Title Page
Abstract
Contents
List of Abbreviations 12
Chapter 1. Research background and objective 14
Chapter 2. Literature review 17
2.1. Importance of biological conversion of methane 18
2.1.1. Methane 18
2.1.2. Biological conversion of methane 19
2.1.3. sMMO and pMMO 20
2.2. Recent developments in methanotrophs 26
2.2.1. Assimilation pathways of aerobic methanotrophs 26
2.2.2. Genetic manipulation tools in methanotrophs 28
2.2.3. Chemicals produced in methanotrophs 29
2.2.4. Methylomonas sp. DH-1 33
2.3. Microbial production of lactic acid 34
2.3.1. Lactic acid 34
2.3.2. LA production by lactic acid bacteria 35
Chapter 3. Materials and methods 41
3.1. Strains and culture conditions 42
3.2. Plasmids 44
3.3. Gene manipulation in Methylomonas sp. DH-1 47
3.4. Adaptive laboratory evolution 47
3.5. Whole genome sequencing analysis 48
3.6. Quantitative transcription PCR (qRTP-PCR) and RNA sequencing analysis 48
3.7. Fermenter culture condition 50
Chapter 4. Production of D-lactate using lactate tolerant strain of Methylomonas sp. DH-1 52
4.1. Introduction 53
4.2. Development of LA tolerant strain of Methylomonas sp. DH-1 55
4.3. Selecting antibiotic resistance genes as selection marker 60
4.4. Expression of heterologous D-lactate dehydrogenase in JHM80 64
4.5. Conclusion 69
Chapter 5. Analysis of LA tolerance mechanism in evolved strains 70
5.1. Introduction 71
5.2. Whole genome sequencing 73
5.3. Functional analysis of mutated genes 76
5.4. RNA sequencing of watR overexpressed strain 79
5.5. RND-type efflux pump contributes to organic acid tolerance in Methylomonas sp. DH-1 83
5.6. Conclusion 89
Chapter 6. Improvement of LA production by fine-tuned expression of LDH and reducing toxic effects 90
6.1. Introduction 91
6.2. Expression of Lm. LDH using different promoters 93
6.3. Evaluation of tac promoter for LA production 99
6.4. Improvement of D-LA production by disruption of glucose 1-phosphate adenylyltransferase 103
6.5. Conclusion 110
Chapter 7. Discussion 111
References 115
Abstract in Korean 130
Table 2.1. Chemicals produced in methanotrophs 31
Table 2.2. Chemicals produced in methanotrophs 39
Table 2.3. Chemicals produced in methanotrophs 40
Table 3.1. Strains used in this study 43
Table 3.2. Strains used in this study. 46
Table 3.3. Primers used for qRT-PCR 51
Table 5.1. Results of whole genome sequencing analysis of the evolved strains 74
Figure 2.1. The structure of Soluble methane monooxygenase (sMMO) 22
Figure 2.2. Three possible mechanisms of methane monooxygenase in pMMO 24
Figure 2.3. Metabolic pathway of Type I methanotroph (RuMP pathway, green) and Type II methantoroph (Serine cycle, blue and EMC pathway, red) 27
Figure 2.4. Structure of stereo-isomers of lactic acid 36
Figure 2.5. Metabolic pathways of LAB for LA production 37
Figure 4.1. Growth profiles of methanotrophic cells. 57
Figure 4.2. Improved LA tolerance in the evolved strains 59
Figure 4.3. Strategy for negative selection in Methylomonas sp. DH-1 62
Figure 4.4. Antibiotic sensitivity of Methylomonas sp. DH-1 63
Figure 4.5. Metabolic pathway for the D-LA production from methane in Metyhlomonas sp. DH-1 66
Figure 4.6. D-LA production by expressing lactate dehydrogenase in JHM80 67
Figure 4.7. Improvement of LA production by neutralization and medium optimization 68
Figure 5.1. Effect of TT deletion on transcription of the downstream operons 75
Figure 5.2. Effect of watR gene deletion on LA tolerance of JHM80 77
Figure 5.3. Effect of watR gene overexpression on LA tolerance 78
Figure 5.4. Effect of fliE gene deletion on LA tolerance of JHM30 79
Figure 5.5. RNA sequencing of JHM80 and JHM11 81
Figure 5.6. RNA sequencing of JHM80 and JHM11 82
Figure 5.7. RNA sequencing of JHM80 and JHM11 84
Figure 5.8. Lactate efflux function of RND-type efflux pump 85
Figure 5.9. Effects of watR gene and efflux pump deletion on organic acid tolerance 88
Figure 6.1. D-LA production by expressing Lm. LDH by constitutive promoters with different strengths. 95
Figure 6.2. Growth inhibitory effect of anhydrotetracyclien in JHM80 97
Figure 6.3. D-LA production by expressing Lm. LDH using inducible promoters 98
Figure 6.4. Evaluation of Tac promoter for LA production 100
Figure 6.5. Effect of culture optimization on LA production in JHM803 101
Figure 6.6. Effect of focA expression on LA production in JHM804 102
Figure 6.7. D-LA production by expressing Lm. LDH by constitutive promoters with different strengths. 105
Figure 6.8. Effect of glgC gene deletion on the growth of JHM804 106
Figure 6.9. Fermenter culture of JHM806 108
Figure 6.10. Improvement of D-LA production by optimizing culture condition 109