제명
- Animal sciences
- 일반주제명
- Microbiology
- 키워드
- Reverse genetics
- 키워드
- Biofuels
- 기타저자
- The Ohio State University Animal Sciences
- 기본자료저록
- Dissertations Abstracts International. 86-11B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■020 ▼a9798314889909
■035 ▼a(MiAaPQ)AAI32111862
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a574
■1001 ▼aOlorunsogbon, Tinuola A.
■24510▼aFunctional Genomics Approach to Identify New Determinants of Butanol Production in Clostridium beijerinckii NCIMB 8052
■260 ▼a[Sl]▼bThe Ohio State University▼c2023
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2023
■300 ▼a501 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 86-11, Section: B.
■500 ▼aAdvisor: Ezeji, Thaddeus C.
■5021 ▼aThesis (Ph.D.)--The Ohio State University, 2023.
■520 ▼aThe finite nature of natural gas, in addition to environmental and health issues arising from the burning of fossil fuels have propelled increased interest in the development of renewable and clean alternative energy sources. Biofuels, and specifically, biobutanol production through ABE fermentation is a promising means of achieving the goal of replacing fossil fuels with a renewable energy source in the short term. However, low yield and productivity of butanol producing fermentation workhorse, Clostridium beijerinckii, is a major impediment to commercialization of biobutanol production. Extensive metabolic engineering efforts have been made to generate an industrially applicable strain; however, success has been limited. The large genome, complex metabolic and regulatory networks, and the abundance of hypothetical proteins in C. beijerinckii, in addition to the limited success obtained with metabolic engineering efforts, indicate there could be new unidentified butanol production determinants in C. beijerinckii. Therefore, this study explored the use of a ribozyme-based approach as a reverse genetics tool to identify unknown genetic determinants of butanol production in C. beijerinckii. Using Gibson assembly, the Escherichia coli-Clostridium shuttle plasmid carrying the E. coli RNase P (M1 RNA) sequence and synthesized external guide sequences (GS) were assembled, to generate a plasmid library of customized M1-based ribozyme-guide sequence (GS) constructs. The M1GS library was generated to target 31 genes that code for hypothetical proteins, which are among the 100 most expressed genes during the transition from acidogenesis to solventogenesis in C. beijerinckii. Generated customized M1GS plasmid library was used to transform C. beijerinckii to generate individual transformants with targeted mRNA degradation. With selective (antibiotics) medium, high performance liquid chromatography and spectrophotometric assays, transformants with various growth and solvent production profiles were isolated and catalogued. Specifically, 23 transformants exhibited butanol and ABE production profiles of interest, producing concentrations that were ⁓ 4 - 126% of the butanol and ⁓ 7 - 142% of ABE produced by the empty plasmid control were obtained. The second objective of this study was to functionally characterize genes encoding hypothetical proteins identified through the knockdown experiments to be involved in growth and ABE production in C. beijerinckii, using direct knockout strategies. Using CRISPR-Cas9 and allele exchange for homologous recombination, Cbei_1177 gene was deleted and overexpressed in C. beijerinckii. Deletion of Cbei_1177 led to ~ 19%, 23%, and 35% increase in growth, butanol, and ABE production, respectively. Surprisingly, overexpression of Cbei_1177 resulted in ~35%, 22%, and 22% reduction in growth, increase in butanol, and ABE production, respectively. Notably, Cbei_1177 deletion resulted in a ⁓ 97% and 17% increase in volume of hydrogen and total gas produced compared to the wildtype, respectively, while overexpression of Cbei_1177 resulted in 5% increase and 19% reduction in volume of hydrogen and total gas produced, respectively, compared to the wildtype. Furthermore, using CRISPR-Cas9 and allele exchange for homologous recombination, Cbei_4587 deletion strain was generated. Interestingly, deletion of Cbei_4587 in C. beijerinckii led to ~34% increase in growth. However, butanol and ABE production decreased by 51% and 45% respectively. Objective 3 explored the application of lantibiotics immunity genes to confer butanol tolerance in C. beijerinckii and improve butanol production. Using allele exchange for homologous recombination, lantibiotics immunity genes, Cbei_3326 (putative lanI) and Cbei_3325-3324-3325 (lanFEG), were overexpressed in C. beijerinckii. Overexpression of lanFEG in C. beijerinckii led to ~31% and 53% increase in butanol and ABE production, respectively, while growth reduced by 16% compared to the wildtype. However, overexpression of lanI resulted in 13% and 22% increase in growth and ABE production, while butanol production remained unchanged.Taken together, this study uncovered previously unknown genetic determinants affecting growth and butanol production in C. beijerinckii, which can be further explored to improve biobutanol production.
■590 ▼aSchool code: 0168.
■650 4▼aBiochemistry
■650 4▼aAlternative energy
■650 4▼aMolecular biology
■650 4▼aAnimal sciences
■650 4▼aMicrobiology
■653 ▼aBiobutanol production
■653 ▼aReverse genetics
■653 ▼aBiofuels
■653 ▼aMetabolic engineering
■653 ▼aClostridium beijerinckii
■690 ▼a0475
■690 ▼a0487
■690 ▼a0363
■690 ▼a0307
■690 ▼a0410
■71020▼aThe Ohio State University▼bAnimal Sciences.
■7730 ▼tDissertations Abstracts International▼g86-11B.
■790 ▼a0168
■791 ▼aPh.D.
■792 ▼a2023
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17358177▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
