Publications

Detailed Information

Substrate channeling and promoter engineering for the production of 2,3-butanediol and cellulosic ethanol in Saccharomyces cerevisiae : Saccharomyces cerevisiae에서2,3-부탄다이올과 섬유소 유래 에탄올 생산을 위한 기질 채널링 및 프로모터의 개발

DC Field Value Language
dc.contributor.advisor한지숙-
dc.contributor.authorSujin Kim-
dc.date.accessioned2017-07-13T08:41:25Z-
dc.date.available2017-07-13T08:41:25Z-
dc.date.issued2015-08-
dc.identifier.other000000066892-
dc.identifier.urihttps://hdl.handle.net/10371/119757-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2015. 8. 한지숙.-
dc.description.abstractSaccharomyces cerevisiae는 연구가 활발히 진행된 진핵 모델 시스템으로서, 연료 및 화학 물질 생산을 위한 미생물 세포 공장으로써의 높은 가능성을 지닌다. 본 연구에서는, 다양한 대사 공학적 전략을 섬유소 유래 에탄올의 생산 및 2,3-부탄다이올의 생산에 적용하여 그 가능성을 확인하였다.
첫 번째로, 통합적 단일 생물 공정용 효모 균주를 개발하기 위하여 Clostridium thermocellum의 셀룰로좀 구조를 S. cerevisiae 세포 표면에 모사하였다. 셀룰로좀은 구조 단백질인 scaffoldin을 구성하는 cohesin 도메인과 효소에 포함되어 있는 dockerin 도메인의 친화력 높은 상호 결합에 의해 이루어진다. Scaffoldin (mini CipA)를 표면에 고정화하는 세포와 CelA, CBHII, BGLI을 분비하는 세 종류의 세포로 세포 컨소시엄을 구성하였고, 셀룰로좀의 기능 및 에탄올 생산을 극대화 할 수 있는 최적 비율을 탐색하였다. 그 결과, mini CipA:CelA:CBHII:BGL1 비율이2:3:3:0.53일 때 가장 효율적이었으며, 94시간 발효 후 1.80 g/L 의 에탄올을 생산하였다. 이는 동일한 비율로 구성된 컨소시엄 (1.48 g/L) 에 비하여 약 20% 향상된 결과이다.
두 번째로, 높은 친화력을 지니는cohesin-dockerin상호 결합을 이용하여 기질 채널링 모듈을 설계하였다. 기질 채널링이란 효소 작용으로 생성된 중간 대사 물질이 외부로 확산되지 않고 다음 효소로 전달되는 현상으로, 반응 속도를 향상시킬 수 있는 방법이다. 2, 3, 7개의 cohesin 도메인으로 구성된 합성 scaffold를 구축하였고, dockerin-융합 단백질과의 상호 결합을 pull-down 기법과 이분자 형광 상보 기법을 이용하여 확인하였다. Dockerin 이 융합된 AlsS, AlsD, Bdh1을 사용하여2,3-부탄다이올 생산에 적용하였으며, 그 결과 scaffold 를 구성하는 cohesin 도메인의 개수가 증가함에 따라 2,3-부탄다이올의 생산이 증가함을 확인하였다.
세 번째로, 물질대사 분기점에서의 기질 채널링의 효과를 확인하기 위하여 피루브산의 물질대사에 기질 채널링을 도입하였다. Cohesin-dockerin상호 결합을 사용하여 피루브산-전환 효소가 피루브산 키나아제 (Pyk1)에 결합 할 수 있게 하였다. 피루브산 키나아제는 해당 과정의 마지막 단계에서 포스포에놀피루브산을 피루브산으로 전환한다. 피루브산 채널링의 기반 균주로서 PYK1-Coh-Myc을 제작하였고, dockerin-융합 단백질과 cohesin-융합 Pyk1의 상호 결합을 면역 침강 기법을 이용하여 확인하였다. 젖산 생산과 2,3-부탄다이올 생산에 적용한 결과, 목적 대사 산물의 생산이 증가하였고 에탄올의 생산은 감소하였다.
네 번째로, Aro80 전사 조절 인자의 결합 위치를 기반으로 방향족 아미노산-유도성 프로모터를 제작하였다. Aro80 결합 위치의 수, 플라스미드 카피 수, 트립토판 처리 농도를 조절함으로써 다양한 발현 세기를 프로모터를 얻을 수 있었다. ARO9 코어 프로모터에 4개의 Aro80 결합 위치를 지니는 합성 프로모터인U4CARO9 를 AlsS와 AlsD의 발현에 도입한 결과, 트립토판 처리 농도에 따라 아세토인 생성이 증가함을 확인하였다. 또한 γ-아미노부틸릭산-유도성 UGA4 프로모터 또한 유도 물질의 농도에 의존적으로 전사가 유도됨을 확인하였다.
마지막으로, 경쟁 경로의 제거 및 산화환원 보조인자의 불균형 해소를 통해 S. cerevisiae에서의 2,3-부탄다이올 생산을 단계적으로 향상시켰다. B. subtilis 유래의2,3-부탄다이올 생합성 경로를 도입하고 에탄올과 글리세롤 생산 경로를 제거함으로써 포도당이2,3-부탄다이올로 성공적으로 전환됨을 확인하였다. 또한, L. lactis 유래의NADH 산화효소 (noxE)를 과발현시킴으로써 2,3-부탄다이올 생산 경로의 도입으로 유발된 산화-환원 보조인자의 불균형을 해소시켰다. AlsS, AlsD, Bdh1, NoxE 가 단일 벡터에서 과발현된 최종 균주 adh1-5Δgpd1Δgpd2Δ 는 최종적으로 72.9 g/L의 2,3-부탄다이올을 생산하였으며, 높은 수율 (0.41 g/g 포도당) 과 생산성 (1.43 g/(L·h))을 나타내었다.
-
dc.description.abstractSaccharomyces cerevisiae is a well-studied eukaryotic model system with great potential as microbial cell factories for the production of fuels and chemicals. In this dissertation, several strategies were developed and applied to produce cellulosic ethanol and 2,3-butanediol in S. cerevisiae.
Firstly, a cellulolytic yeast consortium for consolidated bioprocessing (CBP) was developed based on the surface display of cellulosome structure, mimicking Clostridium thermocellum. The assembly of cellulosome is mediated through high-affinity interaction between cohesin domains in structural scaffoldin and dockerin domains in enzymes. The cellulosome activity and ethanol production of a yeast consortium were optimized by controlling the combination ratio among the four yeast strains capable of either displaying a scaffoldin (mini CipA) or secreting one of the three types of cellulases. As a result, a mixture of cells with the optimized mini CipA:CelA:CBHII:BGLI ratio of 2:3:3:0.53 produced 1.80 g/L ethanol after 94 h, indicating about 20% increase compared with a consortium composed of an equal amount of each cell type (1.48 g/L).
Secondly, substrate channeling modules were designed based on high affinity interaction between cohesin and dockerin domains. Substrate channeling is a process of transferring an intermediate from one enzyme to the next enzyme without diffusion into the bulk phase, thereby leading to an enhanced reaction rate. Synthetic scaffolds containing two, three, or seven cohesin domains were constructed, and the assembly of dockerin-tagged proteins onto the scaffolds was confirmed by pull-down assay and bimolecular fluorescent complementation (BiFC) assay. This system was applied to produce 2,3-butanediol by using dockerin-tagged AlsS, AlsD, and Bdh1 enzymes, resulting in a gradual increase in 2,3-butanediol production depending on the number of cohesin domains in the scaffold.
Thirdly, the effect of substrate channeling was further investigated at a metabolic branch point, focusing on pyruvate metabolism in S. cerevisiae. The cohesin-dockerin interaction was applied to recruit pyruvate-converting enzymes to a pyruvate kinase (Pyk1), which catalyzes the conversion of phosphoenolpyruvate to pyruvate. As a platform strain for pyruvate channeling, PYK1-Coh-Myc strain was constructed, and the assembly of dockerin-tagged enzymes to cohesin-tagged Pyk1 was confirmed by co-immunoprecipitation. In the case of both lactate production and 2,3-butanediol production, pyruvate flux toward the target products was significantly enhanced, coinciding with a decrease in ethanol production.
Fourthly, promoters inducible by aromatic amino acids were constructed based on the binding sites of Aro80 transcription factor. A dynamic range of tryptophan-inducible promoter strengths can be obtained by modulating the number of Aro80 binding sites, plasmid copy numbers, and tryptophan concentrations. The synthetic U4CARO9 promoter, which is composed of four repeats of Aro80 binding half site (CCG) and ARO9 core promoter element, was applied to express AlsS and AlsD for acetoin production, resulting in a gradual increase in acetoin titers depending on tryptophan concentrations. Furthermore, it has been demonstrated that γ-aminobutyrate (GABA)-inducible UGA4 promoter can also be used in metabolic engineering as a dose-dependent inducible promoter.
Lastly, 2,3-butanediol production in S. cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered strain (adh1-5Δgpd1Δgpd2Δ) overexpressing AlsS, AlsD, Bdh1, and NoxE from a single multigene-expression vector, produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.
-
dc.description.tableofcontentsAbstract i
Contents v
List of Figures ix
List of Tables xii
List of Abbreviations xiii

Chapter 1. Research background and objective 1

Chapter 2. Literature review 5
2.1. Metabolic engineeirng 6
2.1.1. Overview of metabolic engineering 6
2.1.2. Metabolic engineering in S. cerevisiae 7
2.1.3. Substrate channeling 10
2.1.4. Promoter engineering 15
2.2. Cellulosic ethanol production in microorganisms 17
2.2.1. Cellulosic biomass 17
2.2.2. Cellulosic bioethanol production in S. cerevisiae 22
2.3. 2,3-Butanediol production in microorganisms 27
2.3.1. 2,3-Butanediol 27
2.3.2. 2,3-Butanediol production in bacteria 29
2.3.3. 2,3-Butanediol production in S. cerevisiae 32

Chapter 3. Materials and methods 36
3.1. Strains and media 37
3.2. Plasmids 42
3.3. Culture conditions 55
3.3.1. Cellulosic ethanol production using a yeast consortium 55
3.3.2. 2,3-Butanediol production using synthetic scaffold-based substrate channeling 55
3.3.3. Redirection of pyruvate flux through enzyme coupling 55
3.3.4. Promoters inducible by aromatic amino acids and γ-aminobutyrate for metabolic engineering applications 56
3.3.5. Efficient production of 2,3-butanediol by eliminating ethanol and glycerol production and redox rebalancing 56
3.4. β-glucosidase activity assay 57
3.5. Endo/exoglucanase activity assays 57
3.6. In vivo GST-pull down assay 58
3.7. Bimolecular fluorescence complementation analysis 58
3.8. Co-immunoprecipitation 59
3.9. RNA preparation and quantitative reverse transcription PCR 59
3.10. Measurement of EGFP fluorescence intensity 60
3.11. Analytic methods 61

Chapter 4. Cellulosic ethanol production using a yeast consortium displaying a minicellulosome and β-glucosidase 62
4.1. Introduction 63
4.2. Construction of a minicellulosome structure on the yeast surface 66
4.3. Cellulosic ethanol production using the optimized cellulosome 72
4.4. Conclusions 77

Chapter 5. Synthetic scaffold based on a cohesin-dockerin interaction for improved production of 2,3-butanediol in Saccharomyces cerevisiae 81
5.1. Introduction 82
5.2. Construction of substrate channeling modules for substrate channeling in the cytosol of S. cerevisiae 83
5.3. The assembly of dockerin-tagged proteins to the scaffold 83
5.4. Substrate channeling effect in 2,3-butanediol synthesis 85
5.5. Conclusions 92

Chapter 6. Redirection of pyruvate flux through metabolite channeling in Saccharomyces cerevisiae 93
6.1. Introduction 94
6.2. Construction of PYK1-Coh strain as a platform strain for the channeling of pyruvate flux 95
6.3. Identification of the interaction between cohesin-fused Pyk1 and dockerin-fused enzyme by co-immunoprecipitation 96
6.4. Lactate production using the pyruvate channeling system 99
6.5. 2,3-Butanediol production using the pyruvate channeling system 100
6.6. Conclusions 106

Chapter 7. Promoters inducible by aromatic amino acids and γ-aminobutyrate for metabolic engineering applications in Saccharomyces cerevisiae 107
7.1. Introduction 108
7.2. Construction of aromatic amino acids-inducible synthetic promoters 111
7.3. Regulation of tryptophan-induced expression levels by plasmid copy numbers and tryptophan concentrations 117
7.4. Acetoin production by using the U4CARO9 promoter 119
7.5. Application of the GABA-inducible UGA4 promoter to metabolic engineering 122
7.6. Conclusions 124

Chapter 8. Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing 125
8.1. Introduction 126
8.2. Construction of 2,3-butanediol biosynthetic pathway in S. cerevisiae 130
8.3. Disruption of competing pathways to improve 2,3-butanediol production 134
8.4. Recovering redox imbalance by expressing water-forming NADH oxidase NoxE 139
8.5. Fed-batch fermentation for 2,3-butanediol production 142
8.6. Conclusions 145

Chapter 9. Overall discussion and recommendations 149

Bibliography 155
Abstract in Korean 172
-
dc.formatapplication/pdf-
dc.format.extent3388130 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectMetabolic engineering-
dc.subjectSubstrate channeling-
dc.subjectPromoter engineering-
dc.subject2-
dc.subject3-Butanediol-
dc.subjectCellulosic ethanol-
dc.subjectSaccharomyces cerevisiae-
dc.subject.ddc660-
dc.titleSubstrate channeling and promoter engineering for the production of 2,3-butanediol and cellulosic ethanol in Saccharomyces cerevisiae-
dc.title.alternativeSaccharomyces cerevisiae에서2,3-부탄다이올과 섬유소 유래 에탄올 생산을 위한 기질 채널링 및 프로모터의 개발-
dc.typeThesis-
dc.contributor.AlternativeAuthor김수진-
dc.description.degreeDoctor-
dc.citation.pagesxiv, 174-
dc.contributor.affiliation공과대학 화학생물공학부-
dc.date.awarded2015-08-
Appears in Collections:
Files in This Item:

Altmetrics

Item View & Download Count

  • mendeley

Items in S-Space are protected by copyright, with all rights reserved, unless otherwise indicated.

Share