| ACKNOWLEDGEMENTS | 第5-11页 |
| LIST OF ABBREVIATIONS | 第11-12页 |
| ABSTRACT | 第12页 |
| 摘要 | 第13-18页 |
| CHAPTER 1 GENERAL INTRODUCTION AND REVIEW OF LITERATURE | 第18-45页 |
| 1.1. INTRODUCTION | 第18-42页 |
| 1.1.1. Yeast (S. cerevisiae) | 第18-24页 |
| 1.1.1.1. S. cerevisiae as ethanol producer | 第19-21页 |
| 1.1.1.2. Factors weakening the activity and growth of yeast | 第21-24页 |
| 1.1.1.2.1. Ethanol stress | 第21-23页 |
| 1.1.1.2.2. Acid stress | 第23页 |
| 1.1.1.2.3. Other stresses | 第23-24页 |
| 1.1.2. The ethanol as alternative biofuel | 第24-33页 |
| 1.1.2.1. Classification of biofuels | 第28-31页 |
| 1.1.2.2. Biological ethanol production | 第31-32页 |
| 1.1.2.3. Ethanol production by other microorganisms | 第32-33页 |
| 1.1.3. Metabolic engineering approaches | 第33-42页 |
| 1.1.3.1. Approaches for phenotype engineering in S. cerevisiae | 第34-36页 |
| 1.1.3.2. Classical strain engineering methodology | 第36-42页 |
| 1.1.3.2.1. Approach of typical and random mutagenesis | 第36-37页 |
| 1.1.3.2.2. Approach of gTME | 第37-42页 |
| 1.2. Statement of the problem and justification of research | 第42-45页 |
| 1.2.1. Objectives of the research | 第42-45页 |
| CHAPTER2 Initial studies to construct random libraries of mutants for enhancing bioethanol production by S. cerevisiae using various molecular biology methods | 第45-68页 |
| 2.1. INTRODUCTION | 第45-46页 |
| 2.2. MATERIAL AND METHOD | 第46-63页 |
| 2.2.1. Microbial strains and culture media | 第46-48页 |
| 2.2.2. Reagents | 第48-49页 |
| 2.2.3. Method of Yeast Mating (hybridization of MAT-a and MAT-α) | 第49-52页 |
| 2.2.4. Target Genes | 第52-54页 |
| 2.2.4.1. Yeast Genomic DNA (g DNA) extraction (Template) | 第53-54页 |
| 2.2.5. Design of primers and vectors | 第54-62页 |
| 2.2.5.1. PCR thermal program | 第56页 |
| 2.2.5.2. Standard (Normal) PCR with Taq enzyme | 第56-58页 |
| 2.2.5.3. Method to add“A”Base for both of SPT15 gene & TAF23 | 第58页 |
| 2.2.5.4. Purification of DNA for sequences for both of genes (SPT15 & TAF23) | 第58-59页 |
| 2.2.5.5. Ligation the DNA product with T-Vector PMD19 (Simple) for bothof genes (SPT15 & TAF23) | 第59页 |
| 2.2.5.6. Thermal transformation Competent Cell (E. coli JM109) for both ofgenes (SPT15 & TAF23) | 第59-60页 |
| 2.2.5.7. Plasmid purification for both of SPT15 & TAF23 genes | 第60-61页 |
| 2.2.5.8. Restriction Enzymes Digested for both of SPT15 gene & TAF23 | 第61页 |
| 2.2.5.9. Verified for the sequences of samples | 第61-62页 |
| 2.2.6. Plasmids construction with the target genes | 第62-63页 |
| 2.3. RESULTS AND DISCUSSION | 第63-67页 |
| 2.3.1.Hybridization, amplification and construction of pMD19-T-Amp-SPT15and pMD19-T-Amp-TAF23 | 第63-67页 |
| 2.3.1.1. Cloning of the target genes | 第64-65页 |
| 2.3.1.2. Stemp and standard (Normal) PCR | 第65页 |
| 2.3.1.4. Method to add“A”Base for both of SPT15 gene & TAF23 | 第65-66页 |
| 2.3.1.5. Restriction Enzymes Digested for both of SPT15 gene & TAF23 | 第66页 |
| 2.3.1.6. Verified for the sequences of samples | 第66-67页 |
| 2.4. Plasmids construction with the target genes | 第67-68页 |
| CHAPTER3 GENETIC ENGINEERING OF S. cerevisiae USING A NOVEL APPROACH GLOBAL TRANSCRIPTION MACHINERY ENGINEERING (GTME) FOR ENHANCING BIOETHANOL PRODUCTION | 第68-92页 |
| 3.1. INTRODUCTION | 第68-71页 |
| 3.2. MATERIALS AND METHODS | 第71-81页 |
| 3.2.1. Media and Reagents | 第71-72页 |
| 3.2.2. Microbial strains | 第72-73页 |
| 3.2.3. Oligonucleotide primers | 第73页 |
| 3.2.4. Extraction, cloning of the target genes | 第73-74页 |
| 3.2.5. Plasmids construction with the target genes | 第74-75页 |
| 3.2.6. Construction of random mutant genes libraries | 第75-76页 |
| 3.2.6.1. Protocol of unbalanced 10XdNTP Mix | 第76页 |
| 3.2.7. Transformation for SPT15-Mu and TAF23-Mu genes into S. cerevisiae | 第76-81页 |
| 3.2.7.1. PCR thermal program for amplification of sequences | 第78页 |
| 3.2.7.2. Verified for the sequences of samples | 第78-81页 |
| 3.3. RESULTS AND DISCUSSIONS | 第81-89页 |
| 3.3.1. Plasmids construction with the target genes | 第81-83页 |
| 3.3.2. Construction of mutants | 第83-89页 |
| 3.3.3. The primary features of the amendment | 第89页 |
| 3.4. CONCLUSION | 第89-92页 |
| CHAPTER OPTIMIZING THE OPTIMAL CONDITIONS TO OBTAIN STABLE MUTANTS TO ENHANCE ETHANOL PRODUCTION OF GLUCOSE USING S. cerevisiae | 第92-117页 |
| 4.1. INTRODUCTION | 第92-94页 |
| 4.2. MATERIALS AND METHODS | 第94-99页 |
| 4.2.1. Microbial strains, culture media and reagents | 第94-95页 |
| 4.2.2. Studies to verify stability and similarity for SPT15-Mu and TAF23-Mugenes to survive after transformation of into S. cerevisiae | 第95-97页 |
| 4.2.3. Investigation of ethanol tolerance of S. cerevisiae mutant strains | 第97页 |
| 4.2.4. Growth rate of mutants | 第97-99页 |
| 4.2.4.1. Mutant’s optical density | 第98-99页 |
| 4.3. RESULTS AND DISCUSSION | 第99-116页 |
| 4.3.1. Studies to verify stability and similarity for SPT15-Mu and TAF23-Mugenes to survive after transformation of into S. cerevisiae | 第99-102页 |
| 4.3.2. Investigation of ethanol tolerance of S. cerevisiae mutant strains | 第102-104页 |
| 4.3.3. Mutant’s optical density | 第104-116页 |
| 4.4. CONCLUSION | 第116-117页 |
| CHAPTER DETERMINE THE BEST MUTANT STRAIN ABLE TO ETHANOL PRODUCTION BETTER THAN R-CONTROL· The Specific objectives for this chapter | 第117-144页 |
| 5.1. INTRODUCTION | 第117-123页 |
| 5.2. MATERIALS AND METHODS | 第123-129页 |
| 5.2.1. Reagents | 第123页 |
| 5.2.2. Hybridization between MAT-a and MAT-α | 第123-126页 |
| 5.2.3. Library construction, sequencing, and functional assay | 第126-128页 |
| 5.2.4. Transformation for SPT15-Mu and TAF23-Mu genes into S. cerevisiae | 第128页 |
| 5.2.5. The concentration of ethanol and glucose by SPT15-Mu aerobic fermentation | 第128-129页 |
| 5.2.5.1. Reaction conditions | 第128页 |
| 5.2.5.2. High-performance liquid chromatography analysis | 第128-129页 |
| 5.3. RESULTS AND DISCUSSIONS | 第129-143页 |
| 5.3.1. Hybridization between MAT-a and MAT-α | 第129页 |
| 5.3.2. Construction of random mutant libraries, Sequencing, and Functional assay | 第129-131页 |
| 5.3.3. The concentration of ethanol and glucose by SPT15-Mu aerobic fermentation | 第131-143页 |
| 5.4. CONCLUSION | 第143-144页 |
| REFERENCES | 第144-152页 |
| CHAPTER GENERAL CONCLUSIONS AND RECOMMENDATIONS | 第152-154页 |
| 6.1. General conclusions | 第152页 |
| 6.2. Key Innovation of thesis | 第152-153页 |
| 6.3. KEY INNOVATION OF THESIS | 第153-154页 |
| LIST OF PUBLICATIONS | 第154-155页 |
| APPENDICES | 第155-170页 |
