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Improving Biochemicals Production using Synthetic Biology Tools in Escherichia coli
- Alternative Title
- Hee Jin Hwang
- Author(s)
- 황희진
- Alternative Author(s)
- Hee Jin Hwang
- Advisor
- 이평천
- Department
- 일반대학원 분자과학기술학과
- Publisher
- The Graduate School, Ajou University
- Publication Year
- 2017-02
- Language
- eng
- Alternative Abstract
- 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.
- Fulltext
- Appears in Collections:
- Graduate School of Ajou University > Department of Molecular Science and Technology > 4. Theses(Ph.D)
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