아주대학교

목차

Chapter 1. General Introduction 1
1.1 Introduction 2
1.2 Laboratory scale long-term adaptive evolution 4
1.3 General introduction of Leuconostoc mesenteroides and Lactobacillus casei 5
1.4 Next generation sequencing (NGS) based comparative genome analysis and transcriptomics 7
1.5 Aims of this study 8
1.6 Reference 11
Chapter 2. Comparative phenotypic studies of the parental strain Leuconostoc mesenteroides ATCC 8293 and five adaptively evolved strains 15
2.1 Abstract 16
2.2 Introduction 17
2.3 Materials and Methods 19
2.3.1. Bacterial Strains and media 19
2.3.2. Lab-scale long-term adaptive evolution and mutant screening 19
2.3.3. Extraction and analysis of cellular fatty acids 22
2.3.4. Microplate scale culture and IC50 analysis by calculating maximum specific growth rate, µmax 22
2.3.5. Serum bottle scale culture 23
2.3.6. Bioreactor scale batch fermentation to assess cell growth and production of lactic acid under natural pH dropping condition 23
2.3.7. Bioreactor scale batch fermentation in acidic condition 24
2.3.8. Salinity test 24
2.3.9. Bioreactor scale batch fermentation under normal condition 24
2.3.10. Analytical methods 25
2.3.11. Statistical analysis 25
2.4. Results and discussion 26
2.4.1. Adaptive evolution and mutant screening 26
2.4.2. Lactic acid production of parental strain Leuconostoc mesenteroides ATCC 8293 and mutant strains in serum bottle culture 30
2.4.3. Assessment of cell growth and lactic acid productivity of Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains without pH buffering and controlling during batch fermentation 34
2.4.4. Batch fermentation of Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains under strictly controlled acidic pH with HCl 39
2.4.5. Comparison IC50 values on lactic acid and EtOH between the wild-type parental strain Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains 43
2.4.6. Batch fermentation of the wild-type strain Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains under optimized condition 50
2.4.7. Analysis of fatty acid composition between Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains in normal and acidic condition 55
2.4.8. Assessment of salinity in a high concentration of NaCl between the wild-type strain Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains 59
2.5 Conclusion 62
2.6 Reference 63
Chapter 3. Next generation sequencing based comparative genomic, and transcriptomic studies of the parental strain Leuconostoc mesenteroides ATCC 8293 and adaptively evolved strains 69
3.1 Abstract 70
3.2 Introduction 71
3.3 Materials and Methods 73
3.3.1. Strains, media, and culture conditions 73
3.3.2. Genome sequencing and annotation of the wild type parental strain Leuconostoc mesenteroides ATCC 8293 and adaptive evolution strains 75
3.3.3. Calculation of average nucleotide identity 76
3.3.4. Orthologous analysis and phylogenetic analysis 76
3.3.5. Protein clustering and construction of the Venn diagram 76
3.3.6. Culture conditions and experimental procedures for RNA-seq based transcriptomics 77
3.3.7. RNA-seq based transcriptome analysis 77
3.3.8. Pathway and network analysis 78
3.3.9. Statistical analysis 78
3.4. Results and discussion 79
3.4.1. NGS-based complete genome sequencing and annotation 79
3.4.2. Complete genome sequence based comparative genomics and phylogenetic analysis 85
3.4.3. Batch fermentation and RNA-sequencing of the wild-type strain Leuconostoc mesenteroides ATCC 8293 and final adaptive evolution strain LMA-100A 101
3.4.4. RNA-seq based clustering analysis of expressed genes 111
3.4.5. Differential expression analysis of Leuconostoc mesenteroides ATCC 8293 and the final adaptive evolution strain LMA-100A at two growth phases 114
3.4.6. Global transcriptome analysis in Leuconostoc mesenteroides ATCC 8293 and the final adaptive evolution strain LMA-100A to investigate acid tolerace mechanisms 131
3.5 Conclusion 140
3.6 Reference 142
Chapter 4. Development of synthetic biology tools in Leuconostoc mesenteroides ATCC 8293 146
4.1 Abstract 147
4.2 Introduction 148
4.3 Materials and Methods 150
4.3.1. Bacterial strains, plasmids, and media 150
4.3.2. Plasmid construction 153
4.3.3. Electroporation method 156
4.3.4. Assessment of plasmid stability 157
4.3.5. Measurement of fluorescence intensity using a microplate reader 157
4.3.6. Microscopic imaging analysis 158
4.3.7. FACS analysis 158
4.3.8. Reverse transcription polymerase chain reaction 158
4.4. Results and discussion 160
4.4.1. Optimization of transformation efficiency in Leuconostoc mesenteroides ATCC 8293 160
4.4.2. Assessment of plasmid stability in Leuconostoc mesenteroides ATCC 8293 by altering the origin of replication and replication initiation proteins in plasmid DNA 169
4.4.3. Fluorescence protein expression in Leuconostoc mesenteroides ATCC 8293 174
4.4.4. Screening and assessment of endogenous promoters in Leuconostoc mesenteroides ATCC 8293 176
4.5 Conclusion 180
4.6 Reference 181
Appendix. In-silico based core region prediction and engineering of endogenous aldolase promoter (PALD) in Lactobacillus casei BL 23 185
A.1 Abstract 186
A.2 Introduction 187
A.3 Materials and Methods 190
A.3.1 Strains and media 190
A.3.2 In-silico promoter region finding and library design 190
A.3.3 Plasmid and promoter library construction in Escherichia coli 191
A.3.4 Library transformation and culture in Lactobacillus casei BL 23 192
A.3.5 Fluorescence-activated cell sorting analysis 192
A.3.6 Measurement of fluorescence intensity 193
A.3.7 Library plasmid DNA extraction and sequencing 193
A.4 Results and discussion 202
A.4.1 Assessment of fluoresence protein expression in Lactobacillus casei BL23 strain 202
A.4.2 Construction of synthetic aldolase promoter library and verification of sequence diversity 211
A.4.3 Fluorescence-activated cell sorting analysis and sorting 216
A.4.4 Assessment of promoter library and measurement of single promoter intensity 220
A.4.5 Sequence analysis of single library promoters 225
A.5 Conclusion 232
A.6 Reference 233
ABSTRACT IN KOREAN 240