Metabolic Reprogramming of Escherichia coli W for Efficient Bioconversion of Sugarcane Molasses into Value-Added Biochemicals = 생화학물질의 효율적 생합성을 위한 대장균 W의 당밀 대사 경로 재설계

다국어 초록 (Multilingual Abstract)

The advancement of microbial cell factories has enabled the sustainable production of diverse value-added compounds. However, challenges such as carbon catabolite repression (CCR), overflow metabolism, and pathway-specific metabolic constraints limit the efficiency and scalability of engineered strains, particularly when using complex industrial feedstocks. This thesis presents a modular synthetic biology framework for optimizing Escherichia coli W as a robust production platform using sugarcane molasses—a renewable substrate composed of sucrose, glucose, and fructose.
In Chapter 1, the hierarchical sugar uptake behavior of native E. coli W was eliminated through targeted disruption of the fructose PTS and CCR regulators, alongside enhancement of sucrose catabolism. These modifications enabled simultaneous co-utilization of mixed sugars, improving fermentation kinetics and biomass formation.
In Chapter 2, this chassis was applied to the biosynthesis of four industrially relevant products—3-hydroxypropionic acid (3-HP), lycopene, 2,3-butanediol (2,3-BDO), and polyhydroxybutyrate (PHB)—each representing distinct metabolic demands. Systematic performance comparisons across engineered strains revealed product-specific interactions with carbon flux and redox balance.
Chapter 3 focuses on controlling acetate overflow metabolism through deletion of poxB and overexpression of acs, individually and in combination. These interventions were evaluated across all production strains to assess their impact on sugar utilization, byproduct formation, and product titers. The results demonstrate that poxB deletion broadly improved performance for 3-HP, lycopene, and 2,3-BDO, while PHB synthesis was favored by acetate re-assimilation through acs. Dual modification frequently imposed metabolic stress, highlighting the need for pathway-specific flux tuning.
Overall, this study establishes a versatile and carbon-efficient E. coli platform tailored for high-performance bioproduction from mixed sugar substrates. The findings contribute to systems metabolic engineering and provide design principles for future strain optimization in industrial biotechnology.

국문 초록 (Abstract)

본 논문은 사탕수수 당밀을 탄소원으로 활용하여 고부가가치 화합물을 효율적으로 생산할 수 있는 견고한 대장균 생산 플랫폼을 구축하고, 이를 대사공학적으로 최적화한 내용을 다루고 있다. 대장균 W 균주는 높은 자당 대사 능력을 지니고 있으나, 탄소 이화작용의 계층적 흡수 및 초과 탄소 흐름에 의한 아세트산 축적 문제가 있어 산업적 활용에 제약이 있었다.
2장에서 본 연구는 먼저 대장균 W의 탄소 계층제어를 해제하고, 포도당, 과당, 자당을 동시에 효율적으로 소비할 수 있는 균주들을 구축하였다. 이를 위해 과당 PTS 시스템의 제거, 합성 자당 수송 및 가수분해 효소의 도입, 그리고 포도당 수송 조절 인자의 변형을 통해 혼합당 동시 이용을 실현하였다.
3장에서는 이러한 균주들을 기반으로 3-하이드록시프로피온산, 라이코펜, 2,3-부탄디올, 폴리하이드록시부티레이트 등 네 가지 고부가가치 화합물을 생산하였다. 각 대사경로는 서로 다른 탄소 및 보조인자 요구성을 가지며, 이를 통해 탄소 흐름, 레독스 균형, 생장 속도, 부산물 축적 등 다양한 생리학적 지표를 종합적으로 분석하였다.
4장에서는 생산 과정에서 공통적으로 관찰된 아세트산 축적 문제를 해결하기 위해 pyruvate oxidase 유전자의 제거와 acetyl-CoA synthetase 유전자의 과발현 전략을 개별 또는 병합하여 적용하였다. 그 결과, poxB 제거는 3-HP, 라이코펜, 2,3-BDO 생산에 긍정적인 영향을 주었으나, PHB의 경우 acetyl-CoA 공급 저해로 인해 생산이 감소하였다. 반면, acs 과발현은 PHB 생산에 효과적이었으며, 두 유전자의 병합 조작은 일부 균주에서 대사 스트레스를 유발하여 생산성과 생장 저하를 초래하였다.
본 연구는 대사 특성에 맞춘 탄소 흐름 조절이 생산성 향상에 필수적임을 강조하며, 복합당 기질을 활용한 친환경 바이오화학물 생산을 위한 대장균 기반 플랫폼 구축에 중요한 기반을 제시한다. 본 논문에서 개발한 모듈형 균주들은 다양한 생합성 경로 및 산업 폐자원 기질로의 확장이 가능하여 차세대 지속가능한 생물공정 기술의 핵심 도구로 활용될 수 있다.

목차 (Table of Contents)

• Table of Contents
• Abstract i
• List of Tables vi
• List of Figures vii
• Chapter 1. Introduction 1
• Chapter 2. Platform Construction ６
• 2.1. Abstract 7
• 2.2. Introduction 9
• 2.3. Materials and Methods 11
• 2.3.1. Bacterial strains, plasmids, and reagents 11
• 2.3.2. Construction of the synthetic expression cassettes 17
• 2.3.3. Media and culture conditions 35
• 2.3.4. Metabolite quantification 35
• 2.4. Results and Discussion 37
• 2.4.1. Enhancing sucrose consumption rate 37
• 2.4.2. Improving glucose-fructose co-utilization 46
• 2.5. Conclusion 54
• Chapter 3. Platform Development - Production of Value-Added Metabolites 55
• 3.1. Abstract 56
• 3.2. Introduction 57
• 3.3. Materials and Methods 60
• 3.3.1. Bacterial strains, plasmids, and reagents 60
• 3.3.2. Media and culture conditions 66
• 3.3.3. Metabolite quantification 66
• 3.3.4. Fermentation conditions 67
• 3.4. Results and Discussion 68
• 3.4.1. 3-Hydroxypropionic acid production 68
• 3.4.1.1. Plasmid construction 68
• 3.4.1.2. Summary of 3-HP production and physiological outcomes 70
• 3.4.1.3. Time-course analysis of substrate utilization and product formation 72
• 3.4.1.4. Sugar-specific utilization: sucrose, glucose, and fructose dynamics 74
• 3.4.1.5. gapA control 76
• 3.4.2. Lycopene production 83
• 3.4.2.1. Plasmid construction 83
• 3.4.2.2. Summary of lycopene production and physiological outcomes 85
• 3.4.2.3. Time-course profiles of lycopene and overflow metabolites 88
• 3.4.2.4. Substrate consumption and sugar utilization dynamics 90
• 3.4.3. 2,3-Butanedial production 92
• 3.4.3.1. Plasmid construction 92
• 3.4.3.2. Summary of 2,3-BDO production and overflow profiles 94
• 3.4.3.3. Time-course of 2,3-BDO production and acetate overflow 97
• 3.4.3.4. Sugar utilization patterns and carbon uptake strategy 99
• 3.4.4. Poly(3-hydroxybutyrate) production 100
• 3.4.4.1. Plasmid construction 100
• 3.4.4.2. Summary of PHB production and quantitative evaluation 102
• 3.4.4.3. Time-course PHB accumulation and acetate overflow 106
• 3.4.4.4. Sugar utilization and flux allocation toward PHB 108
• 3.4.5. Discussion 110
• 3.5. Conclusion 113
• Chapter 4. Pathway-Specific Optimization - Acetate Flux Engineering 115
• 4.1. Abstract 116
• 4.2. Introduction 118
• 4.3. Materials and Methods 120
• 4.3.1. Bacterial strains, plasmids, and reagents 120
• 4.3.2. Media and culture conditions 130
• 4.3.3. Metabolite quantification 130
• 4.3.4. Fermentation conditions 131
• 4.4. Results and Discussion 132
• 4.4.1. Construction of acetate flux control strains via poxB deletion and acs overexpression 132
• 4.4.2. Effects of acetate flux control on 3-HP production 134
• 4.4.3. Effects of acetate flux control on lycopene production 150
• 4.4.4. Effects of acetate flux control on 2,3-BDO production 164
• 4.4.5. Effects of acetate flux control on PHB production 179
• 4.4.4. Discussion 191
• 4.5. Conclusion 194
• Chapter 5. Overall conclusion and further suggestions 196
• References 201
• List of publications 209
• Abstract in Korean 210