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Improving Biochemicals Production using Synthetic Biology Tools in Escherichia coli

초록/요약

In metabolic engineering, a number of heterologous genes are introduced in host strains, such as Escherichia coli and Saccharomyces cerevisiae, for production of natural products, chemicals and fuels. However, microbial cell factories have problems to be solved in order to produce high yields of target products. Especially, when a metabolic pathway is constructed using heterologous genes, which is a non-natural substance, low gene expression and low enzyme activity result in low production yield. Recently, synthetic biology tools have been used to overcome these problems. In this thesis, I have been carried out as followings: 1) securing of Bld mutants to be used to replace AdhE2 in 1,4-butanediol (BDO) biosynthesis pathway, 2) establishing applicability of the nar promoter for metabolic engineering to replace the inducible promoter, and 3) enhancing biochemical production through the synthetic nar promoter. First, adhE2, a gene used in the 1,4-BDO biosynthetic pathway, was replaced with a bld gene. The production of 1,4-butanediol using both Bld and Bdh enzymes was lower than AdhE2. However, 1,4-BDO production was increased due to BldL273I, a mutant of Bld obtained through directed evolution. BldL273T, which is a mutant secured through saturated mutagenesis, produced an approximately 4-fold higher production than wild-type Bld. Genomic editing of E. coli also produced minimal byproducts and increased 1,4-BDO production. In engineered E.coli, AdhE2 produced 180 ± 30 mg/L of 1,4-BDO and BldL273T produced 660 ± 40 mg/L of 1,4-BDO, approximately 3.6-fold higher. Second, a dissolved oxygen (DO) dependent nar promoter was applied to the biosynthesis pathway of the target product in order to overcome the disadvantages of inducible promoters, including chemical inducer cost and abrupt changes of the microbial environment. To confirm the applicability of the nar promoter under micro-aerobic conditions, the D-lactic acid, (R, R)-2,3-butanediol (2,3-BDO) and 1,3-propanediol (1,3-PDO) biosynthesis pathways were used. The production of D-lactic acid, (R, R)-2,3-BDO and 1,3-PDO were 113.12 ± 2.37 g/L, 48.0 ± 8.48 g/L and 15.8 ± 0.62 g/L, respectively. These results were higher production than other reported production in E.coli and, confirmed that the nar promoter could be used for metabolic engineering. Finally, biosynthesis pathway genes under the control of the synthetic nar promoter libraries were fine-tuned to improve biochemical production. The synthetic nar promoter library was selected using GFP as a reporter protein, and three promoters were selected. The pathway enzymes were regulated by each synthetic promoter. Production of D-lactic acid was 105.56 g/L with synthetic strong promoter in fed-batch fermentation. The combination of ilvBN encoding acetohydroxy acid synthase with synthetic strong promoter, BDH1 encoding butanediol dehydrogenase with synthetic strong promoter and aldB encoding acetolactate decarboxylase with synthetic weak promoter produced 87.95 g/L of (R,R)-2,3-BDO in fed-batch fermentation. In conclusion, through DNA assembly, protein engineering and promoter engineering, secured BldL273T produced improved production of 1,4-BDO, and the synthetic nar promoter as an alternative inducible promoter was used to fine-tuning gene expression for increasing biochemical production.

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목차

Chapter 1. General Introduction 1
1.1. Synthetic biology 2
1.2. Synthetic biology tools for metabolic engineering 3
1.2.1. DNA assembly method 3
1.2.2. Genome editing method 3
1.2.3. Promoter engineering 4
1.2.4. Metabolite sensor 5
1.3. Protein engineering 6
1.4. Biochemical production in E.coli 7
1.5. Aims of this study 9
Chapter 2. Protein Engineering of Butyraldehyde Dehydrogenase for Enhancing Production of 1,4-Butanediol 10
2.1. Abstract 11
2.2. Introduction 12
2.3. Materials and methods 15
2.3.1. Bacterial strains, plasmids, and growth conditions 15
2.3.2. Construction of synthetic expression modules 15
2.3.3. Error-prone PCR mutagenesis and screening 16
2.3.4. Site-directed mutagenesis 17
2.3.5. Expression of proteins 17
2.3.6. Purification of proteins 17
2.3.7. Enzyme assays 18
2.3.8. Fermentation conditions 19
2.3.9. Analytical methods 19
2.3.10. Computational methods 20
2.4. Results and discussion 25
2.4.1. Selection of bld and bdh of C. saccharoperbutylacetonicum 25
2.4.2. Directed evolution of Bld 27
2.4.3. Analysis of Bld mutants 30
2.4.4. Second-generated Bld L273X mutants 34
2.4.5. Enzyme assays for wild-type Bld, L273I, and L273T 37
2.4.6. 1.4-BDO Production in knock-out strain 39
2.5. Conclusion 41
Chapter 3. Application of an Oxygen-Inducible nar Promoter System for Production of D-Lactate, 2,3-Butanediol and 1,3-Propanediol 42
3.1. Abstract 43
3.2. Introduction 44
3.3. Materials and Methods 48
3.3.1. Strains and plasmids 48
3.3.2. Media and Cultivation 49
3.3.3. Protein analysis 50
3.3.4. Fluorescence analysis 50
3.3.5. Metabolite analysis 51
3.4. Results and discussion 56
3.4.1. Evaluation of nar promoter using GFPm as a reporter protein 56
3.4.2. Production of D-lactate by the nar promoter 58
3.4.3. Production of (R,R)-2,3-BDO by the nar promoter 61
3.4.4. Production of 1,3-PDO by the nar promoter 64
3.5. Conclusion 67
Chapter 4. Development and Application of Synthetic nar Promoters for Production of D-Lactate and 2,3-Butanediol 68
4.1. Abstract 69
4.2. Introduction 70
4.3. Materials and Methods 72
4.3.1. Strains 72
4.3.2. Construction of synthetic promoter library 72
4.3.3. Manipulation of plasmids 72
4.3.4. Screening of the promoter library 73
4.3.5. Production of D-lactate and 2,3-BDO 74
4.3.6. Transcriptional analysis 75
4.3.7. Protein analysis 75
4.3.8. Fluorescence analysis 76
4.3.9. Metabolite analysis 76
4.4. Results and discussion 83
4.4.1. Construction and screening of the synthetic nar promoter library 83
4.4.2. Promoter strength analysis of the synthetic promoters by GFP fluorescence intensity 86
4.4.3. Characterization of the selected synthetic promoters 88
4.4.4. Production of D-lactate with synthetic promoter 91
4.4.5. Production of acetoin and 2,3-BDO with synthetic promoters 94
4.5. Conclusion 98
CONCLUSIONS 100
REFERENCE 102
ABSTRACT IN KOREAN 114
ACKNOWLEDGEMENTS 117

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