Title Page
ABSTRACT
Contents
ABBREVIATIONS 21
CHAPTER 1. Overall Introduction 23
1.1. The biorefinery and genetic engineering 24
1.2. White Bio and Synthetic Biology 26
1.3. Microbial tolerance and engineering 29
1.4. The overall characteristics of the minicells 35
1.5. Objective 40
CHAPTER 2. Minicell-forming Escherichia coli mutant with increased chemical production capacity and tolerance to toxic compounds 42
2.1. Introduction 43
2.2. Experimental section 47
2.2.1. Genetic manipulation 47
2.2.2. Medium and cultivation 56
2.2.3. Analytical methods 57
2.2.4. Analysis of intracellular NADH/NAD+ ratio 58
2.2.5. Bacterial toxicity assay 59
2.3. Results and discussion 62
2.3.1. Construction of minicell-forming mutants 62
2.3.2. Improved bacterial tolerance in minicell-forming mutants 69
2.3.3. Elevation of NADH/NAD+ ratio in minicell-forming mutants 76
2.3.4. Chemical production in minicell-forming mutants 80
2.3.5. Lycopene production in minicell-forming mutants 86
2.4. Conclusion 91
CHAPTER 3. Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds 93
3.1. Introduction 94
3.2. Experimental section 97
3.2.1. Genetic manipulation 97
3.2.2. Medium and cultivation 109
3.2.3. Purification of minicells 110
3.2.4. Chemical production using minicells 111
3.2.5. Analytical methods 111
3.2.6. Toxic chemical tolerance assay 114
3.3. Results and Discussions 114
3.3.1. Biological Characteristics of Minicell 115
3.3.2. Minicells engineered to concentrate targeted proteins 121
3.3.3. Toxic chemical production in engineered minicells 130
3.3.4. Autoinducer production in engineered minicells 138
3.4. Conclusion 142
CHAPTER 4. Utilization of autoinducers produced by engineered minicells 143
4.1. Introduction 144
4.2. Experimental section 147
4.2.1. Genetic manipulation 147
4.2.2. Medium and cultivation 157
4.2.3. Analytic methods 157
4.3. Results and discussions 158
4.3.1. Regulation of bacterial expression using AI produced from minicells 158
4.3.2. Bacterial growth control by autoinducer producing minicells 162
4.3.3. Simultaneous utilization of two different engineered minicells 167
4.4. Conclusion 172
CHAPTER 5. Overall Conclusion and discussion 173
5.1. Conclusion and discussion 174
References 182
Table 1.1. Examples of tolerance engineering 31
Table 2.1. Bacterial strains and plasmids used in this chapter 49
Table 2.2. Oligonucleotides used in this chapter 53
Table 3.1. Bacterial strains and plasmids used in this chapter 99
Table 3.2. Oligonucleotides used in this chapter 104
Table 3.3. Reported protein production studies in minicell 127
Table 4.1. Bacterial strains and plasmids used in this chapter 148
Table 4.2. Oligonucleotides used in this chapter 153
Figure 1.1. Scheme of the DBTL cycle (Design (D), Build (B), Test (T), and Learning(L)) in microbial cell process systems 28
Figure 1.2. Scheme of vesicles formation about outer membrane vesicle. 36
Figure 1.3. Scheme of vesicles formation about minicell 39
Figure 2.1. Factors causing abnormal cell division. 45
Figure 2.2. Scheme of resistance experiments. 61
Figure 2.3. Factors causing abnormal cell division and their effects on minicell production. 64
Figure 2.4. The results of staining with SYTO9 (a and c) and colony PCR of EMM01 (b). 66
Figure 2.5. The correlation between optical density and dry cell weight in minicells. 68
Figure 2.6. Bacterial growth inhibition by toxic chemicals. 70
Figure 2.7. Biofilm formation and growth inhibition by toxic chemicals. 74
Figure 2.8. Comparison of bacterial growth and NADH/NAD+ ratio during microbial cultivation.[이미지참조] 78
Figure 2.9. Toxic chemical production in minicell-forming mutants on introducing the pathways. 81
Figure 2.10. Lycopene production in n-dodecane-supplemented culture media for 24 h. 89
Figure 3.1. NADH regeneration (a), ATP generation (b) and glucose consumption (c) in minicells. 116
Figure 3.2. Stability of plasmids in bacterial cells and minicells. 118
Figure 3.3. Comparative advantages minicells over bacterial cells in terms of consistent amount of protein expression and stable production. 120
Figure 3.4. Scheme of engineered minicell-forming mutant construction. 122
Figure 3.5. Cardiolipin measurement and ProP linked GFP intensity detection. 124
Figure 3.6. The results of GFP expression in engineered bacterial cells and minicells. 126
Figure 3.7. Comparison of microbial cultivation and chemical production results. 129
Figure 3.8. Comparison of toxic chemical tolerance from C6-C8 aldehyde or alcohol toxicity. 132
Figure 3.9. C6-C10 toxic chemicals production pathway (a-d) and the results of toxic chemical production by separated minicells or... 136
Figure 3.10. The activated methyl cycle (a) and schemes of engineered minicells producing autoinducers (b and c). 139
Figure 3.11. Experimental results to detect production of acyl-homoserine lactones. 141
Figure 4.1. Production of autoinducers and activation of gene circuit through it. 160
Figure 4.2. Changes in cell growth with the expression of ptsH or mazF under the control of the inducible promoter. 165
Figure 4.3. The overall experimental scheme and the dynamic growth results of the expression of mazF in the presence of mazE expression. 168
Figure 4.4. Dynamic bacterial growth regulation depending on the type and concentration of the added minicells (a) and the results of... 171
Figure 5.1. Comparison of NADH/NAD+ ratio in bacterial cells (Orange bar) and minicell (Beige bar) cultivation of EMM01 strain.[이미지참조] 175
Figure 5.2. The hexanol production results of bacterial cells (a) and minicells (b) as exposed to toxic chemicals. 177
Figure 5.3. The effects of storage (a and b) and recycle (c and d) of the cells on toxic chemical production. 179