목차

CHAPTER I: Introduction
1.1. Applications of 3-hydroxypropionic acid and 1,3-propanediol 27
1.1.1. Applications of 3-hydroxypropionic acid 27
1.1.2. Applications of 1,3-PDO 30
1.2. Chemical production of 3-HP and 1,3-PDO 32
1.3. Microbial production of 3-HP and 1,3-PDO 34
1.4. Klebsiella pneumoniae: Host for production of 3-HP and 1,3-PDO 37
1.6. Scope and objective 48
1.7. References 49
CHAPTER II: Development of Klebsiella pneumoniae mutant strains to eliminate 1,3-propanediol during 3-hydroxypropionic acid production from glycerol
2.1. Introduction 55
2.1. Material and methods 57
2.1.1. Materials 57
2.1.2. Identification of putative oxidoreductases by amino acid sequence homology 57
2.1.3. Plasmid construction and expression of putative oxidoreductases in recombinant E. coli 58
2.1.4. Determination of enzyme activities 61
2.1.5. RNA extraction and real-time PCR 61
2.1.6. Development of K. pneumoniae ΔdhaTΔyqhDΔahpF and K. pneumoniae ΔdhaTΔyqhDΔahpFΔadhE mutant strains 62
2.1.7. Culture conditions 63
2.1.8. Analytical methods 64
2.2. Results and Discussion 65
2.2.1. Putative 1,3-propanediol oxidoreductases were identified in Klebsiella pneumoniae 65
2.2.2. Some putative oxidoreductases showed higher mRNA levels when dhaT and yqhD genes were deleted 69
2.2.3. Putative oxidoreductases were expressed in recombinant E. coli BL21 (DE3) and measured for oxidoreductase activity 71
2.2.4. Deletion of ahpF and adhE in K. pneumoniae ΔdhaTΔyqhD neither improved 3-HP production nor eliminated 1,3-PDO production from glycerol 75
2.2.5. Cell-free extracts of KpΔdhaTΔyqhDΔahpF and KpΔdhaTΔyqhDΔahpFΔadhE still showed 1,3-PDOR activity 81
2.3. Conclusion 84
2.4. References 85
CHAPTER III: Evaluation of newly isolated Klebsiella pneumoniae strains for the co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol
3.1. Introduction 89
3.2. Materials and methods 92
3.2.1. Strain and materials 92
3.2.2. Construction of recombinant K. pneumoniae strains overexpressing AldH 92
3.2.3. Culture condition 95
3.2.4. Enzyme activities 95
3.2.5. Analytical methods 96
3.3. Results and discussion 97
3.3.1. Comparison of sedimentation behavior between GSC021 and J2B 97
3.3.2. Comparison of glycerol assimilation and coenzyme B12 synthesis genes in new isolated strains 99
3.3.3. Cell growth and co-production performance with different aeration 102
3.3.4. Cell growth and co-production performance with different temperature 107
3.3.5. Cell growth and co-production performance with different culture media 109
3.3.6. Evaluation of key enzyme activities related to co-production of 3-HP and 1,3-PDO 111
3.4. Conclusion 114
3.5. References 115
CHAPTER 1V: Metabolic engineering of Klebsiella pneumoniae J2B for co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol
4.1. Introduction 119
4.2. Materials and methods 124
4.2.1. Materials 124
4.2.2. Construction of plasmids and mutant strains 124
4.2.3. Culture conditions 127
4.2.4. Enzyme activity assay 127
4.2.5. Analytical methods 128
4.3. Results and discussion 129
4.3.1. Improvement of co-production by deletion of by-products formation pathway 129
4.3.2. Reduction of acetate production by down-regulation of assimilatory glycerol metabolism 132
4.3.3. Reduction of acetate accumulation by upregulating co-production pathway 136
4.3.4. Effect of coenzyme B12 addition on co-production by K. pneumoniae mutants 144
4.3.5. Effect of aeration on co-production in bioreactor scale 146
4.3.6. Evaluation of proficient mutants for co-production in bioreactor scale 150
4.4. Conclusion 155
4.5. References 156
CHAPTER V: Characterization of glycerol metabolism in Klebsiella pneumoniae and reduction of acetate accumulation by activation of tricarboxylic acid cycle
5.1. Introduction 164
5.2. Materials and methods 167
5.2.1. Materials 167
5.2.2. Construction of plasmids and mutant strains 168
5.2.3. Protein expression and gel electrophoresis 170
5.2.4. Culture conditions 170
5.2.5. Determination of enzyme activities 171
5.2.6. Gene expression analysis 171
5.2.7. Analytical methods 172
5.3. Results and discussion 173
5.3.1. Comparison of glycerol metabolism between K. pneumoniae and E. coli 173
5.3.2. Analysis of gene expression and activity of TCA cycle enzymes 178
5.3.3. Operation of TCA/glyoxylate shunt by deletion of IclR and ArcA 182
5.3.4. Overexpression of gltA and ppc related to anaplerotic pathway increase TCA cycle flux 186
5.3.5. Gene expression analysis of electron transport chain components in K. pneumoniae and E. coli 190
5.4. Conclusion 193
5.5. References 194
CHAPTER VI: Coenzyme B12 can be produced by engineered Escherichia coli under both aerobic and anaerobic conditions
6.1. Introduction 200
6.2. Materials and methods 208
6.2.1. P. denitrificans and E. coli strains, pRcob, pCcob, and pAcob plasmids, genetic methods, and materials 208
6.2.2. Cloning of cob genes into expression plasmids 210
6.2.3. Real-time PCR for quantification of mRNA 216
6.2.4. Cob proteins expression and gel electrophoresis 217
6.2.5. Culture medium and conditions to cultivate recombinant E. coli overexpressing coenzyme B12 synthetic pathway 217
6.2.6. Purification of coenzyme B12 218
6.2.7. Analytical methods to quantify cell and protein concentrations 219
6.3. Results 221
6.3.1. Recombinant plasmid constructions with P. denitrificans coenzyme B12 synthetic operons 221
6.3.2. Coenzyme B12 synthesis pathway expression in recombinant E. coli was confirmed by real-time PCR and SDS?PAGE 224
6.3.3. Recombinant E. coli produced larger amounts of coenzyme B12 than wild type P. denitrificans 231
6.3.4. Coenzyme B12 syntheses by recombinant E. coli was improved by varying culture conditions 236
6.4. Discussion 238
6.5. References 242
CHAPTER VII: Epilogue and prospects
7.1. Summary 247
7.2. Prospects 249
Abstract 251