| ABSTRACT | 第5-7页 |
| 摘要 | 第8-16页 |
| 1 INTRODUCTION | 第16-44页 |
| 1.1 Metabolic engineering | 第16-24页 |
| 1.1.1 Definition of metabolic engineering | 第16-17页 |
| 1.1.2 History of metabolic engineering | 第17-18页 |
| 1.1.3 Genome editing | 第18-24页 |
| 1.1.3.1 CRISPR-Cas 9 genome editing | 第18-20页 |
| 1.1.3.2 CRE/LOX P assisted genome editing | 第20-21页 |
| 1.1.3.3 POP-IN/POP-OUT assisted genome editing | 第21-23页 |
| 1.1.3.4 Tandem repeats assisted genome editing | 第23-24页 |
| 1.2 Succinic acid production | 第24-36页 |
| 1.2.1 Enzymes used to enhance succinic acid production | 第24-33页 |
| 1.2.1.1 Phosphoenolpyruvate carboxykinase | 第25-26页 |
| 1.2.1.2 Lactate dehydrogenase | 第26-27页 |
| 1.2.1.3 Pyruvate formate lyase | 第27页 |
| 1.2.1.4 Phosphoglucose isomerase | 第27-28页 |
| 1.2.1.5 Phosphotransferase system | 第28-29页 |
| 1.2.1.6 Phoshotransacetylase-acetate kinase | 第29-30页 |
| 1.2.1.7 Pyruvate oxidase B | 第30页 |
| 1.2.1.8 6-phosphogluconate dehydrogenase | 第30-32页 |
| 1.2.1.9 Phosphoenolpyruvate carboxylase | 第32页 |
| 1.2.1.10 Soluble nucleotide pyridine transhydrogenase | 第32-33页 |
| 1.2.1.11 Muerin Cluster | 第33页 |
| 1.2.2 Succinic acid production from biomass | 第33-34页 |
| 1.2.3 Biomass | 第34-36页 |
| 1.3 Usage of succinic acid | 第36-38页 |
| 1.3.1 Polymer Industry | 第37页 |
| 1.3.2 Pharmaceutical Industry | 第37-38页 |
| 1.3.3 Food, beverage and detergent Industry | 第38页 |
| 1.4 Previous developed succinic acid producing recobinants | 第38-40页 |
| 1.5 Succinic acid production by natural producer | 第40-42页 |
| 1.6 Aims and objectives | 第42-44页 |
| 2 Construction of recombinant Escherichia coli | 第44-52页 |
| 2.1 Targeted modification with Tandem repeat assisted genome editing | 第44-46页 |
| 2.1.1 Transformed pKD46 to target strain | 第44-45页 |
| 2.1.2 Integration of designed fragments into genome | 第45-46页 |
| 2.1.3 Seamless deletion of cat-sacB cassette | 第46页 |
| 2.2 Use of CRISPR-Cas9 system for gene deletion | 第46-48页 |
| 2.2.1 Procedures | 第46-48页 |
| 2.3 Overexpression of Soluble nucleotide pyridine transhydrogenase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase | 第48-49页 |
| 2.4 Chemicals and reagents | 第49-52页 |
| 3 Efficient production of succinic acid from Palmaria palmata hydrolysate by metabolically engineered E. coli | 第52-76页 |
| 3.1 Introduction | 第52-54页 |
| 3.2 Materials and methods | 第54-59页 |
| 3.2.1 Strains and plasmids | 第54-56页 |
| 3.2.2 Medium | 第56页 |
| 3.2.3 Cloning and overexpression of the gene pck | 第56-57页 |
| 3.2.4 Preparation of P. palmata hydrolysate | 第57-58页 |
| 3.2.5 Batch and aerobic fermentation on pure glucose in shaking flask | 第58页 |
| 3.2.6 Dual-phase fermentation on pure glucose or galactose in shaking flask | 第58页 |
| 3.2.7 Dual-phase fermentation of sugar mixture and P. palmata hydrolysate | 第58-59页 |
| 3.3 Analytical methods | 第59页 |
| 3.4 Theory/Calculation | 第59-61页 |
| 3.5 Result and discussion | 第61-74页 |
| 3.5.1 Succinic acid production with different engineered E. coli strains during batch aerobic and anaerobic fermentation | 第61-64页 |
| 3.5.2 Glucose utilization for succinic acid production with different engineered E. coli strains during dual-phase fermentation | 第64-69页 |
| 3.5.3 Galactose utilization for succinic acid production with different engineered E. coli KLPPP strains during dual-phase fermentation | 第69-70页 |
| 3.5.4 Dual-phase fermentation of sugar mixture of glucose and galactose for succinic acid by the engineered E. coli KLPPP | 第70-72页 |
| 3.5.5 Dual-phase fermentation of P. palmata hydrolysate for succinic acid by engineered E.coli KLPPP | 第72-74页 |
| 3.6 Conclusion | 第74-76页 |
| 4 Enhanced production of succinic acid from methanol-organosolv pretreated Strophanthus preussii by recombinant E. coli | 第76-106页 |
| 4.1 Introduction | 第76-78页 |
| 4.2. Methods | 第78-85页 |
| 4.2.1 Strains and plasmids | 第78-81页 |
| 4.2.2 Media | 第81页 |
| 4.2.3 Methanol-oganosolv pretreatment of S. preussii | 第81-82页 |
| 4.2.4 Dilute acid pretreatment of S. preussii | 第82页 |
| 4.2.5 Hot liquid water pretreatment of S. preussii | 第82-83页 |
| 4.2.6 Cloning and overexpression of sthA gene | 第83-84页 |
| 4.2.7 Dual-phase fermentation on pure glucose in shaking flask | 第84页 |
| 4.2.8 Dual-phase fermentation of a modeled sugar mixture and methanol extracts of S.preussii in 5 L bioreactor by engineered E. coli | 第84-85页 |
| 4.3 Analytical methods | 第85页 |
| 4.4 Statistical Analysis | 第85页 |
| 4.5 Results and discussion | 第85-104页 |
| 4.5.1 Methanol organosolv pretreatment of S. preussii biomass | 第85-87页 |
| 4.5.2 Succinate production from sugars with pure or mixed form | 第87-93页 |
| 4.5.3 Succinic acid production by different engineered strains in LB,NBS and M9 media | 第93-98页 |
| 4.5.4 Fermentation of S. preussii by engineered strains in M9 medium using flasks | 第98-100页 |
| 4.5.5 Fermentation of a modeled methanol extracts of S. preussii by E. coli K303 in M9 medium using 5L bioreactor | 第100-101页 |
| 4.5.6 Fermentation of methanol extracts of S. preussii by E. coli K303 in M9 medium using 5L bioreactor | 第101-104页 |
| 4.6 Conclusion | 第104-106页 |
| 5 Effective production of succinic acid from Cocos nucifera water by genetically engineered E. coli | 第106-126页 |
| 5.1 Introduction | 第106-108页 |
| 5.2 Materials and methods | 第108-114页 |
| 5.2.1 Strains and plasmids | 第108-111页 |
| 5.2.2 Media | 第111页 |
| 5.2.3 Cloning and overexpression of gene pyc | 第111-113页 |
| 5.2.4 Dual-phase fermentation of pure sugar in the shaking flasks | 第113页 |
| 5.2.5 Dual-phase fermentation of a modeled sugar mixture and Cocos nucifera water in 5 L bioreactor by engineered E. coli M6PM | 第113-114页 |
| 5.3 Analytical methods and Statistical analysis | 第114页 |
| 5.4 Results and discussion | 第114-124页 |
| 5.4.1 Dual-phase fermentation of different engineered strains using glucose | 第114-115页 |
| 5.4.2 Dual-phase fermentation of different engineered strains using fructose | 第115-116页 |
| 5.4.3 Influence of sucrose substrate on dual-phase fermentations of the different engineered strains | 第116-118页 |
| 5.4.4 Dual-phase fermentation of different engineered strains after 48 h in M9 medium with different carbon sources | 第118-119页 |
| 5.4.5 Dual-phase fermentation of sugar mixture of glucose, fructose and sucrose for succinic acid production using M6PM | 第119-120页 |
| 5.4.6 Effective production of succinic acid from C. nucifera using E. coli M6PM | 第120-124页 |
| 5.5 Conclusion | 第124-126页 |
| 6 Succinate production with metabolically engineered E. coli using elephant grass stalk hydrolysate as carbon sources | 第126-138页 |
| 6.1 Introduction | 第126-127页 |
| 6.2 Materials and methods | 第127-130页 |
| 6.2.1 Strains and plasmids | 第127-128页 |
| 6.2.2 Media | 第128页 |
| 6.2.3 Cloning and overexpression of gene pyc and mdh | 第128-129页 |
| 6.2.4 Pure glucose in the shaking flasks during dual-phase fermentation | 第129页 |
| 6.2.5 Modeled sugar mixture, elephant grass stalk in 5L bioreactor by engineered E. coli M6PM during dual-phase fermentation | 第129-130页 |
| 6.3 Analytical methods | 第130页 |
| 6.4 Results and discussion | 第130-137页 |
| 6.4.1 Succinic acid production with different engineered strains during dual-phase fermentation using glucose | 第130-131页 |
| 6.4.2 Xylose utilization for succinic acid production with different engineered E. coli strain during dual-phase fermentation | 第131-132页 |
| 6.4.3 Sugar mixture utilization for succinic acid production by E.coli M6PM during dual-phase fermentation | 第132-134页 |
| 6.4.4 Elephant grass hydrolysate utilization for succinic acid production by E. coli M6PM during dual-phase fermentation using 5 L bioreactor | 第134-137页 |
| 6.5 Conclusion | 第137-138页 |
| 7 Conclusion and Prospective | 第138-142页 |
| 7.1 Conclusion | 第138-140页 |
| 7.2 Novelty | 第140页 |
| 7.3 Prospective | 第140页 |
| 7.4 Summary of possible mechanisms of succinic acid production | 第140-142页 |
| List of abbreviations | 第142-144页 |
| References | 第144-170页 |
| Supplementary materials | 第170-176页 |
| Curriculum vitae | 第176-180页 |
| Acknowledgement | 第180-181页 |
